STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue.

PubMed

Ilgen, Peter; Stoldt, Stefan; Conradi, Lena-Christin; Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B Michael; Liersch, Torsten; Jakobs, Stefan

2014-01-01

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.

STED Super-Resolution Microscopy of Clinical Paraffin-Embedded Human Rectal Cancer Tissue

PubMed Central

Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B. Michael; Liersch, Torsten; Jakobs, Stefan

2014-01-01

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories. PMID:25025184

A Comparison of Raman Spectral Features of Frozen and Deparaffinized Tissues in Neuroblastoma and Ganglioneuroma

NASA Astrophysics Data System (ADS)

Devpura, Suneetha; Thakur, Jagdish S.; Poulik, Janet M.; Rabah, Raja; Naik, Vaman M.; Naik, Ratna

2012-02-01

We have investigated the cellular regions in neuroblastoma and ganglioneuroma using Raman spectroscopy and compared their spectral characteristics with those of normal adrenal gland. Thin sections from both frozen and deparaffinized tissues, obtained from the same tissue specimen, were studied in conjunction with the pathological examination of the tissues. We found a significant difference in the spectral features of frozen sections of normal adrenal gland, neuroblastoma, and ganglioneuroma when compared to deparaffinized tissues. The quantitative analysis of the Raman data using chemometric methods of principal component analysis and discriminant function analysis obtained from the frozen tissues show a sensitivity and specificity of 100% each. The biochemical identification based on the spectral differences shows that the normal adrenal gland tissues have higher levels of carotenoids, lipids, and cholesterol compared to the neuroblastoma and ganglioneuroma frozen tissues. However, deparaffinized tissues show complete removal of these biochemicals in adrenal tissues. This study demonstrates that Raman spectroscopy combined with chemometric methods can successfully distinguish neuroblastoma and ganglioneuroma at cellular level.

Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

PubMed Central

Fu, Henry L.; Mueller, Jenna L.; Javid, Melodi P.; Mito, Jeffrey K.; Kirsch, David G.; Ramanujam, Nimmi; Brown, J. Quincy

2013-01-01

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy. PMID:23894357

Mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development.

PubMed

Walton, Katherine D; Kolterud, Asa

2014-09-04

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine(1). Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought(1). The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth(2). Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Microscopic neural image registration based on the structure of mitochondria

NASA Astrophysics Data System (ADS)

Cao, Huiwen; Han, Hua; Rao, Qiang; Xiao, Chi; Chen, Xi

2017-02-01

Microscopic image registration is a key component of the neural structure reconstruction with serial sections of neural tissue. The goal of microscopic neural image registration is to recover the 3D continuity and geometrical properties of specimen. During image registration, various distortions need to be corrected, including image rotation, translation, tissue deformation et.al, which come from the procedure of sample cutting, staining and imaging. Furthermore, there is only certain similarity between adjacent sections, and the degree of similarity depends on local structure of the tissue and the thickness of the sections. These factors make the microscopic neural image registration a challenging problem. To tackle the difficulty of corresponding landmarks extraction, we introduce a novel image registration method for Scanning Electron Microscopy (SEM) images of serial neural tissue sections based on the structure of mitochondria. The ellipsoidal shape of mitochondria ensures that the same mitochondria has similar shape between adjacent sections, and its characteristic of broad distribution in the neural tissue guarantees that landmarks based on the mitochondria distributed widely in the image. The proposed image registration method contains three parts: landmarks extraction between adjacent sections, corresponding landmarks matching and image deformation based on the correspondences. We demonstrate the performance of our method with SEM images of drosophila brain.

Three-dimensional fuse deposition modeling of tissue-simulating phantom for biomedical optical imaging

NASA Astrophysics Data System (ADS)

Dong, Erbao; Zhao, Zuhua; Wang, Minjie; Xie, Yanjun; Li, Shidi; Shao, Pengfei; Cheng, Liuquan; Xu, Ronald X.

2015-12-01

Biomedical optical devices are widely used for clinical detection of various tissue anomalies. However, optical measurements have limited accuracy and traceability, partially owing to the lack of effective calibration methods that simulate the actual tissue conditions. To facilitate standardized calibration and performance evaluation of medical optical devices, we develop a three-dimensional fuse deposition modeling (FDM) technique for freeform fabrication of tissue-simulating phantoms. The FDM system uses transparent gel wax as the base material, titanium dioxide (TiO2) powder as the scattering ingredient, and graphite powder as the absorption ingredient. The ingredients are preheated, mixed, and deposited at the designated ratios layer-by-layer to simulate tissue structural and optical heterogeneities. By printing the sections of human brain model based on magnetic resonance images, we demonstrate the capability for simulating tissue structural heterogeneities. By measuring optical properties of multilayered phantoms and comparing with numerical simulation, we demonstrate the feasibility for simulating tissue optical properties. By creating a rat head phantom with embedded vasculature, we demonstrate the potential for mimicking physiologic processes of a living system.

INTRACELLULAR LOCALIZATION AND QUANTITATION OF TRITIATED ANTIGENS IN RETICULOENDOTHELIAL TISSUES OF MICE DURING SECONDARY AND HYPERIMMUNE RESPONSES

PubMed Central

Roberts, Audrey N.; Haurowitz, Felix

1962-01-01

Autoradiography and quantitative radiochemical techniques have been used to determine intracellular localization of tritium and the quantity of tissue-bound tritium, respectively, following injections of H3-aniline azo PGG or H3-arsanilazo PGG to yield hyperimmune or secondary response stimulation in mice. Autoradiography revealed intracytoplasmic localization of grains in macrophages of spleen and lung sections, and in Kupffer cells of liver sections following intravenous and subcutaneous injections of H3-aniline azo PGG. Quantitation of tissue section surface radioactivities in the windowless flow counter and scintillation counter, and of dissolved tissue section activities in the scintillation counter, showed that greatest radioactivity was present in lung tissue, with less in spleen, liver, and mesenteric lymph nodes from these hyperimmunized mice. Autoradiographic studies on tissue sections from mice in secondary response stimulation after subcutaneous foot-pad injections of H3-arsanilazo PGG, showed intracellular and extracellular grains over regional popliteal node sections, with intracytoplasmic grain localization over macrophages and pyroninophilic plasmacytes. Scattered macrophages in spleen and lung sections also contained intracytoplasmic radioactivity. Clusters of antibody-synthesizing cells in the regional lymph nodes were demonstrated with fluorescence microscopy, and these cells were compared to similar cells possessing radioactivity as observed in the section autoradiographs. An occasional Russell body plasma cell containing specific antibody was observed in splenic impressions. Windowless flow counting showed that greatest radioactivity was in regional node sections, with less in spleen and lung, and none in contralateral lymph nodes. A quantitative comparison between windowless flow counting and autoradiography revealed that 20 counts were required to yield one silver grain. PMID:13974279

Comparative Tissue Stainability of Lawsonia inermis (Henna) and Eosin as Counterstains to Hematoxylin in Brain Tissues.

PubMed

Alawa, Judith N; Gideon, Gbenga O; Adetiba, Bamidele; Alawa, Clement B

2015-04-01

We hyposthesized that henna staining could provide an alternative to eosin when used as a counterstain to hematoxylin for understanding basic neurohistological principles. Therefore, this study was aimed at investigating the suitability of henna as counterstain to hematoxylin for the demonstration of the layer stratification and cellular distribution in the brain tissue. Henna stained nervous tissue by reacting with the basic elements in proteins via its amino groups. It stained the neuropil and connective tissue membranes brown and effectively outlined the perikarya of neurons with no visible nuclei demonstrating that it is an acidic dye. Henna as a counterstain to hematoxylin demonstrated reliability as a new neurohistological stain. It facilitated identification of cortical layer stratification and cellular distribution in brain tissue sections from Wistar rats. This was comparable to standard hematoxylin and eosin staining as morphological and morphometrical analyses of stained cells did not show significant differences in size or number. This study presents a method for staining with henna and demonstrates that although henna and eosin belong to different dye groups (anthraquinone and xanthenes, respectively) based on their chromophores, they share similar staining techniques and thus could be used interchangeably in neurohistology.

Mouse hepatitis virus immunofluorescence in formalin- or Bouin's-fixed tissues using trypsin digestion.

PubMed

Brownstein, D G; Barthold, S W

1982-02-01

Mouse hepatitis viral antigens were demonstrated by immunofluorescence in formalin- and Bouin's-fixed tissues processed routinely for histopathology followed by partial digestion with trypsin. Staining was superior in tissues fixed in formalin and was not diminished in tissue sections from paraffin blocks stored at room temperature as long as 2 years. The relative ease of this procedure and the commercial availability of reagents makes this a useful technique for the definitive diagnosis of mouse hepatitis virus infection.

[Immunocytochemical demonstration of astrocytes in brain sections combined with Nissl staining].

PubMed

Korzhevskiĭ, D E; Otellin, V A

2004-01-01

The aim of the present study was to develop an easy and reliable protocol of combined preparation staining, which would unite the advantages of immunocytochemical demonstration of astrocytes with the availability to evaluate functional state of neurons provided by Nissl technique. The presented protocol of paraffin sections processing allows to retain high quality of tissue structure and provides for selective demonstration of astrocytes using the monoclonal antibodies against glial fibrillary acidic protein and contrast Nissl staining of cells. The protocol can be used without any changes for processing of brain sections obtained from the humans and other mammals with the exception of mice and rabbits.

A rapid method for the assessment of bone architecture by confocal microscopy.

PubMed

Zheng, M H; Bruining, H G; Cody, S H; Brankov, B; Wood, D J; Papadimitriou, J M

1997-08-01

Conventional ways of demonstrating and analysing the components of osseous tissue have always been hampered by the difficulty of physically sectioning bone. In this study, we have used Acridine Orange staining of 100-micron-thick unembedded bone slices and then assessed the cellular and tissue architecture by confocal microscopy. The result showed the Acridine Orange, by differential staining of the cellular nucleic acids, permits ready assessment of cell shape and cell organization as well as variations in growth patterns. Our studies have provided a new and relatively easy way of assessing the morphology of bone specimens by rendering unnecessary the need for embedding, decalcification and thin sectioning of the osseous tissue.

Mass spectral analysis and imaging of tissue by ToF-SIMS--The role of buckminsterfullerene, C60+, primary ions

NASA Astrophysics Data System (ADS)

Jones, Emrys A.; Lockyer, Nicholas P.; Vickerman, John C.

2007-02-01

Recent developments in desorption/ionisation mass spectrometry techniques have made their application to biological analysis a realistic and successful proposition. Developments in primary ion source technology, mainly through the advent of polyatomic ion beams, have meant that the technique of secondary ion mass spectrometry (SIMS) can now access the depths of information required to allow biological imaging to be a viable option. Here the role of the primary ion C60+ is assessed with regard to molecular imaging of lipids and pharmaceuticals within tissue sections. High secondary ion yields and low surface damage accumulation are demonstrated on both model and real biological samples, indicating the high secondary ion efficiency afforded to the analyst by this primary ion when compared to other cluster ion beams used in imaging. The newly developed 40 keV C60+ ion source allows the beam to be focused such that high resolution imaging is demonstrated on a tissue sample, and the greater yields allow the molecular signal from the drug raclopride to be imaged within tissue section following in vivo dosing. The localisation shown for this drug alludes to issues regarding the chemical environment affecting the ionisation probability of the molecule; the importance of this effect is demonstrated with model systems and the possibility of using laser post-ionisation as a method for reducing this consequence of bio-sample complexity is demonstrated and discussed.

Fibrinogen Demonstration in Oral Lichen Planus: An Immunofluorescence Study on Archival Tissues.

PubMed

Shirol, Pallavi D; Naik, Veena; Kale, Alka

2015-10-01

Lichen planus is a premalignant condition with minimal diagnostic aids. This study is an attempt to use paraffin embedded sections of lichen planus with immunofluorescein stain and to evaluate the immunofluorescent sections to establish pattern of fibrinogen deposition. Thirty-five paraffin embedded sections of old and new cases of oral lichen planus (study group) and five normal oral mucosa (control group) were chosen. Two sections of each (H & E) case were taken, one was stained with hematoxylin and eosin and another with fluorescein isothiocynate conjugate (FITC) polyclonal rabbit antibody against fibrinogen. Fluorescent findings were examined with a fluorescent microscope. A high statistical significant correlation was found in respect to fluorescence positivity, intensity of fluorescence and distribution of fluorescence each with p < 0.0001 and fluorescence at blood vessel walls (p = 0.0003). This study suggested that paraffin embedded sections can be successfully used in direct immunofluorescence staining in routine set up where only formalin fixed tissues are received. Paraffin embedded sections can be successfully used in direct immunofluorescence staining when only formalin fixed tissues are received.

Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy.

PubMed

Ryan, Duncan P; Gould, Elizabeth A; Seedorf, Gregory J; Masihzadeh, Omid; Abman, Steven H; Vijayaraghavan, Sukumar; Macklin, Wendy B; Restrepo, Diego; Shepherd, Douglas P

2017-09-20

Optical tissue clearing has revolutionized researchers' ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.Optical clearing of tissue has enabled optical imaging deeper into tissue due to significantly reduced light scattering. Here, Ryan et al. tackle first-order defocus, an artefact of a non-uniform refractive index, extending light-sheet microscopy to partially cleared samples.

Quasi-dynamical analysis and real-time tissue temperature monitoring during laser vaporization

NASA Astrophysics Data System (ADS)

Wang, Hui; Ray, Aditi; Jebens, Dave; Chia, Ray; Hasenberg, Tom

2014-03-01

Vaporization and coagulation are two fundamental processes that can be performed during laser-tissue ablation. We demonstrated a method allowing quasi-dynamically observing of the cross-sectional images of tissue response during ablation. The results showed that coagulation depth is relatively constant during vaporization, which supports the excellent hemostasis of green laser benign prostate hyperplasia (BPH) treatment. We also verified a new technology for real-time, in situ tissue temperature monitoring, which may be promising for in vivo tissue vaporization degree feedback during laser ablation to improve the vaporization efficiency and avoid complications.

Spatial mapping of metals in tissue-sections using combination of mass-spectrometry and histology through image registration

NASA Astrophysics Data System (ADS)

Anyz, Jiri; Vyslouzilova, Lenka; Vaculovic, Tomas; Tvrdonova, Michaela; Kanicky, Viktor; Haase, Hajo; Horak, Vratislav; Stepankova, Olga; Heger, Zbynek; Adam, Vojtech

2017-01-01

We describe a new procedure for the parallel mapping of selected metals in histologically characterized tissue samples. Mapping is achieved via image registration of digital data obtained from two neighbouring cryosections by scanning the first as a histological sample and subjecting the second to laser ablation inductively coupled plasma mass spectrometry. This computer supported procedure enables determination of the distribution and content of metals of interest directly in the chosen histological zones and represents a substantial improvement over the standard approach, which determines these values in tissue homogenates or whole tissue sections. The potential of the described procedure was demonstrated in a pilot study that analysed Zn and Cu levels in successive development stages of pig melanoma tissue using MeLiM (Melanoma-bearing-Libechov-Minipig) model. We anticipate that the procedure could be useful for a complex understanding of the role that the spatial distribution of metals plays within tissues affected by pathological states including cancer.

Spatial mapping of metals in tissue-sections using combination of mass-spectrometry and histology through image registration

PubMed Central

Anyz, Jiri; Vyslouzilova, Lenka; Vaculovic, Tomas; Tvrdonova, Michaela; Kanicky, Viktor; Haase, Hajo; Horak, Vratislav; Stepankova, Olga; Heger, Zbynek; Adam, Vojtech

2017-01-01

We describe a new procedure for the parallel mapping of selected metals in histologically characterized tissue samples. Mapping is achieved via image registration of digital data obtained from two neighbouring cryosections by scanning the first as a histological sample and subjecting the second to laser ablation inductively coupled plasma mass spectrometry. This computer supported procedure enables determination of the distribution and content of metals of interest directly in the chosen histological zones and represents a substantial improvement over the standard approach, which determines these values in tissue homogenates or whole tissue sections. The potential of the described procedure was demonstrated in a pilot study that analysed Zn and Cu levels in successive development stages of pig melanoma tissue using MeLiM (Melanoma-bearing-Libechov-Minipig) model. We anticipate that the procedure could be useful for a complex understanding of the role that the spatial distribution of metals plays within tissues affected by pathological states including cancer. PMID:28071735

Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation.

PubMed

Kowtharapu, B S; Marfurt, C; Hovakimyan, M; Will, F; Richter, H; Wree, A; Stachs, O; Guthoff, R F

2017-01-01

Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon-a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70-90 μm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti-neuronal β-III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20-30 μm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Gold internal standard correction for elemental imaging of soft tissue sections by LA-ICP-MS: element distribution in eye microstructures.

PubMed

Konz, Ioana; Fernández, Beatriz; Fernández, M Luisa; Pereiro, Rosario; González, Héctor; Alvarez, Lydia; Coca-Prados, Miguel; Sanz-Medel, Alfredo

2013-04-01

Laser ablation coupled to inductively coupled plasma mass spectrometry has been developed for the elemental imaging of Mg, Fe and Cu distribution in histological tissue sections of fixed eyes, embedded in paraffin, from human donors (cadavers). This work presents the development of a novel internal standard correction methodology based on the deposition of a homogeneous thin gold film on the tissue surface and the use of the (197)Au(+) signal as internal standard. Sample preparation (tissue section thickness) and laser conditions were carefully optimized, and internal normalisation using (197)Au(+) was compared with (13)C(+) correction for imaging applications. (24)Mg(+), (56)Fe(+) and (63)Cu(+) distributions were investigated in histological sections of the anterior segment of the eye (including the iris, ciliary body, cornea and trabecular meshwork) and were shown to be heterogeneously distributed along those tissue structures. Reproducibility was assessed by imaging different human eye sections from the same donor and from ten different eyes from adult normal donors, which showed that similar spatial maps were obtained and therefore demonstrate the analytical potential of using (197)Au(+) as internal standard. The proposed analytical approach could offer a robust tool with great practical interest for clinical studies, e.g. to investigate trace element distribution of metals and their alterations in ocular diseases.

Whole-organ atlas imaged by label-free high-resolution photoacoustic microscopy assisted by a microtome

NASA Astrophysics Data System (ADS)

Wong, Terence T. W.; Zhang, Ruiying; Hsu, Hsun-Chia; Maslov, Konstantin I.; Shi, Junhui; Chen, Ruimin; Shung, K. Kirk; Zhou, Qifa; Wang, Lihong V.

2018-02-01

In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.

An unsupervised MVA method to compare specific regions in human breast tumor tissue samples using ToF-SIMS.

PubMed

Bluestein, Blake M; Morrish, Fionnuala; Graham, Daniel J; Guenthoer, Jamie; Hockenbery, David; Porter, Peggy L; Gamble, Lara J

2016-03-21

Imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA) were used to investigate two sets of pre- and post-chemotherapy human breast tumor tissue sections to characterize lipids associated with tumor metabolic flexibility and response to treatment. The micron spatial resolution imaging capability of ToF-SIMS provides a powerful approach to attain spatially-resolved molecular and cellular data from cancerous tissues not available with conventional imaging techniques. Three ca. 1 mm(2) areas per tissue section were analyzed by stitching together 200 μm × 200 μm raster area scans. A method to isolate and analyze specific tissue regions of interest by utilizing PCA of ToF-SIMS images is presented, which allowed separation of cellularized areas from stromal areas. These PCA-generated regions of interest were then used as masks to reconstruct representative spectra from specifically stromal or cellular regions. The advantage of this unsupervised selection method is a reduction in scatter in the spectral PCA results when compared to analyzing all tissue areas or analyzing areas highlighted by a pathologist. Utilizing this method, stromal and cellular regions of breast tissue biopsies taken pre- versus post-chemotherapy demonstrate chemical separation using negatively-charged ion species. In this sample set, the cellular regions were predominantly all cancer cells. Fatty acids (i.e. palmitic, oleic, and stearic), monoacylglycerols, diacylglycerols and vitamin E profiles were distinctively different between the pre- and post-therapy tissues. These results validate a new unsupervised method to isolate and interpret biochemically distinct regions in cancer tissues using imaging ToF-SIMS data. In addition, the method developed here can provide a framework to compare a variety of tissue samples using imaging ToF-SIMS, especially where there is section-to-section variability that makes it difficult to use a serial hematoxylin and eosin (H&E) stained section to direct the SIMS analysis.

Optically Sectioned Imaging of Microvasculature of In-Vivo and Ex-Vivo Thick Tissue Models with Speckle-illumination HiLo Microscopy and HiLo Image Processing Implementation in MATLAB Architecture

NASA Astrophysics Data System (ADS)

Suen, Ricky Wai

The work described in this thesis covers the conversion of HiLo image processing into MATLAB architecture and the use of speckle-illumination HiLo microscopy for use of ex-vivo and in-vivo imaging of thick tissue models. HiLo microscopy is a wide-field fluorescence imaging technique and has been demonstrated to produce optically sectioned images comparable to confocal in thin samples. The imaging technique was developed by Jerome Mertz and the Boston University Biomicroscopy Lab and has been implemented in our lab as a stand-alone optical setup and a modification to a conventional fluorescence microscope. Speckle-illumination HiLo microscopy combines two images taken under speckle-illumination and standard uniform-illumination to generate an optically sectioned image that reject out-of-focus fluorescence. The evaluated speckle contrast in the images is used as a weighting function where elements that move out-of-focus have a speckle contrast that decays to zero. The experiments shown here demonstrate the capability of our HiLo microscopes to produce optically-sectioned images of the microvasculature of ex-vivo and in-vivo thick tissue models. The HiLo microscope were used to image the microvasculature of ex-vivo mouse heart sections prepared for optical histology and the microvasculature of in-vivo rodent dorsal window chamber models. Studies in label-free surface profiling with HiLo microscopy is also presented.

In vivo monitoring laser tissue interaction using high resolution Fourier-domain optical coherence tomography

NASA Astrophysics Data System (ADS)

Jo, Hang Chan; Shin, Dong Jun; Ahn, Jin-Chul; Chung, Phil-Sang; Kim, DaeYu

2017-02-01

Laser-induced therapies include laser ablation to remove or cut target tissue by irradiating high-power focused laser beam. These laser treatments are widely used tools for minimally invasive surgery and retinal surgical procedures in clinical settings. In this study, we demonstrate laser tissue interaction images of various sample tissues using high resolution Fourier-domain optical coherence tomography (Fd-OCT). We use a Q-switch diode-pumped Nd:YVO4 nanosecond laser (532nm central wavelength) with a 4W maximum output power at a 20 kHz repetition rate to ablate in vitro and in vivo samples including chicken breast and mouse ear tissues. The Fd-OCT system acquires time-series Bscan images at the same location during the tissue ablation experiments with 532nm laser irradiation. The real-time series of OCT cross-sectional (B-scan) images compare structural changes of 532nm laser ablation using same and different laser output powers. Laser tissue ablation is demonstrated by the width and the depth of the tissue ablation from the B-scan images.

Deep UV autofluorescence microscopy for cell biology and tissue histology.

PubMed

Jamme, Frédéric; Kascakova, Slavka; Villette, Sandrine; Allouche, Fatma; Pallu, Stéphane; Rouam, Valérie; Réfrégiers, Matthieu

2013-07-01

Autofluorescence spectroscopy is a powerful tool for molecular histology and for following metabolic processes in biological samples as it does not require labelling. However, at the microscopic scale, it is mostly limited to visible and near infrared excitation of the samples. Several interesting and naturally occurring fluorophores can be excited in the UV and deep UV (DUV), but cannot be monitored in cellulo nor in vivo due to a lack of available microscopic instruments working in this wavelength range. To fulfil this need, we have developed a synchrotron-coupled DUV microspectrofluorimeter which is operational since 2010. An extended selection of endogenous autofluorescent probes that can be excited in DUV, including their spectral characteristics, is presented. The distribution of the probes in various biological samples, including cultured cells, soft tissues, bone sections and maize stems, is shown to illustrate the possibilities offered by this system. In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology. To fulfil this need, we have developed a synchrotron-coupled DUV microspectrofluorimeter which is operational since 2010. An extended selection of endogenous autofluorescent probes that can be excited in DUV, including their spectral characteristics, is presented. The distribution of the probes in various biological samples, including cultured cells, soft tissues, bone sections and maize stems, is shown to illustrate the possibilities offered by this system. In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology. In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology. © 2013 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Study of melanin bleaching after immunohistochemistry of melanin-containing tissues.

PubMed

Shen, Hongwu; Wu, Wenqiao

2015-04-01

Melanin may interfere with immunohistochemical staining. The goal of this study was to investigate the effects of trichloroisocyanuric acid (TCCA) bleaching, potassium permanganate bleaching, and potassium dichromate bleaching on melanin, tissue antigen, and 3,3'-diaminobenzidine (DAB) using melanin-containing and melanin-free tissue samples. Our results demonstrated that all 3 bleaching methods efficiently bleached melanin and partially destroyed tissue antigen. In addition, potassium permanganate bleaching and potassium dichromate bleaching clearly destroyed DAB, whereas TCCA bleaching had no significant effect on DAB. Therefore, neither potassium permanganate nor potassium dichromate is an ideal solution, whereas TCCA might be an ideal solution for melanin bleaching after the immunohistochemical staining of melanin-containing tissues. After immunostaining followed by TCCA bleaching, the melanin could be completely removed in all 120 malignant melanoma tissue sections. Compared with the control, the DAB intensity was clear, and the tissue structure and cellular nuclei were well maintained. It is worth noting that TCCA should be freshly prepared before each experiment, and used within 2 hours of its preparation. In addition, sections should not be incubated with TCCA for over 30 minutes.

Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

NASA Astrophysics Data System (ADS)

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-02-01

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

Cold-Temperature Plastic Resin Embedding of Liver for DNA- and RNA-Based Genotyping

PubMed Central

Finkelstein, Sydney D.; Dhir, Rajiv; Rabinovitz, Mordechai; Bischeglia, Michelle; Swalsky, Patricia A.; DeFlavia, Petrina; Woods, Jeffrey; Bakker, Anke; Becich, Michael

1999-01-01

The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5–1.0 μm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology. PMID:11272904

Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology

PubMed Central

Yin, C.; Glaser, A.K.; Leigh, S. Y.; Chen, Y.; Wei, L.; Pillai, P. C. S.; Rosenberg, M. C.; Abeytunge, S.; Peterson, G.; Glazowski, C.; Sanai, N.; Mandella, M. J.; Rajadhyaksha, M.; Liu, J. T. C.

2016-01-01

There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo clinical use, enables our device to achieve an optical-sectioning thickness and lateral resolution of 2.0 and 1.1 microns respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate the clinical potential of this high-speed optical-sectioning microscopy device. PMID:26977337

The effect of fixatives and temperature on the quality of glycogen demonstration.

PubMed

Zakout, Yosef Mohamed Azzam; Salih, Magdi M; Ahmed, H G

2010-04-01

Glycogen is demonstrated in a number of lesions and is diagnostically significant, particularly in certain tumors. To stain glycogen accurately, it is essential to choose a suitable fixative, temperature and staining method. We used rabbit liver to assess these variables. Specimens were fixed in three fixatives at two temperatures: 10% formalin, neutral buffered formalin (NBF) and Bouin's solution at 37 and 4 degrees C. Seventy-two paraffin sections were prepared and stained with periodic acid-Schiff (PAS), hexamine (methenamine) silver and Best's carmine methods. Negative control sections using diastase digestion were used for all methods to confirm the presence of glycogen. For the PAS reaction, Bouin's fixative gave better results at both temperatures compared to the other fixatives. For hexamine (methenamine) silver, the quality of staining was improved for tissues fixed in both 10% formalin and NBF at 37 degrees C compared to Bouin's solution. Both 10% formalin and NBF at 4 degrees C gave better results than Bouin's solution. For Best's carmine, Bouin's solution gave the best results for tissues fixed at 4 degrees C. Fixation of tissues with NBF at 37 degrees C gave the best quality staining. We concluded that the quality of glycogen staining in paraffin sections is greatly affected by both the fixative and the temperature of fixation.

Raman molecular imaging of brain frozen tissue sections.

PubMed

Kast, Rachel E; Auner, Gregory W; Rosenblum, Mark L; Mikkelsen, Tom; Yurgelevic, Sally M; Raghunathan, Aditya; Poisson, Laila M; Kalkanis, Steven N

2014-10-01

Raman spectroscopy provides a molecular signature of the region being studied. It is ideal for neurosurgical applications because it is non-destructive, label-free, not impacted by water concentration, and can map an entire region of tissue. The objective of this paper is to demonstrate the meaningful spatial molecular information provided by Raman spectroscopy for identification of regions of normal brain, necrosis, diffusely infiltrating glioma and solid glioblastoma (GBM). Five frozen section tissues (1 normal, 1 necrotic, 1 GBM, and 2 infiltrating glioma) were mapped in their entirety using a 300-µm-square step size. Smaller regions of interest were also mapped using a 25-µm step size. The relative concentrations of relevant biomolecules were mapped across all tissues and compared with adjacent hematoxylin and eosin-stained sections, allowing identification of normal, GBM, and necrotic regions. Raman peaks and peak ratios mapped included 1003, 1313, 1431, 1585, and 1659 cm(-1). Tissue maps identified boundaries of grey and white matter, necrosis, GBM, and infiltrating tumor. Complementary information, including relative concentration of lipids, protein, nucleic acid, and hemoglobin, was presented in a manner which can be easily adapted for in vivo tissue mapping. Raman spectroscopy can successfully provide label-free imaging of tissue characteristics with high accuracy. It can be translated to a surgical or laboratory tool for rapid, non-destructive imaging of tumor margins.

Radiologic atlas of rheumatic diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dihlmann, W.

1986-01-01

This book is an ''atlas of rheumatic joint disease'' selected from 20 years of personal experience by the author. The author sets a goal of demonstrating the value of soft-tissue imaging in the diagnosis of early joint disease. This goal is achieved with high quality reproductions, many of which are presented in duplicate to illustrate bone and soft-tissue changes. The contents include an introductory overview of the ''Mosaic of Arthritis'' followed by sections on adult rheumatoid arthritis, seronegative spondyloarthropathies, classic collagen disease, enthesiopathies, and lastly a section on gout and psuedogout. The subject index is specific and indexes figures withmore » boldface type. Each section is introduced by a brief outline or overview of the radiographic spectrum of the joint disorder to be illustrated.« less

Raman spectroscopy of normal oral buccal mucosa tissues: study on intact and incised biopsies

NASA Astrophysics Data System (ADS)

Deshmukh, Atul; Singh, S. P.; Chaturvedi, Pankaj; Krishna, C. Murali

2011-12-01

Oral squamous cell carcinoma is one of among the top 10 malignancies. Optical spectroscopy, including Raman, is being actively pursued as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant, and malignant oral ex vivo tissues. Spectral features showed predominance of lipids and proteins in normal and cancer conditions, respectively, which were attributed to membrane lipids and surface proteins. In view of recent developments in deep tissue Raman spectroscopy, we have recorded Raman spectra from superior and inferior surfaces of 10 normal oral tissues on intact, as well as incised, biopsies after separation of epithelium from connective tissue. Spectral variations and similarities among different groups were explored by unsupervised (principal component analysis) and supervised (linear discriminant analysis, factorial discriminant analysis) methodologies. Clusters of spectra from superior and inferior surfaces of intact tissues show a high overlap; whereas spectra from separated epithelium and connective tissue sections yielded clear clusters, though they also overlap on clusters of intact tissues. Spectra of all four groups of normal tissues gave exclusive clusters when tested against malignant spectra. Thus, this study demonstrates that spectra recorded from the superior surface of an intact tissue may have contributions from deeper layers but has no bearing from the classification of a malignant tissues point of view.

An Automated Platform for High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lanekoff, Ingela T.; Heath, Brandi S.; Liyu, Andrey V.

2012-10-02

An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours. This is achieved by controlling the distance between the sample and the probe by mounting the sample holder onto an automated XYZ stage and defining the tilt of the sample plane. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSImore » QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. High resolution mass analysis combined with MS/MS experiments enabled identification of lipids and metabolites in the tissue section. In addition, high dynamic range and sensitivity of the technique allowed us to generate ion images of low-abundance isobaric lipids. High-spatial resolution image acquired over a small region of the tissue section revealed the spatial distribution of an abundant brain metabolite, creatine, in the white and gray matter that is consistent with the literature data obtained using magnetic resonance spectroscopy.« less

Structural requirements of research tissue banks derived from standardized project surveillance.

PubMed

Herpel, E; Koleganova, N; Schreiber, B; Walter, B; Kalle, C V; Schirmacher, P

2012-07-01

Tissue banks constitute decisive and rate-limiting resource and technology platforms for basic and translational biomedical research, notably in the area of cancer. Thus, it is essential to plan and structure tissue banking and allocate resources according to research needs, but essential requirements are still incompletely defined. The tissue bank of the National Center of Tumor Diseases Heidelberg (NCT) was founded with the intention to provide tissues of optimal quality and to prioritize the realization of research projects. We analysed its structure and prospective project management registration as well as tracking records for all projects of the NCT tissue bank as of its start in 2005 in order to obtain information that may be relevant for tissue bank planning. All project proposals submitted to the NCT tissue bank (n = 681) were included in the study. For a detailed evaluation of provided services, only projects that were completed until July 2011 (n = 605) were analysed. For these 605 projects, NCT tissue bank provided 769 specific services. In all projects/services, we recorded project leader, type and amount of material provided, type of research (basic/translational), work load of project and project completion. Furthermore, all completed projects were tracked after 90 days according to a standard protocol to determine principal investigators' (PI) satisfaction and quality of the provided material. Until July 2011, 605 projects had been successfully completed as documented by material transfer agreement. Of the projects, 72.7 % addressed basic research, 22.3 % were translational research projects and 3 % concerned epidemiological research; 91 % (n = 546) concerned a single PI and the NTC tissue bank. For these projects, 769 specific services were provided. Of these services, 288 concerned providing formalin-fixed and paraffin-embedded (FFPE) tissue (extracts, full size sections), 126 providing fresh frozen materials (including fresh frozen sections), 137 providing tissue micro-array (TMA)-based sections and 199 providing immunohistochemical services. Project tracking demonstrated that all projects had started within 90 days after reception of the material by the PIs, and PI satisfaction with provided material exceeded 97 %. Standardized registration and tracking provides valuable structural information for planning and financing of tissue banks and allocation of resources. The high number of completed projects as well as high user satisfaction demonstrates that structuring of tissue banks should be preferably research-oriented and highly efficient. The comparable number of requests for FFPE and fresh frozen tissue as well as TMA-based services underpins the need for a broad approach in terms of methods and material types in order to fulfil research needs.

Three-dimensional multifunctional optical coherence tomography for skin imaging

NASA Astrophysics Data System (ADS)

Li, En; Makita, Shuichi; Hong, Young-Joo; Kasaragod, Deepa; Sasaoka, Tomoko; Yamanari, Masahiro; Sugiyama, Satoshi; Yasuno, Yoshiaki

2016-02-01

Optical coherence tomography (OCT) visualizes cross-sectional microstructures of biological tissues. Recent developments of multifunctional OCT (MF-OCT) provides multiple optical contrasts which can reveal currently unknown tissue properties. In this contribution we demonstrate multifunctional OCT specially designed for dermatological investigation. And by utilizing it to measure four different body parts of in vivo human skin, three-dimensional scattering OCT, OCT angiography, polarization uniformity tomography, and local birefringence tomography images were obtained by a single scan. They respectively contrast the structure and morphology, vasculature, melanin content and collagen traits of the tissue.

Cytology of cardiac myxomas: presence of Ulex europaeus agglutinin-I (UEA-I) lectin by immunoperoxidase staining.

PubMed

Iwa, N; Yutani, C

1993-12-01

Cytological findings are presented of seven cases of cardiac myxomas. Avidin-biotin-peroxidase complex (ABC) method was employed to demonstrate Ulex europaeus agglutinin-I (UEA-I) lectin in imprint smears as well as in paraffin-embedded tissue sections in cardiac myxomas. The cytology was characterized by tumor cells with polyhedral or stellate and mucinous background with lymphocytes, neutrophils, and hemosiderin-laden macrophages. In smears as well as tissue sections, UEA-I lectin was detected throughout the cytoplasm of myxoma cells. This study established the applicability of the immunoperoxidase staining for cardiac myxoma as an aid in cytopathological diagnosis.

Silver nanoparticle based surface enhanced Raman scattering spectroscopy of diabetic and normal rat pancreatic tissue under near-infrared laser excitation

NASA Astrophysics Data System (ADS)

Huang, H.; Shi, H.; Feng, S.; Lin, J.; Chen, W.; Huang, Z.; Li, Y.; Yu, Y.; Lin, D.; Xu, Q.; Chen, R.

2013-04-01

This paper presents the use of high spatial resolution silver nanoparticle based near-infrared surface enhanced Raman scattering (SERS) from rat pancreatic tissue to obtain biochrmical information about the tissue. A high quality SERS signal from a mixture of pancreatic tissues and silver nanoparticles can be obtained within 10 s using a Renishaw micro-Raman system. Prominent SERS bands of pancreatic tissue were assigned to known molecular vibrations, such as the vibrations of DNA bases, RNA bases, proteins and lipids. Different tissue structures of diabetic and normal rat pancreatic tissues have characteristic features in SERS spectra. This exploratory study demonstrated great potential for using SERS imaging to distinguish diabetic and normal pancreatic tissues on frozen sections without using dye labeling of functionalized binding sites.

Essential tactics of tissue preparation and matrix nano-spotting for successful compound imaging mass spectrometry.

PubMed

Végvári, Akos; Fehniger, Thomas E; Gustavsson, Lena; Nilsson, Anna; Andrén, Per E; Kenne, Kerstin; Nilsson, Johan; Laurell, Thomas; Marko-Varga, György

2010-04-18

The ultimate goal of MALDI-Imaging Mass Spectrometry (MALDI-IMS) is to achieve spatial localization of analytes in tissue sections down to individual tissue compartments or even at the level of a few cells. With compound tissue imaging, it is possible to track the transportation of an unlabelled, inhaled reference compound within lung tissue, through the application of MALDI-IMS. The procedure for isolation and preparation of lung tissues is found to be crucial in order to preserve the anatomy and structure of the pulmonary compartments. To avoid delocalization of analytes within lung tissue compartments we have applied an in-house designed nano-spotter, based on a microdispenser mounted on an XY table, of which movement and spotting functionality were fully computer controlled. We demonstrate the usefulness of this platform in lung tissue sections isolated from rodent in vivo model, applied to compound tissue imaging as exemplified with the determination of the spatial distribution of (1alpha,2beta,4beta,7beta)-7-[(hydroxidi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.0(2,4)]nonane, also known as tiotropium. We provide details on tissue preparation protocols and sample spotting technology for successful identification of drug in mouse lung tissue by using MALDI-Orbitrap instrumentation. Copyright 2010 Elsevier B.V. All rights reserved.

Identification of normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections

NASA Astrophysics Data System (ADS)

Zhou, Yi; Chen, Zhifen; Kang, Deyong; li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Guan, Guoxian; Chen, Jianxin

2016-01-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a potential diagnostic tool is attractive. MPM can effectively provide information about morphological and biochemical changes in biological tissues at the molecular level. In this paper, we attempt to identify normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections (both in transverse and longitudinal sections). The results show that MPM can display different microstructure changes in the transverse and longitudinal sections of colorectal muscularis propria. MPM also can quantitatively describe the alteration of collagen content between normal and cancerous muscle layers. These are important pathological findings that MPM images can bring more detailed complementary information about tissue architecture and cell morphology through observing the transverse and longitudinal sections of colorectal muscularis propria. This work demonstrates that MPM can be better for identifying the microstructural characteristics of normal and cancerous human colorectal muscularis propria in different sections.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

PubMed

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

The direct analysis of drug distribution of rotigotine-loaded microspheres from tissue sections by LESA coupled with tandem mass spectrometry.

PubMed

Xu, Li-Xiao; Wang, Tian-Tian; Geng, Yin-Yin; Wang, Wen-Yan; Li, Yin; Duan, Xiao-Kun; Xu, Bin; Liu, Charles C; Liu, Wan-Hui

2017-09-01

The direct analysis of drug distribution of rotigotine-loaded microspheres (RoMS) from tissue sections by liquid extraction surface analysis (LESA) coupled with tandem mass spectrometry (MS/MS) was demonstrated. The RoMS distribution in rat tissues assessed by the ambient LESA-MS/MS approach without extensive or tedious sample pretreatment was compared with that obtained by a conventional liquid chromatography tandem mass spectrometry (LC-MS/MS) method in which organ excision and subsequent solvent extraction were commonly employed before analysis. Results obtained from the two were well correlated for a majority of the organs, such as muscle, liver, stomach, and hippocampus. The distribution of RoMS in the brain, however, was found to be mainly focused in the hippocampus and striatum regions as shown by the LESA-imaged profiles. The LESA approach we developed is sensitive enough, with an estimated LLOQ at 0.05 ng/mL of rotigotine in brain tissue, and information-rich with minimal sample preparation, suitable, and promising in assisting the development of new drug delivery systems for controlled drug release and protection. Graphical abstract Workflow for the LESA-MS/MS imaging of brain tissue section after intramuscular RoMS administration.

Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

PubMed Central

Robertson, David; Savage, Kay; Reis-Filho, Jorge S; Isacke, Clare M

2008-01-01

Background Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues. Results We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously. Conclusion This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples. PMID:18366689

Resolution enhancement in deep-tissue nanoparticle imaging based on plasmonic saturated excitation microscopy

NASA Astrophysics Data System (ADS)

Deka, Gitanjal; Nishida, Kentaro; Mochizuki, Kentaro; Ding, Hou-Xian; Fujita, Katsumasa; Chu, Shi-Wei

2018-03-01

Recently, many resolution enhancing techniques are demonstrated, but most of them are severely limited for deep tissue applications. For example, wide-field based localization techniques lack the ability of optical sectioning, and structured light based techniques are susceptible to beam distortion due to scattering/aberration. Saturated excitation (SAX) microscopy, which relies on temporal modulation that is less affected when penetrating into tissues, should be the best candidate for deep-tissue resolution enhancement. Nevertheless, although fluorescence saturation has been successfully adopted in SAX, it is limited by photobleaching, and its practical resolution enhancement is less than two-fold. Recently, we demonstrated plasmonic SAX which provides bleaching-free imaging with three-fold resolution enhancement. Here we show that the three-fold resolution enhancement is sustained throughout the whole working distance of an objective, i.e., 200 μm, which is the deepest super-resolution record to our knowledge, and is expected to extend into deeper tissues. In addition, SAX offers the advantage of background-free imaging by rejecting unwanted scattering background from biological tissues. This study provides an inspirational direction toward deep-tissue super-resolution imaging and has the potential in tumor monitoring and beyond.

Ambient Mass Spectrometry in Cancer Research.

PubMed

Takats, Z; Strittmatter, N; McKenzie, J S

2017-01-01

Ambient ionization mass spectrometry was developed as a sample preparation-free alternative to traditional MS-based workflows. Desorption electrospray ionization (DESI)-MS methods were demonstrated to allow the direct analysis of a broad range of samples including unaltered biological tissue specimens. In contrast to this advantageous feature, nowadays DESI-MS is almost exclusively used for sample preparation intensive mass spectrometric imaging (MSI) in the area of cancer research. As an alternative to MALDI, DESI-MSI offers matrix deposition-free experiment with improved signal in the lower (<500m/z) range. DESI-MSI enables the spatial mapping of tumor metabolism and has been broadly demonstrated to offer an alternative to frozen section histology for intraoperative tissue identification and surgical margin assessment. Rapid evaporative ionization mass spectrometry (REIMS) was developed exclusively for the latter purpose by the direct combination of electrosurgical devices and mass spectrometry. In case of the REIMS technology, aerosol particles produced by electrosurgical dissection are subjected to MS analysis, providing spectral information on the structural lipid composition of tissues. REIMS technology was demonstrated to give real-time information on the histological nature of tissues being dissected, deeming it an ideal tool for intraoperative tissue identification including surgical margin control. More recently, the method has also been used for the rapid lipidomic phenotyping of cancer cell lines as it was demonstrated in case of the NCI-60 cell line collection. © 2017 Elsevier Inc. All rights reserved.

Multiphoton fluorescence lifetime imaging of chemotherapy distribution in solid tumors

NASA Astrophysics Data System (ADS)

Carlson, Marjorie; Watson, Adrienne L.; Anderson, Leah; Largaespada, David A.; Provenzano, Paolo P.

2017-11-01

Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.

Development of Raman microspectroscopy for automated detection and imaging of basal cell carcinoma

NASA Astrophysics Data System (ADS)

Larraona-Puy, Marta; Ghita, Adrian; Zoladek, Alina; Perkins, William; Varma, Sandeep; Leach, Iain H.; Koloydenko, Alexey A.; Williams, Hywel; Notingher, Ioan

2009-09-01

We investigate the potential of Raman microspectroscopy (RMS) for automated evaluation of excised skin tissue during Mohs micrographic surgery (MMS). The main aim is to develop an automated method for imaging and diagnosis of basal cell carcinoma (BCC) regions. Selected Raman bands responsible for the largest spectral differences between BCC and normal skin regions and linear discriminant analysis (LDA) are used to build a multivariate supervised classification model. The model is based on 329 Raman spectra measured on skin tissue obtained from 20 patients. BCC is discriminated from healthy tissue with 90+/-9% sensitivity and 85+/-9% specificity in a 70% to 30% split cross-validation algorithm. This multivariate model is then applied on tissue sections from new patients to image tumor regions. The RMS images show excellent correlation with the gold standard of histopathology sections, BCC being detected in all positive sections. We demonstrate the potential of RMS as an automated objective method for tumor evaluation during MMS. The replacement of current histopathology during MMS by a ``generalization'' of the proposed technique may improve the feasibility and efficacy of MMS, leading to a wider use according to clinical need.

Contrast improvement of terahertz images of thin histopathologic sections

PubMed Central

Formanek, Florian; Brun, Marc-Aurèle; Yasuda, Akio

2011-01-01

We present terahertz images of 10 μm thick histopathologic sections obtained in reflection geometry with a time-domain spectrometer, and demonstrate improved contrast for sections measured in paraffin with water. Automated segmentation is applied to the complex refractive index data to generate clustered terahertz images distinguishing cancer from healthy tissues. The degree of classification of pixels is then evaluated using registered visible microscope images. Principal component analysis and propagation simulations are employed to investigate the origin and the gain of image contrast. PMID:21326635

Contrast improvement of terahertz images of thin histopathologic sections.

PubMed

Formanek, Florian; Brun, Marc-Aurèle; Yasuda, Akio

2010-12-03

We present terahertz images of 10 μm thick histopathologic sections obtained in reflection geometry with a time-domain spectrometer, and demonstrate improved contrast for sections measured in paraffin with water. Automated segmentation is applied to the complex refractive index data to generate clustered terahertz images distinguishing cancer from healthy tissues. The degree of classification of pixels is then evaluated using registered visible microscope images. Principal component analysis and propagation simulations are employed to investigate the origin and the gain of image contrast.

Polarization sensitive optical coherence tomography – a review [Invited

PubMed Central

de Boer, Johannes F.; Hitzenberger, Christoph K.; Yasuno, Yoshiaki

2017-01-01

Optical coherence tomography (OCT) is now a well-established modality for high-resolution cross-sectional and three-dimensional imaging of transparent and translucent samples and tissues. Conventional, intensity based OCT, however, does not provide a tissue-specific contrast, causing an ambiguity with image interpretation in several cases. Polarization sensitive (PS) OCT draws advantage from the fact that several materials and tissues can change the light’s polarization state, adding an additional contrast channel and providing quantitative information. In this paper, we review basic and advanced methods of PS-OCT and demonstrate its use in selected biomedical applications. PMID:28663869

Lung and heart pathology in fatal drug addiction. A consecutive autopsy study.

PubMed

Kringsholm, B; Christoffersen, P

1987-01-01

Lung and heart sections from 33 drug addicts submitted for medico-legal autopsy at the Institute of Forensic Medicine in Copenhagen were studied together with tissue sections from 20 'normal' persons. In the drug addict cases focal bleedings in lung tissue were found in 94%, signs of earlier bleedings, haemosiderin containing histiocytes, were seen in 91%, and focal fibrosis in 46%. The bleeding episodes may be due to hypoxia in connection with heroin intake. In 94% of the drug addicts birefringent material in lung tissue was demonstrated, in 58% in granulomas and giant cells, in 27% in giant cells only and in 9% in isolated histiocytes. The material was localized in the wall of pulmonar arteries and/or in the interstitial tissue, undoubtedly depending on the duration of the abuse. In 18% angiothrombosis was seen, in all cases granulomas/giant cells were observed in the wall of the vessel concerned. The results indicate periodical intravenous injection of dissolved tablets in addition to heroin. Histological signs of pulmonary hypertension were not seen, possibly due to the fact that abuse of central stimulants is very rare in Denmark. Regarding heart alterations no significant differences were demonstrated between drug addicts and controls. The only note-worthy finding was focal infiltration of lymphocytes in the atrio-ventricular bundle in two drug addicts, the meaning of which is uncertain.

Autofluorescence and non-specific immunofluorescent labeling in frozen bovine intestinal tissue sections: Solutions for multi-color immunofluorescence experiments

USDA-ARS?s Scientific Manuscript database

Autofluorescence and non-specific immunofluorescent labeling are common challenges associated with immunofluorescence experiments. Autofluorescence typically demonstrates a broad emission spectrum, increasing the potential for overlap with experiments that utilize multiple fluorophores. During immun...

COMPLEMENT FIXATION IN DISEASED TISSUES

PubMed Central

Burkholder, Peter M.

1961-01-01

An immunohistologic complement fixation test has been used in an effort to detect immune complexes in sections of kidney from rats injected with rabbit anti-rat kidney serum and in sections of biopsied kidneys from four humans with membranous glomerulonephritis. Sections of the rat and human kidneys were treated with fluorescein-conjugated anti-rabbit globulin or antihuman globulin respectively. Adjacent sections in each case were incubated first with fresh guinea pig serum and then in a second step were treated with fluorescein-conjugated antibodies against fixed guinea pig complement to detect sites of fixation of the complement. It was demonstrated that the sites of rabbit globulin in glomerular capillary walls of the rat kidneys and the sites of localized human globulin in thickened glomerular capillary walls and swollen glomerular endothelial cells of the human kidneys were the same sites in which guinea pig complement was fixed in vitro. It was concluded from these studies that rabbit nephrotoxic antibodies localize in rat glomeruli in complement-fixing antigen-antibody complexes. Furthermore, it was concluded that the deposits of human globulin in the glomeruli of the human kidneys behaved like antibody globulin in complement-fixing antigen-antibody complexes. The significance of demonstrating complement-fixing immune complexes in certain diseased tissues is discussed in regard to determination of the causative role of allergic reactions in disease. PMID:19867205

Automated assessment of breast tissue density in non-contrast 3D CT images without image segmentation based on a deep CNN

NASA Astrophysics Data System (ADS)

Zhou, Xiangrong; Kano, Takuya; Koyasu, Hiromi; Li, Shuo; Zhou, Xinxin; Hara, Takeshi; Matsuo, Masayuki; Fujita, Hiroshi

2017-03-01

This paper describes a novel approach for the automatic assessment of breast density in non-contrast three-dimensional computed tomography (3D CT) images. The proposed approach trains and uses a deep convolutional neural network (CNN) from scratch to classify breast tissue density directly from CT images without segmenting the anatomical structures, which creates a bottleneck in conventional approaches. Our scheme determines breast density in a 3D breast region by decomposing the 3D region into several radial 2D-sections from the nipple, and measuring the distribution of breast tissue densities on each 2D section from different orientations. The whole scheme is designed as a compact network without the need for post-processing and provides high robustness and computational efficiency in clinical settings. We applied this scheme to a dataset of 463 non-contrast CT scans obtained from 30- to 45-year-old-women in Japan. The density of breast tissue in each CT scan was assigned to one of four categories (glandular tissue within the breast <25%, 25%-50%, 50%-75%, and >75%) by a radiologist as ground truth. We used 405 CT scans for training a deep CNN and the remaining 58 CT scans for testing the performance. The experimental results demonstrated that the findings of the proposed approach and those of the radiologist were the same in 72% of the CT scans among the training samples and 76% among the testing samples. These results demonstrate the potential use of deep CNN for assessing breast tissue density in non-contrast 3D CT images.

Feasibility of in Vivo SAXS Imaging for Detection of Alzheiemer's Disease

NASA Astrophysics Data System (ADS)

Choi, Mina

Small-angle x-ray scattering (SAXS) imaging has been proposed as a technique to characterize and selectively image structures based on electron density structure which allows for discriminating materials based on their scatter cross sections. This dissertation explores the feasibility of SAXS imaging for the detection of Alzheimer's disease (AD) amyloid plaques. The inherent scatter cross sections of amyloid plaque serve as biomarkers in vivo without the need of injected molecular tags. SAXS imaging can also assist in a better understanding of how these biomarkers play a role in Alzheimer's disease which in turn can lead to the development of more effective disease-modifying therapies. I implement simulations of x-ray transport using Monte Carlo methods for SAXS imaging enabling accurate calculation of radiation dose and image quality in SAXS-computed tomography (CT). I describe SAXS imaging phantoms with tissue-mimicking material and embedded scatter targets as a way of demonstrating the characteristics of SAXS imaging. I also performed a comprehensive study of scattering cross sections of brain tissue from measurements of ex-vivo sections of a wild-type mouse brain and reported generalized cross sections of gray matter, white matter, and corpus callosum obtained and registered by planar SAXS imaging. Finally, I demonstrate the ability of SAXS imaging to locate an amyloid fibril pellet within a brain section. This work contributes to novel application of SAXS imaging for Alzheimer's disease detection and studies its feasibility as an imaging tool for AD biomarkers.

Adaptation of the ammoniacal silver reaction to cytochemical demonstration of myelin basic protein.

PubMed

Staykova, M; Jordanov, J; Goranov, I

1978-01-01

A modification of Black and Ansley's ammoniacal silver reaction (ASR) for histones is proposed for visualizing myelin basic protien (MBP) in the nervous system. The reaction is performed on histological sections of tissues fixed in neutralized formalin-alcohol and delipidized in the course of the routine paraffin embedding. The deparaffinized sections are again treated with formalin in order to make the "unmasked" by the delipidization basic groups of MBP reactive to ammoniacal silver. After treatment with this reagent MBP of the myelin sheaths of the nerve fibres is impregnated brownish-black. Deparaffinized sections subjected to an extraction of MBP with hydrochloric acid exhibit a negative reaction at the level of the myelin sheaths the same reaction being preserved at the level of the nuclear histones. The reaction is positive in paper spots of nervous tissue extracts obtained with the same acid. These assays indicate the specificity of the modified ASR. The method can be used for studies on the processes of myelination and demylination in normal histogenesis and in pathology of the nervous tissue.

Fibre tip pH sensor for tumor detection during surgery

NASA Astrophysics Data System (ADS)

Henderson, Matthew R.; Schartner, Erik P.; Callen, David F.; Gill, P. Grantley; Monro, Tanya M.

2015-05-01

Surgery on tumours commonly involves a lumpectomy method, where a section of tissue containing the tumour is removed, to improve cosmetic outcomes and quality of life. Following surgery, the margins of the removed section are checked by pathology tests to ensure that the entire tumour has been removed. Unfortunately, approximately 15-20% of margins show incomplete removal and require a subsequent operation to remove the remaining tumour. Tumour detection during surgery could allow the removed section to be enlarged appropriately, reducing the likelihood of requiring subsequent surgery. A change in the extracellular pH in the vicinity of a tumour, when compared to normal tissue, has been shown previously in literature. We have fabricated an optical fibre tip pH sensor by embedding a fluorophore within a photopolymerised acrylamide polymer on the tip of a 200 micron diameter silica fibre. Preliminary measurements of human melanoma samples have shown a significant difference in the measured pH values between tumour and normal tissue. This demonstration paves to way to highly accurate margin detection during surgery.

Neuromedin B receptor in esophagus: evidence for subtypes of bombesin receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Von Schrenck, T.; Heinz-Erian, P.; Moran, T.

1989-04-01

To identify receptors for bombesin-related peptides in the rat esophagus, we measured binding of 125I-Bolton-Hunter neuromedin B (125I-BH-neuromedin B) and 125I-(Tyr4)bombesin to tissue sections from the rat esophagus and compared the results with those for rat pancreas. Esophagus bound both tracers, whereas pancreas bound only 125I-(Tyr4)bombesin. In each tissue binding was saturable, dependent on pH, on time, and on temperature, reversible, and specific. Autoradiography demonstrated binding of both tracers only to the muscularis mucosae of the esophagus and binding of 125I-(Tyr4)bombesin diffusely over pancreatic acini. In the esophagus, the relative potencies for inhibition of binding of both tracers were asmore » follows: neuromedin B greater than bombesin greater than GRP = neuromedin C; similar relative potencies were found for causing contraction of muscle strips from whole esophagus and from the isolated muscularis mucosae. In pancreas tissue sections and dispersed acini, the relative potencies for inhibition of binding of 125I-(Tyr4)bombesin were as follows: bombesin greater than GRP = neuromedin C much greater than neuromedin B. Similar relative potencies were found for stimulation of enzyme secretion from dispersed pancreatic acini. Computer analysis in both tissues demonstrated only a single binding site. The present study demonstrates that rat esophagus muscle possesses specific receptors for bombesin-related peptides. Furthermore, this study shows that the esophageal bombesin receptors represent a previously unidentified class of bombesin receptors in that they have a higher affinity for neuromedin B than for bombesin. In contrast, the pancreatic bombesin receptors have, like all other bombesin receptors described to date, a high affinity for bombesin, but low affinity for neuromedin B.« less

Mapping of neuron soma size as an effective approach to delineate differences between neural populations.

PubMed

Lingley, Alexander J; Bowdridge, Joshua C; Farivar, Reza; Duffy, Kevin R

2018-04-30

A single histological marker applied to a slice of tissue often reveals myriad cytoarchitectonic characteristics that can obscure differences between neuron populations targeted for study. Isolation and measurement of a single feature from the tissue is possible through a variety of approaches, however, visualizing the data numerically or through graphs alone can preclude being able to identify important features and effects that are not obvious from direct observation of the tissue. We demonstrate an efficient, effective, and robust approach to quantify and visualize cytoarchitectural features in histologically prepared brain sections. We demonstrate that this approach is able to reveal small differences between populations of neurons that might otherwise have gone undiscovered. We used stereological methods to record the cross-sectional soma area and in situ position of neurons within sections of the cat, monkey, and human visual system. The two-dimensional coordinate of every measured cell was used to produce a scatter plot that recapitulated the natural spatial distribution of cells, and each point in the plot was color-coded according to its respective soma area. The final graphic display was a multi-dimensional map of neuron soma size that revealed subtle differences across neuron aggregations, permitted delineation of regional boundaries, and identified small differences between populations of neurons modified by a period of sensory deprivation. This approach to collecting and displaying cytoarchitectonic data is simple, efficient, and provides a means of investigating small differences between neuron populations. Copyright © 2018. Published by Elsevier B.V.

High axial resolution imaging system for large volume tissues using combination of inclined selective plane illumination and mechanical sectioning

PubMed Central

Zhang, Qi; Yang, Xiong; Hu, Qinglei; Bai, Ke; Yin, Fangfang; Li, Ning; Gang, Yadong; Wang, Xiaojun; Zeng, Shaoqun

2017-01-01

To resolve fine structures of biological systems like neurons, it is required to realize microscopic imaging with sufficient spatial resolution in three dimensional systems. With regular optical imaging systems, high lateral resolution is accessible while high axial resolution is hard to achieve in a large volume. We introduce an imaging system for high 3D resolution fluorescence imaging of large volume tissues. Selective plane illumination was adopted to provide high axial resolution. A scientific CMOS working in sub-array mode kept the imaging area in the sample surface, which restrained the adverse effect of aberrations caused by inclined illumination. Plastic embedding and precise mechanical sectioning extended the axial range and eliminated distortion during the whole imaging process. The combination of these techniques enabled 3D high resolution imaging of large tissues. Fluorescent bead imaging showed resolutions of 0.59 μm, 0.47μm, and 0.59 μm in the x, y, and z directions, respectively. Data acquired from the volume sample of brain tissue demonstrated the applicability of this imaging system. Imaging of different depths showed uniform performance where details could be recognized in either the near-soma area or terminal area, and fine structures of neurons could be seen in both the xy and xz sections. PMID:29296503

Simple and robust image-based autofocusing for digital microscopy.

PubMed

Yazdanfar, Siavash; Kenny, Kevin B; Tasimi, Krenar; Corwin, Alex D; Dixon, Elizabeth L; Filkins, Robert J

2008-06-09

A simple image-based autofocusing scheme for digital microscopy is demonstrated that uses as few as two intermediate images to bring the sample into focus. The algorithm is adapted to a commercial inverted microscope and used to automate brightfield and fluorescence imaging of histopathology tissue sections.

Using transmission electron microscopy and 3View® to determine collagen fibril size and three-dimensional organization

PubMed Central

Mironov, Aleksandr; Cootes, Timothy F.; Holmes, David F.; Kadler, Karl E.

2017-01-01

Collagen fibrils are the major tensile element in vertebrate tissues where they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess formation of the fibrils, predominantly by measuring fibril diameter. Here we describe an enhanced protocol for measuring fibril diameter as well as fibril-volume-fraction, mean fibril length, fibril cross-sectional shape, and fibril 3D organization that are also major determinants of tissue function. Serial section TEM (ssTEM) has been used to visualize fibril 3D-organization in vivo. However, serial block face-scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View®). We demonstrate the power of 3View® for studying collagen fibril organization in vivo and show how to find and track individual fibrils. Time scale: ~8 days from isolating the tissue to having a 3D image stack. PMID:23807286

Volume holographic reflection endoscope for in-vivo ovarian cancer clinical studies

NASA Astrophysics Data System (ADS)

Howlett, I. D.; Gordon, M.; Brownlee, J. W.; Barton, J. K.; Kostuk, R. K.

2014-03-01

We present the design for an endoscopic system capable of imaging tissues of the ovary at two selected imaging depths simultaneously. The method utilizes a multiplexed volume hologram to select wavefronts from different depths within the tissue. It is the first demonstration of an endoscopic volume holographic imaging system. The endoscope uses both gradient index (GRIN) optical components and off the shelf singlet lenses to relay an image from the distal tip to the proximal end. The endoscope has a minimum diameter of 3.75 mm. The system length is 30 cm which is connected to a handle that includes the holographic components and optics that relay the image to a camera. Preliminary evaluation of the endoscope was performed with tissue phantoms and calibrated targets, which shows lateral resolution ≍ 4 μm at an operating wavelength of 660 nm. The hologram is recorded in phenanthraquinone doped poly methacrylate and is designed to produce images from two tissue depths. One image is obtained at the tissue surface and the second 70 μm below the surface. This method requires no mechanical scanning and acquires an image at the camera frame rate. The preliminary ex-vivo results show good correlation with histology sections of the same tissue sections.

Automatic segmentation of colon glands using object-graphs.

PubMed

Gunduz-Demir, Cigdem; Kandemir, Melih; Tosun, Akif Burak; Sokmensuer, Cenk

2010-02-01

Gland segmentation is an important step to automate the analysis of biopsies that contain glandular structures. However, this remains a challenging problem as the variation in staining, fixation, and sectioning procedures lead to a considerable amount of artifacts and variances in tissue sections, which may result in huge variances in gland appearances. In this work, we report a new approach for gland segmentation. This approach decomposes the tissue image into a set of primitive objects and segments glands making use of the organizational properties of these objects, which are quantified with the definition of object-graphs. As opposed to the previous literature, the proposed approach employs the object-based information for the gland segmentation problem, instead of using the pixel-based information alone. Working with the images of colon tissues, our experiments demonstrate that the proposed object-graph approach yields high segmentation accuracies for the training and test sets and significantly improves the segmentation performance of its pixel-based counterparts. The experiments also show that the object-based structure of the proposed approach provides more tolerance to artifacts and variances in tissues.

Evaluation of osteoarthritis progression using polarization-sensitive optical coherence tomography

NASA Astrophysics Data System (ADS)

Nassif, Nader A.; Pierce, Mark C.; Park, B. Hyle; Cense, Barry; de Boer, Johannes F.

2004-07-01

Osteoarthritis is a prevalent medical condition that presents a diagnostic and therapeutic challenge to physicians today because of the inability to assess the integrity of the articular cartilage early in the disease. Polarization sensitive optical coherence tomography (PS-OCT) is a high resolution, non-contact imaging modality that provides cross-sectional images with additional information regarding the integrity of the collagen matrix. Using PS-OCT to image provides information regarding thickness of the articular cartilage and gives an index of biochemical changes based on alterations in optical properties (i.e. birefringence) of the tissue. We demonstrate initial experiments performed on specimens collected following total knee replacement surgery. Articular cartilage was imaged using a 1310 nm PS-OCT system where both intensity and phase images were acquired. PS-OCT images were compared with histology, and the changes in tissue optical properties were characterized. Analysis of the intensity images demonstrates differences between healthy and diseased cartilage surface and thickness. Phase maps of the tissue demonstrated distinct differences between healthy and diseased tissue. PS-OCT was able to image a gradual loss of birefringence as the tissue became more diseased. In this way, determining the rate of change of the phase provides a quantitative measure of pathology. Thus, imaging and evaluation of osteoarthritis using PS-OCT can be a useful means of quantitative assessment of the disease.

Influence of sample preparation and reliability of automated numerical refocusing in stain-free analysis of dissected tissues with quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

2015-05-01

Digital holographic microscopy (DHM) has been demonstrated to be a versatile tool for high resolution non-destructive quantitative phase imaging of surfaces and multi-modal minimally-invasive monitoring of living cell cultures in-vitro. DHM provides quantitative monitoring of physiological processes through functional imaging and structural analysis which, for example, gives new insight into signalling of cellular water permeability and cell morphology changes due to toxins and infections. Also the analysis of dissected tissues quantitative DHM phase contrast prospects application fields by stain-free imaging and the quantification of tissue density changes. We show that DHM allows imaging of different tissue layers with high contrast in unstained tissue sections. As the investigation of fixed samples represents a very important application field in pathology, we also analyzed the influence of the sample preparation. The retrieved data demonstrate that the quality of quantitative DHM phase images of dissected tissues depends strongly on the fixing method and common staining agents. As in DHM the reconstruction is performed numerically, multi-focus imaging is achieved from a single digital hologram. Thus, we evaluated the automated refocussing feature of DHM for application on different types of dissected tissues and revealed that on moderately stained samples highly reproducible holographic autofocussing can be achieved. Finally, it is demonstrated that alterations of the spatial refractive index distribution in murine and human tissue samples represent a reliable absolute parameter that is related of different degrees of inflammation in experimental colitis and Crohn's disease. This paves the way towards the usage of DHM in digital pathology for automated histological examinations and further studies to elucidate the translational potential of quantitative phase microscopy for the clinical management of patients, e.g., with inflammatory bowel disease.

Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method.

PubMed

Namimatsu, Shigeki; Ghazizadeh, Mohammad; Sugisaki, Yuichi

2005-01-01

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.

Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

PubMed Central

Aboushousha, Tarek; Mamdouh, Samah; Hamdy, Hussam; Helal, Noha; Khorshed, Fatma; Safwat, Gehan; Seleem, Mohamed

2018-01-01

Objective: To investigate the expression of TTF-1, RAGE, GLUT1 and SOX2 in HCV-associated HCCs and in surrounding non-tumorous liver tissue. Material and Methods: Tissue material from partial hepatectomy cases for HCC along with corresponding serum samples and 30 control serum samples from healthy volunteers were studied. Biopsies were classified into: non-tumor hepatic tissue (36 sections); HCC (33 sections) and liver cell dysplasia (LCD) (15 sections). All cases were positive for HCV. Immunohistochemistry (IHC), gene extraction and quantitative real-time reverse-transcription assays (qRT-PCR) were applied. Results: By IHC, LCD and HCC showed significantly high percentages of positive cases with all markers. SOX2 showed significant increase with higher HCC grades, while RAGE demonstrated an inverse relation and GLUT-1 and TTF-1 lacked any correlation. In nontumorous-HCV tissue, we found significantly high TTF-1, low RAGE and negative SOX2 expression. RAGE, GLUT-1 and SOX2 show non-significant elevation positivity in high grade HCV compared to low grade lesions. TTF-1, RAGE and SOX2 exhibited low expression in cirrhosis compared to fibrosis. Biochemical studies on serum and tissue extracts revealed significant down-regulation of RAGE, GLUT-1 and SOX2 genes, as well as significant up-regulation of the TTF-1 gene in HCC cases compared to controls. All studied genes show significant correlation with HCC grade. In non-tumor tissue, only TTF-1 gene expression had a significant correlation with the fibrosis score. Conclusion: Higher expression of TTF-1, RAGE, GLUT-1 and SOX2 in HCC and dysplasia compared to non-tumor tissues indicates up-regulation of these markers as early events during the development of HCV-associated HCC. PMID:29373917

Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

PubMed

Aboushousha, Tarek; Mamdouh, Samah; Hamdy, Hussam; Helal, Noha; Khorshed, Fatma; Safwat, Gehan; Seleem, Mohamed

2018-01-27

Objective: To investigate the expression of TTF-1, RAGE, GLUT1 and SOX2 in HCV-associated HCCs and in surrounding non-tumorous liver tissue. Material and Methods: Tissue material from partial hepatectomy cases for HCC along with corresponding serum samples and 30 control serum samples from healthy volunteers were studied. Biopsies were classified into: non-tumor hepatic tissue (36 sections); HCC (33 sections) and liver cell dysplasia (LCD) (15 sections). All cases were positive for HCV. Immunohistochemistry (IHC), gene extraction and quantitative real-time reverse-transcription assays (qRT-PCR) were applied. Results: By IHC, LCD and HCC showed significantly high percentages of positive cases with all markers. SOX2 showed significant increase with higher HCC grades, while RAGE demonstrated an inverse relation and GLUT-1 and TTF-1 lacked any correlation. In nontumorous-HCV tissue, we found significantly high TTF-1, low RAGE and negative SOX2 expression. RAGE, GLUT-1 and SOX2 show non-significant elevation positivity in high grade HCV compared to low grade lesions. TTF-1, RAGE and SOX2 exhibited low expression in cirrhosis compared to fibrosis. Biochemical studies on serum and tissue extracts revealed significant down-regulation of RAGE, GLUT-1 and SOX2 genes, as well as significant up-regulation of the TTF-1 gene in HCC cases compared to controls. All studied genes show significant correlation with HCC grade. In non-tumor tissue, only TTF-1 gene expression had a significant correlation with the fibrosis score. Conclusion: Higher expression of TTF-1, RAGE, GLUT-1 and SOX2 in HCC and dysplasia compared to non-tumor tissues indicates up-regulation of these markers as early events during the development of HCV-associated HCC. Creative Commons Attribution License

An immunohistochemical identification key for cell types in adult mouse prostatic and urethral tissue sections

PubMed Central

Turco, Anne E.; Gottschalk, Adam; Halberg, Richard B.; Guo, Jinjin; McMahon, Jill A.; McMahon, Andrew P.

2017-01-01

Though many methods can be used to identify cell types contained in complex tissues, most require cell disaggregation and destroy information about where cells reside in relation to their microenvironment. Here, we describe a polytomous key for cell type identification in intact sections of adult mouse prostate and prostatic urethra. The key is organized as a decision tree and initiates with one round of immunostaining for nerve, epithelial, fibromuscular/hematolymphoid, or vascular associated cells. Cell identities are recursively eliminated by subsequent staining events until the remaining pool of potential cell types can be distinguished by direct comparison to other cells. We validated our identification key using wild type adult mouse prostate and urethra tissue sections and it currently resolves sixteen distinct cell populations which include three nerve fiber types as well as four epithelial, five fibromuscular/hematolymphoid, one nerve-associated, and three vascular-associated cell types. We demonstrate two uses of this novel identification methodology. We first used the identification key to characterize prostate stromal cell type changes in response to constitutive phosphatidylinositide-3-kinase activation in prostate epithelium. We then used the key to map cell lineages in a new reporter mouse strain driven by Wnt10aem1(cre/ERT2)Amc. The identification key facilitates rigorous and reproducible cell identification in prostate tissue sections and can be expanded to resolve additional cell types as new antibodies and other resources become available. PMID:29145476

Region-specific tritium enrichment, and not differential beta-absorption, is the major cause of 'quenching' in film autoradiography.

PubMed

McEachron, D L; Nissanov, J; Tretiak, O J

1997-06-01

Tritium quenching refers to the situation in which estimates of tritium content generated by film autoradiography depend on the chemical composition of the tissue as well as on the concentration of the radioisotope. When analysing thin brain sections, for example, regions rich in lipid content generate reduced optical densities on x-ray film compared with lipid-poor regions even when the total tissue concentration of tritium in those regions is identical. We hypothesize that the dried thickness of regions within sections depends upon the relative concentrations and types of lipid within the regions. Areas low in white matter dry thinner than areas high in white matter, leading to a relative enrichment of tritium in the thinner regions. To test this model, a series of brain pastes were made with different concentrations of grey and white matter and impregnated with equal amounts of tritium. The thickness of dried sections was compared with percentage of white matter and apparent radioactive content as determined by autoradiogram analysis. The results demonstrated that thickness increased, and apparent radioactivity decreased, with higher percentages of white matter. In the second experiment, thickness measurements from dried sections were successfully used to correct the apparent radioisotope content of autoradiograms created from tritium containing white- and grey-matter tissue slices. We conclude that within-section thickness variation is the major physical cause for 'tritium quenching'.

Investigation of Thermal Effects of Photocoagulation on Retinal Tissue Using Fine-Motion-Sensitive Dynamic Optical Coherence Tomography.

PubMed

Kurokawa, Kazuhiro; Makita, Shuichi; Yasuno, Yoshiaki

2016-01-01

To enable an objective evaluation of photocoagulation, we characterize thermal tissue changes induced by laser irradiation with different laser parameters using optical coherence tomography (OCT). Spectral-domain OCT with a newly developed image processing method was used to monitor the thermal changes of ex vivo porcine retina. A sequence of OCT B-scans was obtained at the same retinal position simultaneously with the photocoagulation. Cross-sectional tissue displacement maps with respect to an OCT image taken before laser irradiation were computed for images taken before, during, and after laser irradiation, by using a correlation-based custom algorithm. Cross-sectional correlation maps (OCT correlation maps) were also computed from an OCT image taken before laser irradiation as a base-line to visualize alterations of tissue microstructure induced by laser irradiation. By systematically controlling laser power and exposure times, tissue displacements and structural changes of 200 retinal regions of 10 porcine eyes were characterized. Thermal tissue changes were characterized by B-scan images, OCT correlation maps, and tissue displacement maps. Larger tissue deformation was induced with higher laser power and shorter exposure time, while the same total laser energy (10 mJ) was applied. The measured tissue displacements revealed the complicated dynamics of tissue displacements. Three types of dynamics were observed; lateral expansion, lateral constriction, and a type showing more complicated dynamics. The results demonstrated the ability of this OCT-based method to evaluate retinal changes induced by laser irradiation. This evaluation could lead to further understanding of thermal effects, and increasing reproducibility of photocoagulation therapy.

Ultrastructural identification of noradrenergic nerve terminals and vasopressin-containing neurons of the paraventricular nucleus in the same thin section.

PubMed

Silverman, A J; Hou-Yu, A; Oldfield, B J

1983-09-01

Since many peptidergic cell groups receive a diverse and complex monoaminergic innervation, we have developed a double-label procedure to visualize a peptide and a catecholamine in the same ultrathin section. Radiolabeled norepinephrine (NE) is applied locally and its reuptake into NE terminals is demonstrated by ultrastructural radioautography. Controls in this and other studies demonstrate that the NE labels only NE (and possibly epinephrine) terminals and not dopaminergic or serotonergic terminals. In the same tissue, vasopressin is localized by immunocytochemistry on unembedded sections that are subsequently embedded in epoxy resins for thin sectioning. The procedure as described here shows that NE terminals in the periventricular zone of the paraventricular nucleus of the hypothalamus innervate both vasopressin-positive and vasopressin-negative structures. This technique is useful in determining the chemical connectivity of the hypothalamus.

Growth hormone mRNA in mammary gland tumors of dogs and cats.

PubMed Central

Mol, J A; van Garderen, E; Selman, P J; Wolfswinkel, J; Rijinberk, A; Rutteman, G R

1995-01-01

We have shown recently that in the dog progestin administration results in mammary production of immunoreactive growth hormone (GH). At present we demonstrate the expression of the gene encoding GH in the mammary gland of dogs and cats using reverse-transcriptase PCR. GH mRNA was found in the great majority of normal mammary tissues as well as benign and malignant mammary tumors of the dog and was associated with the presence of immunoreactive GH in cryostat sections. The mammary PCR product proved to be identical to that of the pituitary. The highest expression levels were found after prolonged treatment with progestins. In carcinomas GH mRNA was also found in progesterone receptor-negative tissue samples, indicating that after malignant transformation GH gene expression may become progestin independent. GH mRNA was also present in mammary tissues of cats with progestin-induced fibroadenomatous changes. It is concluded that GH gene expression occurs in normal, hyperplastic, and neoplastic mammary tissue of the dog. The expression in normal tissue is stimulated by progestins and might mediate the progestin-stimulated development of canine mammary tumors. The demonstration of progestin-stimulated GH expression in mammary tissue of cats indicates that the phenomenon is more generalized among mammals. Images PMID:7738169

High-resolution breast tomography at high energy: a feasibility study of phase contrast imaging on a whole breast

NASA Astrophysics Data System (ADS)

Sztrókay, A.; Diemoz, P. C.; Schlossbauer, T.; Brun, E.; Bamberg, F.; Mayr, D.; Reiser, M. F.; Bravin, A.; Coan, P.

2012-05-01

Previous studies on phase contrast imaging (PCI) mammography have demonstrated an enhancement of breast morphology and cancerous tissue visualization compared to conventional imaging. We show here the first results of the PCI analyser-based imaging (ABI) in computed tomography (CT) mode on whole and large (>12 cm) tumour-bearing breast tissues. We demonstrate in this work the capability of the technique of working at high x-ray energies and producing high-contrast images of large and complex specimens. One entire breast of an 80-year-old woman with invasive ductal cancer was imaged using ABI-CT with monochromatic 70 keV x-rays and an area detector of 92×92 µm2 pixel size. Sagittal slices were reconstructed from the acquired data, and compared to corresponding histological sections. Comparison with conventional absorption-based CT was also performed. Five blinded radiologists quantitatively evaluated the visual aspects of the ABI-CT images with respect to sharpness, soft tissue contrast, tissue boundaries and the discrimination of different structures/tissues. ABI-CT excellently depicted the entire 3D architecture of the breast volume by providing high-resolution and high-contrast images of the normal and cancerous breast tissues. These results are an important step in the evolution of PCI-CT towards its clinical implementation.

Serial Section Scanning Electron Microscopy (S3EM) on Silicon Wafers for Ultra-Structural Volume Imaging of Cells and Tissues

PubMed Central

Horstmann, Heinz; Körber, Christoph; Sätzler, Kurt; Aydin, Daniel; Kuner, Thomas

2012-01-01

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S3EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 µm3 volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S3EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S3EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation. PMID:22523574

Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues.

PubMed

Horstmann, Heinz; Körber, Christoph; Sätzler, Kurt; Aydin, Daniel; Kuner, Thomas

2012-01-01

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S(3)EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 µm(3) volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S(3)EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S(3)EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.

FIB-SEM imaging of carbon nanotubes in mouse lung tissue.

PubMed

Købler, Carsten; Saber, Anne Thoustrup; Jacobsen, Nicklas Raun; Wallin, Håkan; Vogel, Ulla; Qvortrup, Klaus; Mølhave, Kristian

2014-06-01

Ultrastructural characterisation is important for understanding carbon nanotube (CNT) toxicity and how the CNTs interact with cells and tissues. The standard method for this involves using transmission electron microscopy (TEM). However, in particular, the sample preparation, using a microtome to cut thin sample sections for TEM, can be challenging for investigation of regions with agglomerations of large and stiff CNTs because the CNTs cut with difficulty. As a consequence, the sectioning diamond knife may be damaged and the uncut CNTs are left protruding from the embedded block surface excluding them from TEM analysis. To provide an alternative to ultramicrotomy and subsequent TEM imaging, we studied focused ion beam scanning electron microscopy (FIB-SEM) of CNTs in the lungs of mice, and we evaluated the applicability of the method compared to TEM. FIB-SEM can provide serial section volume imaging not easily obtained with TEM, but it is time-consuming to locate CNTs in the tissue. We demonstrate that protruding CNTs after ultramicrotomy can be used to locate the region of interest, and we present FIB-SEM images of CNTs in lung tissue. FIB-SEM imaging was applied to lung tissue from mice which had been intratracheally instilled with two different multiwalled CNTs; one being short and thin, and the other longer and thicker. FIB-SEM was found to be most suitable for detection of the large CNTs (Ø ca. 70 nm), and to be well suited for studying CNT agglomerates in biological samples which is challenging using standard TEM techniques.

Hard X-ray submicrometer tomography of human brain tissue at Diamond Light Source

NASA Astrophysics Data System (ADS)

Khimchenko, A.; Bikis, C.; Schulz, G.; Zdora, M.-C.; Zanette, I.; Vila-Comamala, J.; Schweighauser, G.; Hench, J.; Hieber, S. E.; Deyhle, H.; Thalmann, P.; Müller, B.

2017-06-01

There is a lack of the necessary methodology for three-dimensional (3D) investigation of soft tissues with cellular resolution without staining or tissue transformation. Synchrotron radiation based hard X-ray in-line phase contrast tomography using single-distance phase reconstruction (SDPR) provides high spatial resolution and density contrast for the visualization of individual cells using a standard specimen preparation and data reconstruction. In this study, we demonstrate the 3D characterization of a formalin-fixed paraffin-embedded (FFPE) human cerebellum specimen by SDPR at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, UK) at pixel sizes down to 0.45 μm. The approach enables visualization of cerebellar layers (Stratum moleculare and Stratum granulosum), the 3D characterization of individual cells (Purkinje, stellate and granule cells) and can even resolve some subcellular structures (nucleus and nucleolus of Purkinje cells). The tomographic results are qualitatively compared to hematoxylin and eosin (H&E) stained histological sections. We demonstrate the potential benefits of hard X-ray microtomography for the investigations of biological tissues in comparison to conventional histology.

Magnetic resonance imaging of intraoral hard and soft tissues using an intraoral coil and FLASH sequences.

PubMed

Flügge, Tabea; Hövener, Jan-Bernd; Ludwig, Ute; Eisenbeiss, Anne-Kathrin; Spittau, Björn; Hennig, Jürgen; Schmelzeisen, Rainer; Nelson, Katja

2016-12-01

To ascertain the feasibility of MRI as a non-ionizing protocol for routine dentomaxillofacial diagnostic imaging. Wireless coils were used for MRI of intraoral hard and soft tissues. FLASH MRI was applied in vivo with a mandible voxel size of 250 × 250 × 500 μm 3 , FOV of 64 × 64 × 28 mm 3 and acquisition time of 3:57 min and with a maxilla voxel size of 350 μm 3 and FOV of 34 cm 3 in 6:40 min. Ex vivo imaging was performed in 4:38 min, with a resolution of 200 μm 3 and FOV of 36.5 cm 3 . Cone beam (CB) CT of the mandible and subjects were acquired. MRI was compared to CBCT and histological sections. Deviations were calculated with intraclass correlation coefficient (ICC) and coefficient of variation (c v ). A high congruence between CBCT, MRI and specimens was demonstrated. Hard and soft tissues including dental pulp, periodontium, gingiva, cancellous bone and mandibular canal contents were adequately displayed with MRI. Imaging of select intraoral tissues was achieved using custom MRI protocols with an easily applicable intraoral coil in a clinically acceptable acquisition time. Comparison with CBCT and histological sections helped demonstrate dimensional accuracy of the MR images. The course of the mandibular canal was accurately displayed with CBCT and MRI. • MRI is a clinically available diagnostic tool in dentistry • Intraoral hard and soft tissues can be imaged with a high resolution with MRI • The dimensional accuracy of MRI is comparable to cone beam CT.

THE LOCALIZATION OF HOMOLGOUS PLASMA PROTEINS IN THE TISSUES OF YOUNG HUMAN BEINGS AS DEMONSTRATED WITH FLUORESCENT ANTIBODIES

PubMed Central

Gitlin, David; Landing, Benjamin H.; Whipple, Ann

1953-01-01

Employing fluorescent antibodies for the detection of homologous plasma proteins in tissue sections, the distribution of plasma albumin, γ-globulin, β-lipoprotein, β1-metal-combining globulin, and fibrinogen has been studied in the tissues of infants and children. Plasma albumin, γ-globulin, and β1-metal-combining globulin were found in many cells and particularly cell nuclei, connective tissues and interstitial spaces, lymphatics, and blood vessels. β-Lipoprotein was found mostly in the nuclei of all cell types while fibrinogen was restricted largely to the lymphatic and vascular channels, connective tissues and the interstitial spaces. The widespread distribution of these plasma proteins in cells and connective tissues indicates the magnitude of the extravascular plasma protein pool which is in equilibrium with circulating plasma. Unfortunately, these results do not permit accurate localization of the sites of production of these plasma proteins, but do give some idea of their intimate relationship to the tissues. PMID:13022871

Differentiation of Normal and Malignant Breast Tissues using Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Mehrotra, Ranjana; Jangir, Deepak Kumar; Gupta, Alka; Kandpal, H. C.

2008-11-01

Infrared spectra of carcinomatous and their normal fore bearing tissues were collected in the 600 cm-1 to 4000 cm-1 region. Fourier Transform Infrared (FTIR) data of infiltrating ductal carcinoma of breast with different grades of malignancy from patients of different age groups were analyzed. Infrared spectra demonstrate significant spectral differences between the tumor sections of normal and the malignant breast tissues. In particular, changes in frequency and intensity in the spectra of protein, nucleic acid and glycogen were observed. This allows to make a qualitative and semi quantitative evaluation of the changes in proliferation activities from normal to diseased tissue. The findings establish a framework for additional studies, which may enable us to establish a relation of the diseased state with its infrared spectra.

[Radiographic and histological study of a case of apexification in a human molar].

PubMed

Sahli, C C

1989-01-01

A case of apexification in a lower right second molar is described. Radiographs demonstrate apical closure with a different morphological pattern from that of the lower left second molar. Following extraction, after 15 months, serial histologic sections show calcified tissue obturating the apical foramen, well adapted to the initial dentin and cementum walls. Inside some small areas containing connective tissue with capillaries can be observed. The histologic and radiographic observations indicate that apical closure occurs as a result of differentiation of periodontal apical cells.

Diattenuation of brain tissue and its impact on 3D polarized light imaging

PubMed Central

Menzel, Miriam; Reckfort, Julia; Weigand, Daniel; Köse, Hasan; Amunts, Katrin; Axer, Markus

2017-01-01

3D-polarized light imaging (3D-PLI) reconstructs nerve fibers in histological brain sections by measuring their birefringence. This study investigates another effect caused by the optical anisotropy of brain tissue – diattenuation. Based on numerical and experimental studies and a complete analytical description of the optical system, the diattenuation was determined to be below 4 % in rat brain tissue. It was demonstrated that the diattenuation effect has negligible impact on the fiber orientations derived by 3D-PLI. The diattenuation signal, however, was found to highlight different anatomical structures that cannot be distinguished with current imaging techniques, which makes Diattenuation Imaging a promising extension to 3D-PLI. PMID:28717561

Histological methods to determine blood flow distribution with fluorescent microspheres.

PubMed

Luchtel, D L; Boykin, J C; Bernard, S L; Glenny, R W

1998-11-01

We evaluated several histological methods and determined their advantages and disadvantages for histological studies of tissues and organs perfused with fluorescent microspheres. Microspheres retained their fluorescence in 7-10 microm serial sections with a change in the antimedium from toluene when samples were fixed in formalin and embedded in paraffin. Several antimedia allowed both wax infiltration of tissue and preservation of microsphere fluorescence. Histoclear II was the best substitute for toluene. When samples were fixed in formalin and embedded in glycol methacrylate, thinner (3-5 microm) sections provided greater histological detail but had fewer microspheres per section. Air dried lung tissue followed by Vibratome sectioning provided thick sections (100 microm) that facilitated rapid survey of large volumes of tissue for microspheres but limited histological detail, and the air drying procedure was restricted to lung tissue. Samples fixed in formalin followed by Vibratome sectioning of unembedded tissue provided better histological detail of lung tissue and was also useful for other organs. These sections were more difficult to handle and to mount on slides compared to air dried tissue, whereas fixed tissue embedded in gelatin provided better tissue support for Vibratome sectioning. Rapid freezing followed by cryo-microtome sectioning resulted in frozen sections that were relatively difficult to handle compared to embedded or unembedded tissue; they also deteriorated relatively rapidly with time. Paraffin sections were stained with hematoxylin and eosin or with aqueous methyl green, although tissue autofluorescence by itself was usually sufficient to identify histological features. Methacrylate sections quenched tissue autofluorescence, and Lee's stain or Richardson's stain were used for staining sections. Toluene based mountants such as Cytoseal quenched fluorescence, particularly the red fluorescent microspheres. Aqueous based mountants such as Aquamount, Crystal/Mount, Fluoromount-G were substituted, although such preparations were not as permanent as Cytoseal mounted coverglasses and tended to cause fading of stained sections.

Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.

PubMed Central

Campagnola, Paul J; Millard, Andrew C; Terasaki, Mark; Hoppe, Pamela E; Malone, Christian J; Mohler, William A

2002-01-01

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)-nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices. PMID:11751336

Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death Events and Fibrosis in Tissue Sections

PubMed Central

Krajewska, Maryla; Smith, Layton H.; Rong, Juan; Huang, Xianshu; Hyer, Marc L.; Zeps, Nikolajs; Iacopetta, Barry; Linke, Steven P.; Olson, Allen H.; Reed, John C.; Krajewski, Stan

2009-01-01

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections. (J Histochem Cytochem 57:649–663, 2009) PMID:19289554

Superresolution Imaging of Aquaporin-4 Cluster Size in Antibody-Stained Paraffin Brain Sections

PubMed Central

Smith, Alex J.; Verkman, Alan S.

2015-01-01

The water channel aquaporin-4 (AQP4) forms supramolecular clusters whose size is determined by the ratio of M1- and M23-AQP4 isoforms. In cultured astrocytes, differences in the subcellular localization and macromolecular interactions of small and large AQP4 clusters results in distinct physiological roles for M1- and M23-AQP4. Here, we developed quantitative superresolution optical imaging methodology to measure AQP4 cluster size in antibody-stained paraffin sections of mouse cerebral cortex and spinal cord, human postmortem brain, and glioma biopsy specimens. This methodology was used to demonstrate that large AQP4 clusters are formed in AQP4−/− astrocytes transfected with only M23-AQP4, but not in those expressing only M1-AQP4, both in vitro and in vivo. Native AQP4 in mouse cortex, where both isoforms are expressed, was enriched in astrocyte foot-processes adjacent to microcapillaries; clusters in perivascular regions of the cortex were larger than in parenchymal regions, demonstrating size-dependent subcellular segregation of AQP4 clusters. Two-color superresolution imaging demonstrated colocalization of Kir4.1 with AQP4 clusters in perivascular areas but not in parenchyma. Surprisingly, the subcellular distribution of AQP4 clusters was different between gray and white matter astrocytes in spinal cord, demonstrating regional specificity in cluster polarization. Changes in AQP4 subcellular distribution are associated with several neurological diseases and we demonstrate that AQP4 clustering was preserved in a postmortem human cortical brain tissue specimen, but that AQP4 was not substantially clustered in a human glioblastoma specimen despite high-level expression. Our results demonstrate the utility of superresolution optical imaging for measuring the size of AQP4 supramolecular clusters in paraffin sections of brain tissue and support AQP4 cluster size as a primary determinant of its subcellular distribution. PMID:26682810

A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

NASA Astrophysics Data System (ADS)

Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

1981-05-01

Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

PubMed Central

Scott, Anna E.; Vasilescu, Dragos M.; Seal, Katherine A. D.; Keyes, Samuel D.; Mavrogordato, Mark N.; Hogg, James C.; Sinclair, Ian; Warner, Jane A.; Hackett, Tillie-Louise; Lackie, Peter M.

2015-01-01

Background Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data. Methods FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging. Results The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections. Conclusion We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis. PMID:26030902

A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy

PubMed Central

Zhang, Yuying; Akins, Meredith L.; Murari, Kartikeya; Xi, Jiefeng; Li, Ming-Jun; Luby-Phelps, Katherine; Mahendroo, Mala; Li, Xingde

2012-01-01

We report the development of an all-fiber-optic scanning endomicroscope capable of high-resolution second harmonic generation (SHG) imaging of biological tissues and demonstrate its utility for monitoring the remodeling of cervical collagen during gestation in mice. The endomicroscope has an overall 2.0 mm diameter and consists of a single customized double-clad fiber, a compact rapid two-dimensional beam scanner, and a miniature compound objective lens for excitation beam delivery, scanning, focusing, and efficient SHG signal collection. Endomicroscopic SHG images of murine cervical tissue sections at different stages of normal pregnancy reveal progressive, quantifiable changes in cervical collagen morphology with resolution similar to that of bench-top SHG microscopy. SHG endomicroscopic imaging of ex vivo murine and human cervical tissues through intact epithelium has also been performed. Our findings demonstrate the feasibility of SHG endomicroscopy technology for staging normal pregnancy, and suggest its potential application as a minimally invasive tool for clinical assessment of abnormal cervical remodeling associated with preterm birth. PMID:22826263

Effective melanin depigmentation of human and murine ocular tissues: an improved method for paraffin and frozen sections.

PubMed

Manicam, Caroline; Pitz, Susanne; Brochhausen, Christoph; Grus, Franz H; Pfeiffer, Norbert; Gericke, Adrian

2014-01-01

The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues. Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4) with oxalic acid, and the second 10% hydrogen peroxide (H2O2). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation. Optimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage. Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.

Handling Golgi-impregnated tissue for light microscopy.

PubMed

Berbel, P J; Fairén, A

1983-08-08

The use of cyanocrylic glue to fix pieces of Golgi-stained nervous tissue on a paraffin blank is proposed for obtaining thick sections of unembedded tissue with a sliding microtome. This procedure makes correct orientation of the tissue easy during sectioning and makes it possible to obtain tissue sections quickly. The sections are flat-mounted using epoxy resin, resulting in permanent preparations with excellent optical properties and enabling further thin-sectioning for light and electron microscopic studies.

Tumor margin assessment in Mohs surgery using reflectance, fluorescence and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Nguyen, Hieu T. M.; Moy, Austin J.; Zhang, Yao; Feng, Xu; Reichenberg, Jason S.; Fox, Matthew; Tunnell, James W.

2017-02-01

Mohs surgery is the current gold standard to treat large, aggressive or high-risk non-melanoma skin cancer (NMSC) cases. While Mohs surgery is an effective treatment, the procedure is time-consuming and expensive for physicians as well as burdensome for patients as they wait for frozen section histology. Our group has recently demonstrated high diagnostic accuracy using a noninvasive "spectral biopsy" (combination of diffuse reflectance (DRS), fluorescence (FS) and Raman spectroscopy (RS)) to classify NMSC vs. normal lesion in a screening setting of intact tissue. Here, we examine the sensitivity of spectral biopsy to pathology in excised Mohs sections. The system is designed with three modalities integrated into one fiber probe, which is utilized to measure DRS, FS, and RS of freshly excised skin from patients with various NMSC pathologies including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), where each measurement location is correlated to histopathology. The spectral biopsy provides complimentary physiological information including the reduced scattering coefficient, hemoglobin content and oxygen saturation from DRS, NADH and collagen contribution from FS and information regarding multiple proteins and lipids from RS. We then apply logistic regression model to the extracted physiological parameters to classify NMSC vs. normal tissue. The results on the excised tissue are generally consistent with in vivo measurements showing decreased scattering within the tumor and reduced fluorescence. Due to the high sensitivity of RS to lipids, subcutaneous fat often dominates the RS signal. This pilot study demonstrates the potential for a spectral biopsy to classify NMSC vs. normal tissue, indicating the opportunity to guide Mohs excisions.

Regulation of cellular marker modulated upon irradiation of low power laser light in burn injured mice

NASA Astrophysics Data System (ADS)

Rathnakar, Bharath; Prabhu, Vijendra; Rao, Bola Sadashiva Satish; Chandra, Subhash; Rai, Sharada; Mahato, Krishna Kishore

2016-12-01

The present study intends to understand the importance of cellular marker in tissue regeneration regulated upon irradiation of low power laser light in burn inflicted mice. Under anesthetic conditions, the thermal injury was induced on Swiss albino mice of either sex. Following injury, the animals were randomly divided into three groups; i. e., un-illuminated control, the group treated with 5% Povidone iodine (reference standard) and single exposure of 3 J/cm2 (830 nm). Burn tissue samples from each group were excised at day 6 post burn injury upon euthanization and used for histological and immunohistochemical analysis. Haematoxylin and Eosin (H and E) staining was performed on the selected sections to asses proliferation and angiogenesis at day 6 post-injury. For immunohistochemical analysis, tissue sections from all the three treatment groups on day 6 were stained using specific antibody against Proliferating cell nuclear antigen (PCNA). The results of the histological and immunohistochemical analysis showed improved tissue restoration in animals treated with optimal laser influence as compared to un-illuminated controls. The findings of present study clearly demonstrated the beneficial effects of 830 nm laser in burn wound healing and its influence in regulating the cellular marker.

Analysis of human tissues by total reflection X-ray fluorescence. Application of chemometrics for diagnostic cancer recognition

NASA Astrophysics Data System (ADS)

Benninghoff, L.; von Czarnowski, D.; Denkhaus, E.; Lemke, K.

1997-07-01

For the determination of trace element distributions of more than 20 elements in malignant and normal tissues of the human colon, tissue samples (approx. 400 mg wet weight) were digested with 3 ml of nitric acid (sub-boiled quality) by use of an autoclave system. The accuracy of measurements has been investigated by using certified materials. The analytical results were evaluated by using a spreadsheet program to give an overview of the element distribution in cancerous samples and in normal colon tissues. A further application, cluster analysis of the analytical results, was introduced to demonstrate the possibility of classification for cancer diagnosis. To confirm the results of cluster analysis, multivariate three-way principal component analysis was performed. Additionally, microtome frozen sections (10 μm) were prepared from the same tissue samples to compare the analytical results, i.e. the mass fractions of elements, according to the preparation method and to exclude systematic errors depending on the inhomogeneity of the tissues.

Quantitative diagnosis of tongue cancer from histological images in an animal model

NASA Astrophysics Data System (ADS)

Lu, Guolan; Qin, Xulei; Wang, Dongsheng; Muller, Susan; Zhang, Hongzheng; Chen, Amy; Chen, Zhuo G.; Fei, Baowei

2016-03-01

We developed a chemically-induced oral cancer animal model and a computer aided method for tongue cancer diagnosis. The animal model allows us to monitor the progress of the lesions over time. Tongue tissue dissected from mice was sent for histological processing. Representative areas of hematoxylin and eosin stained tissue from tongue sections were captured for classifying tumor and non-tumor tissue. The image set used in this paper consisted of 214 color images (114 tumor and 100 normal tissue samples). A total of 738 color, texture, morphometry and topology features were extracted from the histological images. The combination of image features from epithelium tissue and its constituent nuclei and cytoplasm has been demonstrated to improve the classification results. With ten iteration nested cross validation, the method achieved an average sensitivity of 96.5% and a specificity of 99% for tongue cancer detection. The next step of this research is to apply this approach to human tissue for computer aided diagnosis of tongue cancer.

Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts

PubMed Central

Ozbay, Baris N.; Losacco, Justin T.; Cormack, Robert; Weir, Richard; Bright, Victor M.; Gopinath, Juliet T.; Restrepo, Diego; Gibson, Emily A.

2015-01-01

We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2 g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ~12 µm and an axial scan range of ~80 µm. The lateral field-of-view is 300 µm, and the lateral resolution is 1.8 µm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP). PMID:26030555

Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by Widefield Epifluorescence, super-resolution light, and immunoelectron microscopy.

PubMed

Killingsworth, Murray C; Lai, Ken; Wu, Xiaojuan; Yong, Jim L C; Lee, C Soon

2012-11-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy.

Quantum Dot Immunocytochemical Localization of Somatostatin in Somatostatinoma by Widefield Epifluorescence, Super-resolution Light, and Immunoelectron Microscopy

PubMed Central

Lai, Ken; Wu, Xiaojuan; Yong, Jim L. C.; Lee, C. Soon

2012-01-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy. PMID:22899862

[Method of determination of fat deposit in chickens].

PubMed

Bakhnev, D

1975-01-01

The method described is based on the histochemical demonstration of fats with the use of Sudan stains and the clearing of tissues in an alkaline medium. It is employed to investigate whole birds, thick sections or individual organs to determine the morphotopographic localization of the fat depots. The procedure comprises the alcohol fixation of tissues, the staining of lipids with Sudan black, the clearing of tissues in a solution of potassium hydroxide, and the final mounting of the cleared materials in glycerine. This treatment results in that the sites of fat deposition assume a blue coloration, the bones are grey, and all remaining tissues and organs (if properly bled) are colorless and transparent. The method can also be successfully used with materials that have been preliminary fixed with formalin but their clearing has been considerably worse.

Histochemical Demonstration of Protein-Bound Alpha-Acylamido Carboxyl Groups

PubMed Central

Barrnett, Russell J.; Seligman, Arnold M.

1958-01-01

A method has been developed to demonstrate the alpha-acylamido carboxyl groups of protein, taking advantage of the fact that acylamido carboxyl groups are converted to ketonic carbonyls by the action of acetic anhydride and absolute pyridine. The method utilizes deparaffinized sections of tissues fixed in a variety of fixatives. Following the conversion of carboxyls to the methyl ketones, the latter are stained with 2-hydroxy-3-naphthoic acid hydrazide. Control experiments have indicated that methylation of carboxyls prevented staining, as did carbonyl reagents after the carboxyls were transformed to methyl ketones. Leucofuchsin did not stain the ketonic carbonyls, and only elastic tissue stained with 2-hydroxy-3-naphthoic acid hydrazide without the previous use of the catalyzed reaction with anhydride. A brief survey of the reaction on various tissues of the albino rat was made, and the effects of various fixatives were assayed. Of particular interest were certain sites, such as acidophiles of the anterior pituitary gland, where an intense reaction occurred. The possibility exists that certain specific proteins rich in terminal acylamido carboxyl groups, by virtue of their protein side chains or low molecular weight, may be demonstrated by this method. PMID:13525430

Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits

PubMed Central

Posé, Sara; Paniagua, Candelas; Cifuentes, Manuel; Blanco-Portales, Rosario; Quesada, Miguel A.; Mercado, José A.

2013-01-01

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell–cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit. PMID:23873994

Actinomyces israelii in osteoradionecrosis of the jaws. Histopathologic and immunocytochemical study of five cases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Happonen, R.P.; Viander, M.; Pelliniemi, L.

1983-06-01

Five surgically treated patients with osteoradionecrosis of the jaws are presented. The clinical history of the disease varied from 3 to 17 years. In three cases the progression of the disease was enhanced by surgical procedures performed in the irradiated area causing exfoliation of the premaxillary area in one case and spontaneous mandibular fracture in two cases. Actinomyces israelii was demonstrated in tissue sections of all five cases by using FITC-labeled specific antiserum and additionally with peroxidase-antiperoxidase method in one case. Candida was found in histologic sections of three cases. Radiation damage in the oral soft tissues and jawbones makesmore » the atmosphere favorable for anaerobic microorganisms. The present results indicate that the role of A. israelii in the pathogenesis of osteoradionecrosis of the jaws has not been fully appreciated.« less

Boiling histotripsy lesion characterization on a clinical magnetic resonance imaging-guided high intensity focused ultrasound system

PubMed Central

Eranki, Avinash; Farr, Navid; Partanen, Ari; V. Sharma, Karun; Chen, Hong; Rossi, Christopher T.; Kothapalli, Satya V. V. N.; Oetgen, Matthew; Kim, AeRang; H. Negussie, Ayele; Woods, David; J. Wood, Bradford; C. W. Kim, Peter; S. Yarmolenko, Pavel

2017-01-01

Purpose High intensity focused ultrasound (HIFU) is a non-invasive therapeutic technique that can thermally ablate tumors. Boiling histotripsy (BH) is a HIFU approach that can emulsify tissue in a few milliseconds. Lesion volume and temperature effects for different BH sonication parameters are currently not well characterized. In this work, lesion volume, temperature distribution, and area of lethal thermal dose were characterized for varying BH sonication parameters in tissue-mimicking phantoms (TMP) and demonstrated in ex vivo tissues. Methods The following BH sonication parameters were varied using a clinical MR-HIFU system (Sonalleve V2, Philips, Vantaa, Finland): acoustic power, number of cycles/pulse, total sonication time, and pulse repetition frequency (PRF). A 3×3×3 pattern was sonicated inside TMP’s and ex vivo tissues. Post sonication, lesion volumes were quantified using 3D ultrasonography and temperature and thermal dose distributions were analyzed offline. Ex vivo tissues were sectioned and stained with H&E post sonication to assess tissue damage. Results Significant increase in lesion volume was observed while increasing the number of cycles/pulse and PRF. Other sonication parameters had no significant effect on lesion volume. Temperature full width at half maximum at the end of sonication increased significantly with all parameters except total sonication time. Positive correlation was also found between lethal thermal dose and lesion volume for all parameters except number of cycles/pulse. Gross pathology of ex vivo tissues post sonication displayed either completely or partially damaged tissue at the focal region. Surrounding tissues presented sharp boundaries, with little or no structural damage to adjacent critical structures such as bile duct and nerves. Conclusion Our characterization of effects of HIFU sonication parameters on the resulting lesion demonstrates the ability to control lesion morphologic and thermal characteristics with a clinical MR-HIFU system in TMP’s and ex vivo tissues. We demonstrate that this system can produce spatially precise lesions in both phantoms and ex vivo tissues. The results provide guidance on a preliminary set of BH sonication parameters for this system, with a potential to facilitate BH translation to the clinic. PMID:28301597

Solitary fibrous tumour of the cheek: An unusual presentation of a rare soft tissue tumour

PubMed Central

Jones, JL; Jones, AV; Drage, NA; Bhatia, S; Hourihan, MD

2014-01-01

This case report discusses the unusual presentation and ultrasound features of a solitary fibrous tumour of the face. Solitary fibrous tumour is an uncommon form of soft tissue tumour which, although seen predominantly within the lung pleura, can occur throughout the body in sites such as the peritoneum, mediastinum and head and neck. Ultrasound is an excellent imaging modality in the assessment of soft tissue masses in the head and neck. The ultrasound features demonstrated by this example of solitary fibrous tumour are reviewed. This report also highlights that ultrasound alone is ultimately limited in reaching a definitive diagnosis. The roles of other investigations such as ultrasound-guided biopsy and cross-sectional imaging are discussed. PMID:27433225

3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

PubMed Central

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2017-01-01

High-throughput sectioning and optical imaging of tissue samples using traditional immunohistochemical techniques can be costly and inaccessible in resource-limited areas. We demonstrate three-dimensional (3D) imaging and phenotyping in optically transparent tissue using lens-free holographic on-chip microscopy as a low-cost, simple, and high-throughput alternative to conventional approaches. The tissue sample is passively cleared using a simplified CLARITY method and stained using 3,3′-diaminobenzidine to target cells of interest, enabling bright-field optical imaging and 3D sectioning of thick samples. The lens-free computational microscope uses pixel super-resolution and multi-height phase recovery algorithms to digitally refocus throughout the cleared tissue and obtain a 3D stack of complex-valued images of the sample, containing both phase and amplitude information. We optimized the tissue-clearing and imaging system by finding the optimal illumination wavelength, tissue thickness, sample preparation parameters, and the number of heights of the lens-free image acquisition and implemented a sparsity-based denoising algorithm to maximize the imaging volume and minimize the amount of the acquired data while also preserving the contrast-to-noise ratio of the reconstructed images. As a proof of concept, we achieved 3D imaging of neurons in a 200-μm-thick cleared mouse brain tissue over a wide field of view of 20.5 mm2. The lens-free microscope also achieved more than an order-of-magnitude reduction in raw data compared to a conventional scanning optical microscope imaging the same sample volume. Being low cost, simple, high-throughput, and data-efficient, we believe that this CLARITY-enabled computational tissue imaging technique could find numerous applications in biomedical diagnosis and research in low-resource settings. PMID:28819645

Probing neural tissue with airy light-sheet microscopy: investigation of imaging performance at depth within turbid media

NASA Astrophysics Data System (ADS)

Nylk, Jonathan; McCluskey, Kaley; Aggarwal, Sanya; Tello, Javier A.; Dholakia, Kishan

2017-02-01

Light-sheet microscopy (LSM) has received great interest for fluorescent imaging applications in biomedicine as it facilitates three-dimensional visualisation of large sample volumes with high spatiotemporal resolution whilst minimising irradiation of, and photo-damage to the specimen. Despite these advantages, LSM can only visualize superficial layers of turbid tissues, such as mammalian neural tissue. Propagation-invariant light modes have played a key role in the development of high-resolution LSM techniques as they overcome the natural divergence of a Gaussian beam, enabling uniform and thin light-sheets over large distances. Most notably, Bessel and Airy beam-based light-sheet imaging modalities have been demonstrated. In the single-photon excitation regime and in lightly scattering specimens, Airy-LSM has given competitive performance with advanced Bessel-LSM techniques. Airy and Bessel beams share the property of self-healing, the ability of the beam to regenerate its transverse beam profile after propagation around an obstacle. Bessel-LSM techniques have been shown to increase the penetration-depth of the illumination into turbid specimens but this effect has been understudied in biologically relevant tissues, particularly for Airy beams. It is expected that Airy-LSM will give a similar enhancement over Gaussian-LSM. In this paper, we report on the comparison of Airy-LSM and Gaussian-LSM imaging modalities within cleared and non-cleared mouse brain tissue. In particular, we examine image quality versus tissue depth by quantitative spatial Fourier analysis of neural structures in virally transduced fluorescent tissue sections, showing a three-fold enhancement at 50 μm depth into non-cleared tissue with Airy-LSM. Complimentary analysis is performed by resolution measurements in bead-injected tissue sections.

Localization of cortical tissue optical changes during seizure activity in vivo with optical coherence tomography

PubMed Central

Eberle, Melissa M.; Hsu, Mike S.; Rodriguez, Carissa L.; Szu, Jenny I.; Oliveira, Michael C.; Binder, Devin K.; Park, B. Hyle

2015-01-01

Optical coherence tomography (OCT) is a high resolution, minimally invasive imaging technique, which can produce depth-resolved cross-sectional images. In this study, OCT was used to detect changes in the optical properties of cortical tissue in vivo in mice during the induction of global (pentylenetetrazol) and focal (4-aminopyridine) seizures. Through the use of a confidence interval statistical method on depth-resolved volumes of attenuation coefficient, we demonstrated localization of regions exhibiting both significant positive and negative changes in attenuation coefficient, as well as differentiating between global and focal seizure propagation. PMID:26137382

Laser Desorption/Ionization Mass Spectrometric Imaging of Endogenous Lipids from Rat Brain Tissue Implanted with Silver Nanoparticles

NASA Astrophysics Data System (ADS)

Muller, Ludovic; Baldwin, Kathrine; Barbacci, Damon C.; Jackson, Shelley N.; Roux, Aurélie; Balaban, Carey D.; Brinson, Bruce E.; McCully, Michael I.; Lewis, Ernest K.; Schultz, J. Albert; Woods, Amina S.

2017-08-01

Mass spectrometry imaging (MSI) of tissue implanted with silver nanoparticulate (AgNP) matrix generates reproducible imaging of lipids in rodent models of disease and injury. Gas-phase production and acceleration of size-selected 8 nm AgNP is followed by controlled ion beam rastering and soft landing implantation of 500 eV AgNP into tissue. Focused 337 nm laser desorption produces high quality images for most lipid classes in rat brain tissue (in positive mode: galactoceramides, diacylglycerols, ceramides, phosphatidylcholines, cholesteryl ester, and cholesterol, and in negative ion mode: phosphatidylethanolamides, sulfatides, phosphatidylinositol, and sphingomyelins). Image reproducibility in serial sections of brain tissue is achieved within <10% tolerance by selecting argentated instead of alkali cationized ions. The imaging of brain tissues spotted with pure standards was used to demonstrate that Ag cationized ceramide and diacylglycerol ions are from intact, endogenous species. In contrast, almost all Ag cationized fatty acid ions are a result of fragmentations of numerous lipid types having the fatty acid as a subunit. Almost no argentated intact fatty acid ions come from the pure fatty acid standard on tissue.

Automated gastric cancer diagnosis on H&E-stained sections; ltraining a classifier on a large scale with multiple instance machine learning

NASA Astrophysics Data System (ADS)

Cosatto, Eric; Laquerre, Pierre-Francois; Malon, Christopher; Graf, Hans-Peter; Saito, Akira; Kiyuna, Tomoharu; Marugame, Atsushi; Kamijo, Ken'ichi

2013-03-01

We present a system that detects cancer on slides of gastric tissue sections stained with hematoxylin and eosin (H&E). At its heart is a classi er trained using the semi-supervised multi-instance learning framework (MIL) where each tissue is represented by a set of regions-of-interest (ROI) and a single label. Such labels are readily obtained because pathologists diagnose each tissue independently as part of the normal clinical work ow. From a large dataset of over 26K gastric tissue sections from over 12K patients obtained from a clinical load spanning several months, we train a MIL classi er on a patient-level partition of the dataset (2/3 of the patients) and obtain a very high performance of 96% (AUC), tested on the remaining 1/3 never-seen before patients (over 8K tissues). We show this level of performance to match the more costly supervised approach where individual ROIs need to be labeled manually. The large amount of data used to train this system gives us con dence in its robustness and that it can be safely used in a clinical setting. We demonstrate how it can improve the clinical work ow when used for pre-screening or quality control. For pre-screening, the system can diagnose 47% of the tissues with a very low likelihood (< 1%) of missing cancers, thus halving the clinicians' caseload. For quality control, compared to random rechecking of 33% of the cases, the system achieves a three-fold increase in the likelihood of catching cancers missed by pathologists. The system is currently in regular use at independent pathology labs in Japan where it is used to double-check clinician's diagnoses. At the end of 2012 it will have analyzed over 80,000 slides of gastric and colorectal samples (200,000 tissues).

Comparison of multivariate analysis methods for extracting the paraffin component from the paraffin-embedded cancer tissue spectra for Raman imaging

NASA Astrophysics Data System (ADS)

Meksiarun, Phiranuphon; Ishigaki, Mika; Huck-Pezzei, Verena A. C.; Huck, Christian W.; Wongravee, Kanet; Sato, Hidetoshi; Ozaki, Yukihiro

2017-03-01

This study aimed to extract the paraffin component from paraffin-embedded oral cancer tissue spectra using three multivariate analysis (MVA) methods; Independent Component Analysis (ICA), Partial Least Squares (PLS) and Independent Component - Partial Least Square (IC-PLS). The estimated paraffin components were used for removing the contribution of paraffin from the tissue spectra. These three methods were compared in terms of the efficiency of paraffin removal and the ability to retain the tissue information. It was found that ICA, PLS and IC-PLS could remove the paraffin component from the spectra at almost the same level while Principal Component Analysis (PCA) was incapable. In terms of retaining cancer tissue spectral integrity, effects of PLS and IC-PLS on the non-paraffin region were significantly less than that of ICA where cancer tissue spectral areas were deteriorated. The paraffin-removed spectra were used for constructing Raman images of oral cancer tissue and compared with Hematoxylin and Eosin (H&E) stained tissues for verification. This study has demonstrated the capability of Raman spectroscopy together with multivariate analysis methods as a diagnostic tool for the paraffin-embedded tissue section.

Comparing The Efficacy of Hematoxylin and Eosin, Periodic Acid Schiff and Fluorescent Periodic Acid Schiff-Acriflavine Techniques for Demonstration of Basement Membrane in Oral Lichen Planus: A Histochemical Study

PubMed Central

Pujar, Ashwini; Pereira, Treville; Tamgadge, Avinash; Bhalerao, Sudhir; Tamgadge, Sandhya

2015-01-01

Background: Basement membrane (BM) is a thick sheet of extracellular matrix molecules, upon which epithelial cells attach. Various immunohistochemical studies in the past have been carried out but these advanced staining techniques are expensive and not feasible in routine laboratories. Although hematoxylin and eosin (H-E) is very popular among pathologists for looking at biopsies, the method has some limitations. This is where special stains come handy. Aims and Objectives: The aim of the present study was to demonstrate and compare the efficacy of H-E, periodic acid Schiff (PAS) and fluorescent periodic acid–acriflavine staining techniques for the basement membrane and to establish a histochemical stain which could be cost effective, less time consuming, and unambiguous for observation of the basement membrane zone. Materials and Methods: A total number of 40 paraffin-embedded tissue sections of known basement membrane containing tissues including 10 – Normal oral mucosa (NOM) and 30 – oral lichen planus (OLP) were considered in the study. Four-micron-thick sections of each block were cut and stained with H-E stain, PAS and fluorescent periodic acid–acriflavine stain. Sections were evaluated by three oral pathologists independently for continuity, contrast and pattern. Results: Though all the three stains showed favorable features at different levels, acriflavine stain was better than the other stains in demonstrating BM continuity, contrast and also the pattern followed by PAS stain. Acriflavine stain was the better in demonstrating a fibrillar pattern of a BM. Acriflavine stains a BM distinctly and is less time consuming and easy to carry out using readily available dyes as compared to other stains. Conclusion: The continuity and contrast along with the homogenous pattern and the afibrillar pattern of the BM was better demonstrated by acriflavine followed by the PAS stain. PMID:26538690

Influence of Desorption Conditions on Analyte Sensitivity and Internal Energy in Discrete Tissue or Whole Body Imaging by IR-MALDESI

NASA Astrophysics Data System (ADS)

Rosen, Elias P.; Bokhart, Mark T.; Ghashghaei, H. Troy; Muddiman, David C.

2015-06-01

Analyte signal in a laser desorption/postionization scheme such as infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is strongly coupled to the degree of overlap between the desorbed plume of neutral material from a sample and an orthogonal electrospray. In this work, we systematically examine the effect of desorption conditions on IR-MALDESI response to pharmaceutical drugs and endogenous lipids in biological tissue using a design of experiments approach. Optimized desorption conditions have then been used to conduct an untargeted lipidomic analysis of whole body sagittal sections of neonate mouse. IR-MALDESI response to a wide range of lipid classes has been demonstrated, with enhanced lipid coverage received by varying the laser wavelength used for mass spectrometry imaging (MSI). Targeted MS2 imaging (MS2I) of an analyte, cocaine, deposited beneath whole body sections allowed determination of tissue-specific ion response factors, and CID fragments of cocaine were monitored to comment on wavelength-dependent internal energy deposition based on the "survival yield" method.

A Novel Application for the Cavalieri Principle: A Stereological and Methodological Study

PubMed Central

Altunkaynak, Berrin Zuhal; Altunkaynak, Eyup; Unal, Deniz; Unal, Bunyamin

2009-01-01

Objective The Cavalieri principle was applied to consecutive pathology sections that were photographed at the same magnification and used to estimate tissue volumes via superimposing a point counting grid on these images. The goal of this study was to perform the Cavalieri method quickly and practically. Materials and Methods In this study, 10 adult female Sprague Dawley rats were used. Brain tissue was removed and sampled both systematically and randomly. Brain volumes were estimated using two different methods. First, all brain slices were scanned with an HP ScanJet 3400C scanner, and their images were shown on a PC monitor. Brain volume was then calculated based on these images. Second, all brain slices were photographed in 10× magnification with a microscope camera, and brain volumes were estimated based on these micrographs. Results There was no statistically significant difference between the volume measurements of the two techniques (P>0.05; Paired Samples t Test). Conclusion This study demonstrates that personal computer scanning of serial tissue sections allows for easy and reliable volume determination based on the Cavalieri method. PMID:25610077

A novel application for the cavalieri principle: a stereological and methodological study.

PubMed

Altunkaynak, Berrin Zuhal; Altunkaynak, Eyup; Unal, Deniz; Unal, Bunyamin

2009-08-01

The Cavalieri principle was applied to consecutive pathology sections that were photographed at the same magnification and used to estimate tissue volumes via superimposing a point counting grid on these images. The goal of this study was to perform the Cavalieri method quickly and practically. In this study, 10 adult female Sprague Dawley rats were used. Brain tissue was removed and sampled both systematically and randomly. Brain volumes were estimated using two different methods. First, all brain slices were scanned with an HP ScanJet 3400C scanner, and their images were shown on a PC monitor. Brain volume was then calculated based on these images. Second, all brain slices were photographed in 10× magnification with a microscope camera, and brain volumes were estimated based on these micrographs. There was no statistically significant difference between the volume measurements of the two techniques (P>0.05; Paired Samples t Test). This study demonstrates that personal computer scanning of serial tissue sections allows for easy and reliable volume determination based on the Cavalieri method.

Ultrasonographic anatomy of the dorsal and abaxial aspects of the equine fetlock.

PubMed

Denoix, J M; Jacot, S; Bousseau, B; Perrot, P

1996-01-01

This paper describes normal ultrasound images of the soft tissues of the dorsal and abaxial aspects of the equine fetlock. The palmar aspect of the fetlock is not discussed because it is related to the suspensory apparatus and flexor tendon anatomy which has been previously described. Ultrasound scanning was performed with 7.5 MHz linear or 10 MHz sector probes and recorded on 7.5 cm U-matic videocassettes allowing further retrospective data analysis, computer manipulation and good image reproducibility. Sagittal, parasagittal, frontal and transverse ultrasound scans of 13 lameness free mature horses were compared to anatomically dissected leg specimens, anatomical sections and Magnetic Resonance Imaging scans of isolated limbs. The results are focused on the comparison between anatomical sections and ultrasonograms performed on the legs of nonlame horses. Ultrasonography was demonstrated to be a very accurate imaging procedure for soft tissue structures at the dorsal and abaxial aspects of the equine fetlock. Under clinical conditions, a thorough knowledge of normal ultrasonographic anatomy is critical for an accurate diagnosis of fetlock soft tissue injury.

Correlation of two bioadhesion assays: the everted sac technique and the CAHN microbalance.

PubMed

Santos, C A; Jacob, J S; Hertzog, B A; Freedman, B D; Press, D L; Harnpicharnchai, P; Mathiowitz, E

1999-08-27

This contribution correlates two in vitro methods utilized to determine bioadhesion. One method, the everted intestinal sac technique, is a passive test for bioadhesion involving several polymer microspheres and a section of everted intestinal tissue. The other method, the CAHN microbalance, employs a CAHN dynamic contact angle analyzer with modified software to record the tensile forces measured as a single polymer microsphere is pulled from intestinal tissue. This study demonstrates that CAHN and everted sac experiments yield similar results when used to quantify the bioadhesive nature of polymer microsphere systems. A polymer showing high adhesion in one method also demonstrates high bioadhesion in the other method; polymers that exhibit high fracture strength and tensile work measurements with the CAHN microbalance also yield high binding percentages with the everted sac method. The polymers tested and reported here are poly(caprolactone) and different copolymer ratios of poly(fumaric-co-sebacic anhydride). The results of this correlation demonstrate that each method alone is a valuable indicator of bioadhesion.

SIMPLE: a sequential immunoperoxidase labeling and erasing method.

PubMed

Glass, George; Papin, Jason A; Mandell, James W

2009-10-01

The ability to simultaneously visualize expression of multiple antigens in cells and tissues can provide powerful insights into cellular and organismal biology. However, standard methods are limited to the use of just two or three simultaneous probes and have not been widely adopted for routine use in paraffin-embedded tissue. We have developed a novel approach called sequential immunoperoxidase labeling and erasing (SIMPLE) that enables the simultaneous visualization of at least five markers within a single tissue section. Utilizing the alcohol-soluble peroxidase substrate 3-amino-9-ethylcarbazole, combined with a rapid non-destructive method for antibody-antigen dissociation, we demonstrate the ability to erase the results of a single immunohistochemical stain while preserving tissue antigenicity for repeated rounds of labeling. SIMPLE is greatly facilitated by the use of a whole-slide scanner, which can capture the results of each sequential stain without any information loss.

Detection of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms.

PubMed

Miller, J; Jenny, A; Rhyan, J; Saari, D; Suarez, D

1997-07-01

A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk (Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria (M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.

Review: history of the amyloid fibril.

PubMed

Sipe, J D; Cohen, A S

2000-06-01

Rudolph Virchow, in 1854, introduced and popularized the term amyloid to denote a macroscopic tissue abnormality that exhibited a positive iodine staining reaction. Subsequent light microscopic studies with polarizing optics demonstrated the inherent birefringence of amyloid deposits, a property that increased intensely after staining with Congo red dye. In 1959, electron microscopic examination of ultrathin sections of amyloidotic tissues revealed the presence of fibrils, indeterminate in length and, invariably, 80 to 100 A in width. Using the criteria of Congophilia and fibrillar morphology, 20 or more biochemically distinct forms of amyloid have been identified throughout the animal kingdom; each is specifically associated with a unique clinical syndrome. Fibrils, also 80 to 100 A in width, have been isolated from tissue homogenates using differential sedimentation or solubility. X-ray diffraction analysis revealed the fibrils to be ordered in the beta pleated sheet conformation, with the direction of the polypeptide backbone perpendicular to the fibril axis (cross beta structure). Because of the similar dimensions and tinctorial properties of the fibrils extracted from amyloid-laden tissues and amyloid fibrils in tissue sections, they have been assumed to be identical. However, the spatial relationship of proteoglycans and amyloid P component (AP), common to all forms of amyloid, to the putative protein only fibrils in tissues, has been unclear. Recently, it has been suggested that, in situ, amyloid fibrils are composed of proteoglycans and AP as well as amyloid proteins and thus resemble connective tissue microfibrils. Chemical and physical definition of the fibrils in tissues will be needed to relate the in vitro properties of amyloid protein fibrils to the pathogenesis of amyloid fibril formation in vivo. Copyright 2000 Academic Press.

Stiffness mapping prostate biopsy samples using a tactile sensor.

PubMed

Peng, Qiyu; Omata, Sadao; Peehl, Donna M; Constantinou, Chris E

2011-01-01

Previous studies have demonstrated that the stiffness of cancerous cells reflects their pathological stage and progression rates, with increased cancerous cell stiffness associated with increased aggressiveness. Therefore, the elasticity of the cancerous cells has the potential to be used as an indicator of the cancer's aggressiveness. However, the sensitivity and resolution of current palpation and imaging techniques are not sufficient to detect small cancerous tissues. In previous studies, we developed a tactile-based device to map with high resolution the stiffness of a tissue section. The purpose of this study is to evaluate this device using different tissues (BPH, Cancer and PZ) collected from human prostates. The preliminary results show that the tactile device is sensitive enough to tell the differences of the stiffness of different tissues. The results also disclosed the factors (humidity, temperature and tissue degradation) which could dramatically affect the results of stiffness mapping. The tactile technology described in this paper has the potential to help disclose the underlying mechanical mechanisms that lead to increased stiffness in prostate tumors.

On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS)*

PubMed Central

Quanico, Jusal; Franck, Julien

2016-01-01

Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues. PMID:27512083

Penetration of immunoreagents in Vibratome-sectioned brain: a light and electron microscopic study.

PubMed

Piekut, D T; Casey, S M

1983-05-01

Immunocytochemical studies on the localization of peptides at the ultrastructural level have most frequently involved the application of the peroxidase--antiperoxidase (PAP) method of immunocytochemistry and the use of the preembedding or postembedding staining procedures. The present study was designed to determine the depth of penetration of Vibratome tissue sections by immunoreagents used in the preembedding method in which immunostaining of unembedded fixed tissue sections is accomplished prior to tissue dehydration and embedment. Our data indicate that penetration of immunoreagents is restricted to the superficial 8-9 micrometers of a 80-micrometers thick Vibratome tissue section of hypothalamus of brain using antisera generated against arginine vasopressin. The final immunoreaction product visualized in a Vibratome tissue section may reflect only a fraction of the amount of hormone contained within the thickness of the tissue section.

Morphological Characterisation of Unstained and Intact Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography

NASA Astrophysics Data System (ADS)

Walton, Lucy A.; Bradley, Robert S.; Withers, Philip J.; Newton, Victoria L.; Watson, Rachel E. B.; Austin, Clare; Sherratt, Michael J.

2015-05-01

Characterisation and quantification of tissue structures is limited by sectioning-induced artefacts and by the difficulties of visualising and segmenting 3D volumes. Here we demonstrate that, even in the absence of X-ray contrast agents, X-ray computed microtomography (microCT) and nanotomography (nanoCT) can circumvent these problems by rapidly resolving compositionally discrete 3D tissue regions (such as the collagen-rich adventitia and elastin-rich lamellae in intact rat arteries) which in turn can be segmented due to their different X-ray opacities and morphologies. We then establish, using X-ray tomograms of both unpressurised and pressurised arteries that intra-luminal pressure not only increases lumen cross-sectional area and straightens medial elastic lamellae but also induces profound remodelling of the adventitial layer. Finally we apply microCT to another human organ (skin) to visualise the cell-rich epidermis and extracellular matrix-rich dermis and to show that conventional histological and immunohistochemical staining protocols are compatible with prior X-ray exposure. As a consequence we suggest that microCT could be combined with optical microscopy to characterise the 3D structure and composition of archival paraffin embedded biological materials and of mechanically stressed dynamic tissues such as the heart, lungs and tendons.

Morphological Characterisation of Unstained and Intact Tissue Micro-architecture by X-ray Computed Micro- and Nano-Tomography

PubMed Central

Walton, Lucy A.; Bradley, Robert S.; Withers, Philip J.; Newton, Victoria L.; Watson, Rachel E. B.; Austin, Clare; Sherratt, Michael J.

2015-01-01

Characterisation and quantification of tissue structures is limited by sectioning-induced artefacts and by the difficulties of visualising and segmenting 3D volumes. Here we demonstrate that, even in the absence of X-ray contrast agents, X-ray computed microtomography (microCT) and nanotomography (nanoCT) can circumvent these problems by rapidly resolving compositionally discrete 3D tissue regions (such as the collagen-rich adventitia and elastin-rich lamellae in intact rat arteries) which in turn can be segmented due to their different X-ray opacities and morphologies. We then establish, using X-ray tomograms of both unpressurised and pressurised arteries that intra-luminal pressure not only increases lumen cross-sectional area and straightens medial elastic lamellae but also induces profound remodelling of the adventitial layer. Finally we apply microCT to another human organ (skin) to visualise the cell-rich epidermis and extracellular matrix-rich dermis and to show that conventional histological and immunohistochemical staining protocols are compatible with prior X-ray exposure. As a consequence we suggest that microCT could be combined with optical microscopy to characterise the 3D structure and composition of archival paraffin embedded biological materials and of mechanically stressed dynamic tissues such as the heart, lungs and tendons. PMID:25975937

Quantitative Time-Resolved Fluorescence Imaging of Androgen Receptor and Prostate-Specific Antigen in Prostate Tissue Sections.

PubMed

Krzyzanowska, Agnieszka; Lippolis, Giuseppe; Helczynski, Leszek; Anand, Aseem; Peltola, Mari; Pettersson, Kim; Lilja, Hans; Bjartell, Anders

2016-05-01

Androgen receptor (AR) and prostate-specific antigen (PSA) are expressed in the prostate and are involved in prostate cancer (PCa). The aim of this study was to develop reliable protocols for reproducible quantification of AR and PSA in benign and malignant prostate tissue using time-resolved fluorescence (TRF) imaging techniques. AR and PSA were detected with TRF in tissue microarrays from 91 PCa patients. p63/ alpha-methylacyl-CoA racemase (AMACR) staining on consecutive sections was used to categorize tissue areas as benign or cancerous. Automated image analysis was used to quantify staining intensity. AR intensity was significantly higher in AMACR+ and lower in AMACR- cancer areas as compared with benign epithelium. The PSA intensity was significantly lower in cancer areas, particularly in AMACR- glands. The AR/PSA ratio varied significantly in the AMACR+ tumor cells as compared with benign glands. There was a trend of more rapid disease progression in patients with higher AR/PSA ratios in the AMACR- areas. This study demonstrates the feasibility of developing reproducible protocols for TRF imaging and automated image analysis to study the expression of AR and PSA in benign and malignant prostate. It also highlighted the differences in AR and PSA protein expression within AMACR- and AMACR+ cancer regions. © 2016 The Histochemical Society.

Biological tissue imaging with a position and time sensitive pixelated detector.

PubMed

Jungmann, Julia H; Smith, Donald F; MacAleese, Luke; Klinkert, Ivo; Visser, Jan; Heeren, Ron M A

2012-10-01

We demonstrate the capabilities of a highly parallel, active pixel detector for large-area, mass spectrometric imaging of biological tissue sections. A bare Timepix assembly (512 × 512 pixels) is combined with chevron microchannel plates on an ion microscope matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI TOF-MS). The detector assembly registers position- and time-resolved images of multiple m/z species in every measurement frame. We prove the applicability of the detection system to biomolecular mass spectrometry imaging on biologically relevant samples by mass-resolved images from Timepix measurements of a peptide-grid benchmark sample and mouse testis tissue slices. Mass-spectral and localization information of analytes at physiologic concentrations are measured in MALDI-TOF-MS imaging experiments. We show a high spatial resolution (pixel size down to 740 × 740 nm(2) on the sample surface) and a spatial resolving power of 6 μm with a microscope mode laser field of view of 100-335 μm. Automated, large-area imaging is demonstrated and the Timepix' potential for fast, large-area image acquisition is highlighted.

In situ PCR detection of phytoplasma DNA in embryos from coconut palms with lethal yellowing disease.

PubMed

Cordova, Ivan; Jones, Phil; Harrison, Nigel A; Oropeza, Carlos

2003-03-01

SUMMARY DNA of the lethal yellowing (LY) phytoplasma was detected in 13 of 72 embryos from fruits of four diseased Atlantic tall coconut palms by polymerase chain reaction (PCR) assays employing phytoplasma universal rRNA primer pair P1/P7, nested LY group-specific rRNA primer pair 503f/LY16Sr or LY phytoplasma-specific nonribosomal primer pair LYF1/R1. Phytoplasma distribution in sectioned tissues from six PCR positive embryos was determined by in situ PCR and digoxigenin-11-deoxy-UTP (Dig) labelling of amplification products. Dig-labeled DNA products detected by colourimetric assay were clearly evident on sections from the same three embryos investigated in detail by in situ PCRs employing primer pairs P1/P7 or LYF1/R1. Deposition of blue-green stain on sections as a result of each assay was restricted to areas of the embryos corresponding to the plumule and cells ensheathing it. By comparison, similarly treated embryo sections derived from fruits of a symptomless Atlantic tall coconut palm were consistently devoid of any stain. Presence of phytoplasma DNA in embryo tissues suggests the possible potential for seed transmission which remains to be demonstrated.

In vivo preventive effects of insect tea on buccal mucosa cancer in ICR mice.

PubMed

Zhao, Xin; Wang, Rui; Qian, Yu; Li, Guijie; Zhou, Yalin; Sun, Peng

2014-01-01

Insect tea is a particular drink or health product in China and it is also used as Chinese medicine now. Its functional effects need to be proved. The ICR mice buccal mucosa cancer model was established by injecting the mice with U14 cells and mice was treated with insect tea. Tumor volumes and lymph node metastasis rates were determined. And the buccal mucosa tissues and cancer cervical lymph node were also checked by histology test, real-time polymerase chain reaction, and western blot assays. The tumor volumes for the group treated with insect tea mice was smaller than those from the control mice. The sections of buccal mucosa cancer tissue showed that the canceration of insect tea mice was weaker than control mice. Insect tea significantly induced apoptosis in buccal mucosa tissues by upregulating Bax, caspases, and downregulating Bcl-2. Nuclear factor-κB, inducible nitric oxide synthase, and COX-2 gene, was significantly downregulated by insect tea, demonstrating its anti-inflammatory properties. Insect tea also exerted a great anti-metastasis effect on tissues as demonstrated by decreased expression of Matrix metalloproteinases genes and increased expression of tissue inhibitors of matrix metalloproteinases. The highest concentration of 1600 mg/kg oral gavage and 400 mg/mL smear insect tea showed the best anticancer effects. Based on the results, insect tea showed the strong in vivo buccal mucosa cancer preventive effects.

Label-free biomolecular characterization of human breast cancer tissue with stimulated Raman scattering (SRS) spectral imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lu, Fa-Ke F.; Calligaris, David; Suo, Yuanzhen; Santagata, Sandro; Golby, Alexandra J.; Xie, X. Sunney; Mallory, Melissa A.; Golshan, Mehra; Dillon, Deborah A.; Agar, Nathalie Y. R.

2017-02-01

Stimulated Raman scattering (SRS) microscopy has been used for rapid label-free imaging of various biomolecules and drugs in living cells and tissues (Science, doi:10.1126/science.aaa8870). Our recent work has demonstrated that lipid and protein mapping of cancer tissue renders pathology-like images, providing essential histopathological information with subcellular resolution of the entire specimen (Cancer Research, doi: 10.1158/0008-5472.CAN-16-027). We have also established the first SRS imaging Atlas of human brain tumors (Harvard Dataverse, doi: (doi:10.7910/DVN/EZW4EK). SRS imaging of tissue could provide invaluable information for cancer diagnosis and surgical guidance in two aspects: rapid surgical pathology and quantitative biomolecular characterization. In this work, we present the use of SRS microscopy for characterization of a few essential biomolecules in breast cancer. Human breast cancer tissue specimens at the tumor core, tumor margin and normal area (5 cm away from the tumor) from surgical cases will be imaged with SRS at multiple Raman shifts, including the peaks for lipid, protein, blood (absorption), collagen, microcalcification (calcium phosphates and calcium oxalate) and carotenoids. Most of these Raman shifts have relatively strong Raman cross sections, which ensures high-quality and fast imaging. This proof-of-principle study is sought to demonstrate the feasibility and potential of SRS imaging for ambient diagnosis and surgical guidance of breast cancer.

Optical Coherence Tomography

NASA Astrophysics Data System (ADS)

Huang, David

Optical coherence tomography (OCT) is a new method for noninvasive cross-sectional imaging in biological systems. In OCT, the longitudinal locations of tissue structures are determined by measuring the time-of-flight delays of light backscattered from these structures. The optical delays are measured by low coherence interferometry. Information on lateral position is provided by transverse scanning of the probe beam. The two dimensional map of optical scattering from internal tissue microstructures is then represented in a false-color or grayscale image. OCT is the optical analog of ultrasonic pulse-echo imaging, but with greatly improved spatial resolutions (a few microns). This thesis describes the development of this new high resolution tomographic imaging technology and the demonstration of its use in a variety of tissues under both in vitro and in vivo conditions. In vitro OCT ranging and imaging studies were performed using human ocular and arterial tissues, two clinically relevant examples of transparent and turbid media, respectively. In the anterior eye, precise measurements of cornea and anterior chamber dimensions were made. In the arterial specimens, the differentiation between fatty -calcified and fibromuscular tissues was demonstrated. In vivo OCT imaging in the retina and optic nerve head in human subjects was also performed. The delineation of retinal layers, which has not been possible with other noninvasive imaging techniques, is demonstrated in these OCT images. OCT has high spatial resolution but limited penetration into turbid tissue. It has potential for diagnostic applications where high resolution is needed and optical access is available, such as in the eye, skin, surgically exposed tissues, and surfaces that can be reached by various catheters and endoscopic probes. In particular, the measurement of fine retinal structures promises improvements in the diagnosis and management of glaucoma, macular edema and other vitreo-retinal diseases. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253 -1690.).

Bioreactor-induced mesenchymal progenitor cell differentiation and elastic fiber assembly in engineered vascular tissues.

PubMed

Lin, Shigang; Mequanint, Kibret

2017-09-01

In vitro maturation of engineered vascular tissues (EVT) requires the appropriate incorporation of smooth muscle cells (SMC) and extracellular matrix (ECM) components similar to native arteries. To this end, the aim of the current study was to fabricate 4mm inner diameter vascular tissues using mesenchymal progenitor cells seeded into tubular scaffolds. A dual-pump bioreactor operating either in perfusion or pulsatile perfusion mode was used to generate physiological-like stimuli to promote progenitor cell differentiation, extracellular elastin production, and tissue maturation. Our data demonstrated that pulsatile forces and perfusion of 3D tubular constructs from both the lumenal and ablumenal sides with culture media significantly improved tissue assembly, effectively inducing mesenchymal progenitor cell differentiation to SMCs with contemporaneous elastin production. With bioreactor cultivation, progenitor cells differentiated toward smooth muscle lineage characterized by the expression of smooth muscle (SM)-specific markers smooth muscle alpha actin (SM-α-actin) and smooth muscle myosin heavy chain (SM-MHC). More importantly, pulsatile perfusion bioreactor cultivation enhanced the synthesis of tropoelastin and its extracellular cross-linking into elastic fiber compared with static culture controls. Taken together, the current study demonstrated progenitor cell differentiation and vascular tissue assembly, and provides insights into elastin synthesis and assembly to fibers. Incorporation of elastin into engineered vascular tissues represents a critical design goal for both mechanical and biological functions. In the present study, we seeded porous tubular scaffolds with multipotent mesenchymal progenitor cells and cultured in dual-pump pulsatile perfusion bioreactor. Physiological-like stimuli generated by bioreactor not only induced mesenchymal progenitor cell differentiation to vascular smooth muscle lineage but also actively promoted elastin synthesis and fiber assembly. Gene expression and protein synthesis analyses coupled with histological and immunofluorescence staining revealed that elastin-containing vascular tissues were fabricated. More importantly, co-localization and co-immunoprecipitation experiments demonstrated that elastin and fibrillin-1 were abundant throughout the cross-section of the tissue constructs suggesting a process of elastin protein crosslinking. This study paves a way forward to engineer elastin-containing functional vascular substitutes from multipotent progenitor cells in a bioreactor. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

PubMed

Lauterbach, Marcel A; Ronzitti, Emiliano; Sternberg, Jenna R; Wyart, Claire; Emiliani, Valentina

2015-01-01

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy

PubMed Central

Sternberg, Jenna R.; Wyart, Claire; Emiliani, Valentina

2015-01-01

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes. PMID:26625116

Tissue-tissue interaction-triggered calcium elevation is required for cell polarization during Xenopus gastrulation.

PubMed

Shindo, Asako; Hara, Yusuke; Yamamoto, Takamasa S; Ohkura, Masamichi; Nakai, Junichi; Ueno, Naoto

2010-02-02

The establishment of cell polarity is crucial for embryonic cells to acquire their proper morphologies and functions, because cell alignment and intracellular events are coordinated in tissues during embryogenesis according to the cell polarity. Although much is known about the molecules involved in cell polarization, the direct trigger of the process remains largely obscure. We previously demonstrated that the tissue boundary between the chordamesoderm and lateral mesoderm of Xenopus laevis is important for chordamesodermal cell polarity. Here, we examined the intracellular calcium dynamics during boundary formation between two different tissues. In a combination culture of nodal-induced chordamesodermal explants and a heterogeneous tissue, such as ectoderm or lateral mesoderm, the chordamesodermal cells near the boundary frequently displayed intracellular calcium elevation; this frequency was significantly less when homogeneous explants were used. Inhibition of the intracellular calcium elevation blocked cell polarization in the chordamesodermal explants. We also observed frequent calcium waves near the boundary of the dorsal marginal zone (DMZ) dissected from an early gastrula-stage embryo. Optical sectioning revealed that where heterogeneous explants touched, the chordamesodermal surface formed a wedge with the narrow end tucked under the heterogeneous explant. No such configuration was seen between homogeneous explants. When physical force was exerted against a chordamesodermal explant with a glass needle at an angle similar to that created in the explant, or migrating chordamesodermal cells crawled beneath a silicone block, intracellular calcium elevation was frequent and cell polarization was induced. Finally, we demonstrated that a purinergic receptor, which is implicated in mechano-sensing, is required for such frequent calcium elevation in chordamesoderm and for cell polarization. This study raises the possibility that tissue-tissue interaction generates mechanical forces through cell-cell contact that initiates coordinated cell polarization through a transient increase in intracellular calcium.

Tissue Distribution of the Ehrlichia muris-Like Agent in a Tick Vector

PubMed Central

Lynn, Geoffrey E.; Oliver, Jonathan D.; Nelson, Curtis M.; Felsheim, Roderick F.; Kurtti, Timothy J.; Munderloh, Ulrike G.

2015-01-01

Human pathogens transmitted by ticks undergo complex life cycles alternating between the arthropod vector and a mammalian host. While the latter has been investigated to a greater extent, examination of the biological interactions between microbes and the ticks that carry them presents an equally important opportunity for disruption of the disease cycle. In this study, we used in situ hybridization to demonstrate infection by the Ehrlichia muris-like organism, a newly recognized human pathogen, of Ixodes scapularis ticks, a primary vector for several important human disease agents. This allowed us to assess whole sectioned ticks for the patterns of tissue invasion, and demonstrate generalized dissemination of ehrlichiae in a variety of cell types and organs within ticks infected naturally via blood feeding. Electron microscopy was used to confirm these results. Here we describe a strong ehrlichial affinity for epithelial cells, neuronal cells of the synganglion, salivary glands, and male accessory glands. PMID:25781930

Quantitative detection of multiple fluorophore sites as a tool for diagnosis and monitoring disease progression in salivary glands

NASA Astrophysics Data System (ADS)

Gannot, Israel; Bonner, Robert F.; Gannot, Gallya; Fox, Philip C.; You, Joon S.; Waynant, Ronald W.; Gandjbakhche, Amir H.

1997-08-01

A series of fluorescent surface images were obtained from physical models of localized fluorophores embedded at various depths and separations in tissue phantoms. Our random walk theory was applied to create an analytical model of multiple flurophores embedded in tissue-like phantom. Using this model, from acquired set of surface images, the location of the fluorophores was reconstructed and compared it to their known 3-D distributions. A good correlation was found, and the ability to resolve fluorophores as a function of depth and separation was determined. In parallel in in-vitro study, specific coloring of sections of minor salivary glands was also demonstrated. These results demonstrate the possibility of using inverse methods to reconstruct unknown locations and concentrations of optical probes specifically bound to infiltrating lymphocytes in minor salivary glands of patients with Sjogren's syndrome.

Application of immunogold-silver staining and immunoenzymatic methods in multiple labelling of human pancreatic Langerhans islet cells.

PubMed

Krenács, T; Lászik, Z; Dobó, E

1989-01-01

The use of immunogold-silver staining (IGSS) combined with immunoperoxidase and/or immunoalkaline phosphatase methods for the simultaneous demonstration of pancreatic islet cell hormones on routinely fixed paraffin-embedded human tissue sections was examined. If IGSS was applied first, the black colour of silver-enhanced colloidal gold on doubly immunostained sections contrasted with the colours of most of the chromogens used generally in the 2 immunoenzymatic methods. If IGSS was followed by immunoalkaline phosphatase and immunoperoxidase techniques in optional sequence, 3 different hormone-containing cell types could be stained simultaneously without non-specific cross-reactions. IGSS and immunoalkaline phosphatase methods, together with 2 kinds of non-cross-reacting immunoperoxidase systems, permitted the detection of 4 distinct antigens on the same tissue section. Multiple immunohistochemical labelling of the endocrine pancreas provides an opportunity for the correct and rapid analysis of the topographic and morphometric relationships between different hormone-producing cell populations under both normal and pathological conditions. IGSS is of great potential for the simultaneous immunolabelling of antigens situated within separate cells.

Exercise-Induced Changes in the Cortical Bone of Growing Mice Are Bone and Gender Specific

PubMed Central

Wallace, Joseph M.; Rajachar, Rupak M.; Allen, Matthew R.; Bloomfield, Susan A.; Robey, Pamela G.; Young, Marian F.; Kohn, David H.

2009-01-01

Fracture risk and mechanical competence of bone are functions of bone mass and tissue quality, which in turn are dependent on the bone’s mechanical environment. Male mice have a greater response to non weight-bearing exercise than females, resulting in larger, stronger bones compared with control animals. The aim of this study was to test the hypothesis that short-term weight-bearing running during growth (21 days starting at 8 weeks of age; 30 minutes/day; 12 meters/minute; 5° incline; 7 days/week) would similarly have a greater impact on cross sectional geometry and mechanical competence in the femora and tibiae of male mice versus females. Based on the orientation of the legs during running and the proximity of the tibia to the point of impact, this response was hypothesized to be greatest in the tibia. Exercise-related changes relative to controls were assayed by four-point bending tests, while volumetric bone mineral density and cross-sectional geometry were also assessed. The response to running was bone and gender-specific, with male tibiae demonstrating the greatest effects. In male tibiae, periosteal perimeter, endocortical perimeter, cortical area, medial-lateral width and bending moment of inertia increased versus control mice suggesting that while growth is occurring in these mice between 8 and 11 weeks of age, exercise accelerated this growth resulting in a greater increase in bone tissue over the 3 weeks of the study. Exercise increased tissue-level strain-to-failure and structural post-yield deformation in the male tibiae, but these post-yield benefits came at the expense of decreased yield deformation, structural and tissue-level yield strength and tissue-level ultimate strength. These results suggest that exercise superimposed upon growth accelerated growth-related increases in tibial cross-sectional dimensions. Exercise also influenced the quality of this forming bone, significantly impacting structural and tissue-level mechanical properties. PMID:17240210

Breast Cancer Diagnosis Using a Microfluidic Multiplexed Immunohistochemistry Platform

PubMed Central

Kim, Minseok S.; Kim, Taemin; Kong, Sun-Young; Kwon, Soim; Bae, Chae Yun; Choi, Jaekyu; Kim, Chul Hwan; Lee, Eun Sook; Park, Je-Kyun

2010-01-01

Background Biomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness. Methodology/Principal Findings By creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendall's coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies. Conclusions/Significance The microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy. PMID:20454672

Optical properties of mouse brain tissue after optical clearing with FocusClear™

NASA Astrophysics Data System (ADS)

Moy, Austin J.; Capulong, Bernard V.; Saager, Rolf B.; Wiersma, Matthew P.; Lo, Patrick C.; Durkin, Anthony J.; Choi, Bernard

2015-09-01

Fluorescence microscopy is commonly used to investigate disease progression in biological tissues. Biological tissues, however, are strongly scattering in the visible wavelengths, limiting the application of fluorescence microscopy to superficial (<200 μm) regions. Optical clearing, which involves incubation of the tissue in a chemical bath, reduces the optical scattering in tissue, resulting in increased tissue transparency and optical imaging depth. The goal of this study was to determine the time- and wavelength-resolved dynamics of the optical scattering properties of rodent brain after optical clearing with FocusClear™. Light transmittance and reflectance of 1-mm mouse brain sections were measured using an integrating sphere before and after optical clearing and the inverse adding doubling algorithm used to determine tissue optical scattering. The degree of optical clearing was quantified by calculating the optical clearing potential (OCP), and the effects of differing OCP were demonstrated using the optical histology method, which combines tissue optical clearing with optical imaging to visualize the microvasculature. We observed increased tissue transparency with longer optical clearing time and an analogous increase in OCP. Furthermore, OCP did not vary substantially between 400 and 1000 nm for increasing optical clearing durations, suggesting that optical histology can improve ex vivo visualization of several fluorescent probes.

Penetration of Chlorhexidine into Human Skin ▿

PubMed Central

Karpanen, T. J.; Worthington, T.; Conway, B. R.; Hilton, A. C.; Elliott, T. S. J.; Lambert, P. A.

2008-01-01

This study evaluated a model of skin permeation to determine the depth of delivery of chlorhexidine into full-thickness excised human skin following topical application of 2% (wt/vol) aqueous chlorhexidine digluconate. Skin permeation studies were performed on full-thickness human skin using Franz diffusion cells with exposure to chlorhexidine for 2 min, 30 min, and 24 h. The concentration of chlorhexidine extracted from skin sections was determined to a depth of 1,500 μm following serial sectioning of the skin using a microtome and analysis by high-performance liquid chromatography. Poor penetration of chlorhexidine into skin following 2-min and 30-min exposures to chlorhexidine was observed (0.157 ± 0.047 and 0.077 ± 0.015 μg/mg tissue within the top 100 μm), and levels of chlorhexidine were minimal at deeper skin depths (less than 0.002 μg/mg tissue below 300 μm). After 24 h of exposure, there was more chlorhexidine within the upper 100-μm sections (7.88 ± 1.37 μg/mg tissue); however, the levels remained low (less than 1 μg/mg tissue) at depths below 300 μm. There was no detectable penetration through the full-thickness skin. The model presented in this study can be used to assess the permeation of antiseptic agents through various layers of skin in vitro. Aqueous chlorhexidine demonstrated poor permeation into the deeper layers of the skin, which may restrict the efficacy of skin antisepsis with this agent. This study lays the foundation for further research in adopting alternative strategies for enhanced skin antisepsis in clinical practice. PMID:18676882

Inactivation of infectious virus and serological detection of virus antigen in Rift Valley fever virus-exposed mosquitoes fixed with paraformaldehyde.

PubMed

Kading, Rebekah; Crabtree, Mary; Miller, Barry

2013-04-01

Formaldehyde is routinely used to fix tissues in preparation for pathology studies, however concerns remain that treatment of tissues with cellular fixatives may not entirely inactivate infectious virus particles. This concern is of particular regulatory importance for research involving viruses that are classified as select agents such as Rift Valley fever virus (RVFV). Therefore, the specific aims of this study were to (1) assay RVFV-exposed Aedes aegypti mosquitoes fixed in 4% paraformaldehyde for the presence of infectious RVFV particles at various time points following infection and (2) demonstrate the utility of immunofluorescence assay (IFA) for the detection of RVFV antigen in various tissues of paraformaldehyde-fixed mosquitoes. Mosquitoes were administered an infectious blood meal containing one of two strains of RVFV, harvested at various time points following infection, intrathoracically inoculated with 4% paraformaldehyde, and fixed overnight at 4°C. The infection status of a subset of mosquitoes was verified by IFA on leg tissues prior to fixation, and infectivity of RVFV in fixed mosquito carcasses was determined by Vero cell plaque assay. Paraformaldehyde-fixed mosquitoes harvested 14 days post infection were also paraffin-embedded and sectioned for detection of RVFV antigen to particular tissues by IFA. None of the RVFV-exposed mosquitoes tested by Vero cell plaque assay contained infectious RVFV after fixation. Furthermore, incubation of mosquito sections with trypsin prior to antibody staining is recommended for optimal visualization of RVFV antigen in infected mosquito tissues by IFA. Published by Elsevier B.V.

Non-destructive optical clearing technique enhances optical coherence tomography (OCT) for real-time, 3D histomorphometry of brain tissue (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paul, Akshay; Chang, Theodore H.; Chou, Li-Dek; Ramalingam, Tirunelveli S.

2016-03-01

Evaluation of neurodegenerative disease often requires examination of brain morphology. Volumetric analysis of brain regions and structures can be used to track developmental changes, progression of disease, and the presence of transgenic phenotypes. Current standards for microscopic investigation of brain morphology are limited to detection of superficial structures at a maximum depth of 300μm. While histological techniques can provide detailed cross-sections of brain structures, they require complicated tissue preparation and the ultimate destruction of the sample. A non-invasive, label-free imaging modality known as Optical Coherence Tomography (OCT) can produce 3-dimensional reconstructions through high-speed, cross-sectional scans of biological tissue. Although OCT allows for the preservation of intact samples, the highly scattering and absorbing properties of biological tissue limit imaging depth to 1-2mm. Optical clearing agents have been utilized to increase imaging depth by index matching and lipid digestion, however, these contemporary techniques are expensive and harsh on tissues, often irreversibly denaturing proteins. Here we present an ideal optical clearing agent that offers ease-of-use and reversibility. Similar to how SeeDB has been effective for microscopy, our fructose-based, reversible optical clearing technique provides improved OCT imaging and functional immunohistochemical mapping of disease. Fructose is a natural, non-toxic sugar with excellent water solubility, capable of increasing tissue transparency and reducing light scattering. We will demonstrate the improved depth-resolving performance of OCT for enhanced whole-brain imaging of normal and diseased murine brains following a fructose clearing treatment. This technique potentially enables rapid, 3-dimensional evaluation of biological tissues at axial and lateral resolutions comparable to histopathology.

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models.

PubMed

Blutke, Andreas; Wanke, Rüdiger

2018-03-06

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the "Orientator" and "Isector" methods, and vertical uniform random (VUR) sections.

Rapid In Situ Profiling of Lipid C═C Location Isomers in Tissue Using Ambient Mass Spectrometry with Photochemical Reactions.

PubMed

Tang, Fei; Guo, Chengan; Ma, Xiaoxiao; Zhang, Jian; Su, Yuan; Tian, Ran; Shi, Riyi; Xia, Yu; Wang, Xiaohao; Ouyang, Zheng

2018-05-01

Rapid and in situ profiling of lipids using ambient mass spectrometry (AMS) techniques has great potential for clinical diagnosis, biological studies, and biomarker discovery. In this study, the online photochemical reaction involving carbon-carbon double bonds was coupled with a surface sampling technique to develop a direct tissue-analysis method with specificity to lipid C═C isomers. This method enabled the in situ analysis of lipids from the surface of various tissues or tissue sections, which allowed the structural characterization of lipid isomers within 2 min. Under optimized reaction conditions, we have established a method for the relative quantitation of lipid C═C location isomers by comparing the abundances of the diagnostic ions arising from each isomer, which has been proven effective through the established linear relationship ( R 2 = 0.999) between molar ratio and diagnostic ion ratio of the FA 18:1 C═C location isomers. This method was then used for the rapid profiling of unsaturated lipid C═C isomers in the sections of rat brain, lung, liver, spleen, and kidney, as well as in normal and diseased rat tissues. Quantitative information on FA 18:1 and PC 16:0-18:1 C═C isomers was obtained, and significant differences were observed between different samples. To the best of our knowledge, this is the first study to report the direct analysis of lipid C═C isomers in tissues using AMS. Our results demonstrated that this method can serve as a rapid analytical approach for the profiling of unsaturated lipid C═C isomers in biological tissues and should contribute to functional lipidomics and clinical diagnosis.

Phase Contrast Microscopy Analysis of Breast Tissue

PubMed Central

Wells, Wendy A.; Wang, Xin; Daghlian, Charles P.; Paulsen, Keith D.; Pogue, Brian W.

2010-01-01

OBJECTIVE To assess how optical scatter properties in breast tissue, as measured by phase contrast microscopy and interpreted pathophysiologically, might be exploited as a diagnostic tool to differentiate cancer from benign tissue. STUDY DESIGN We evaluated frozen human breast tissue sections of adipose tissue, normal breast parenchyma, benign fibroadenoma tumors and noninvasive and invasive malignant cancers by phase contrast microscopy through quantification of grayscale values, using multiple regions of interest (ROI). Student’s t tests were performed on phase contrast measures across diagnostic categories testing data from individual cases; all ROI data were used as separate measures. RESULTS Stroma demonstrated significantly higher scatter intensity than did epithelium, with lower scattering in tumor-associated stroma as compared with normal or benign-associated stroma. Measures were comparable for invasive and noninvasive malignant tumors but were higher than those found in benign tumors and were lowest in adipose tissue. CONCLUSION Significant differences were found in scatter coefficient properties of epithelium and stroma across diagnostic categories of breast tissue, particularly between benign and malignant-associated stroma. Improved understanding of how scatter properties correlate with morphologic criteria used in routine pathologic diagnoses could have a significant clinical impact as developing optical technology allows macroscopic in situ phase contrast imaging. PMID:19736867

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033

Multiview hyperspectral topography of tissue structural and functional characteristics

NASA Astrophysics Data System (ADS)

Liu, Peng; Huang, Jiwei; Zhang, Shiwu; Xu, Ronald X.

2016-01-01

Accurate and in vivo characterization of structural, functional, and molecular characteristics of biological tissue will facilitate quantitative diagnosis, therapeutic guidance, and outcome assessment in many clinical applications, such as wound healing, cancer surgery, and organ transplantation. We introduced and tested a multiview hyperspectral imaging technique for noninvasive topographic imaging of cutaneous wound oxygenation. The technique integrated a multiview module and a hyperspectral module in a single portable unit. Four plane mirrors were cohered to form a multiview reflective mirror set with a rectangular cross section. The mirror set was placed between a hyperspectral camera and the target biological tissue. For a single image acquisition task, a hyperspectral data cube with five views was obtained. The five-view hyperspectral image consisted of a main objective image and four reflective images. Three-dimensional (3-D) topography of the scene was achieved by correlating the matching pixels between the objective image and the reflective images. 3-D mapping of tissue oxygenation was achieved using a hyperspectral oxygenation algorithm. The multiview hyperspectral imaging technique was validated in a wound model, a tissue-simulating blood phantom, and in vivo biological tissue. The experimental results demonstrated the technical feasibility of using multiview hyperspectral imaging for 3-D topography of tissue functional properties.

A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis

PubMed Central

Cabrera, Javier; Olmo, Rocio; Ruiz-Ferrer, Virginia; Hermans, Christian; Martinez-Argudo, Isabel; Escobar, Carolina

2018-01-01

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells’ area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions. PMID:29389847

A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis.

PubMed

Cabrera, Javier; Olmo, Rocio; Ruiz-Ferrer, Virginia; Abreu, Isidro; Hermans, Christian; Martinez-Argudo, Isabel; Fenoll, Carmen; Escobar, Carolina

2018-02-01

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells' area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 ( CTL1 ) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species ( Arabidopsis , cucumber or Medicago ). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions.

Freezing without Ice Crystal Damage: Semithin and Ultrathin Frozen Sections of Ethanol-Infiltrated Tissue for Microscopy, with Applications to Immunocytochemistry

NASA Astrophysics Data System (ADS)

Christensen, A. Kent; Lowry, Terry B.

1995-10-01

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about [minus sign]117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at [minus sign]155 to [minus sign]170°C to produce semithin frozen sections (0.5 to 3 [mu]m thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope.

PubMed

Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D

2013-02-01

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0-400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument.

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope

PubMed Central

Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J.; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D.

2013-01-01

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0–400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument. PMID:23412564

Quadratus lumborum asymmetry and lumbar spine injury in cricket fast bowlers.

PubMed

Kountouris, Alex; Portus, Marc; Cook, Jill

2012-09-01

Previous studies have demonstrated quadratus lumborum asymmetry in cricket fast bowlers, but there has been conflicting evidence regarding the relationship to lumbar spine injury, particularly vertebral bone stress injuries. This study investigated the relationship between quadratus lumborum asymmetry and lumbar spine injury in adolescent cricket fast bowlers. The study was a prospective cohort design. Magnetic resonance imaging of 38 adolescent cricket fast bowlers was completed prior to a cricket season, and the cross sectional area of the quadratus lumborum muscle was measured at each lumbar spinal level. The bowlers were followed through the cricket season and those that reported lumbar spine injuries were investigated and classified as either having a soft tissue injury or a bone stress injury. The pre-season cross sectional area of quadratus lumborum was associated with injury status at the conclusion of the cricket season. Twenty-one percent of the cohort developed lumbar bone stress injuries during the cricket season. There was no significant relationship between lumbar spine injury and quadratus lumborum cross sectional area. A high incidence of lumbar bone stress injuries was demonstrated in adolescent fast bowlers. Unlike previous research that demonstrated a link between lumbar spine bone stress injuries and quadratus lumborum cross-sectional area, no such relationship was found. Copyright © 2012 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

Imaging deep skeletal muscle structure using a high-sensitivity ultrathin side-viewing optical coherence tomography needle probe

PubMed Central

Yang, Xiaojie; Lorenser, Dirk; McLaughlin, Robert A.; Kirk, Rodney W.; Edmond, Matthew; Simpson, M. Cather; Grounds, Miranda D.; Sampson, David D.

2013-01-01

We have developed an extremely miniaturized optical coherence tomography (OCT) needle probe (outer diameter 310 µm) with high sensitivity (108 dB) to enable minimally invasive imaging of cellular structure deep within skeletal muscle. Three-dimensional volumetric images were acquired from ex vivo mouse tissue, examining both healthy and pathological dystrophic muscle. Individual myofibers were visualized as striations in the images. Degradation of cellular structure in necrotic regions was seen as a loss of these striations. Tendon and connective tissue were also visualized. The observed structures were validated against co-registered hematoxylin and eosin (H&E) histology sections. These images of internal cellular structure of skeletal muscle acquired with an OCT needle probe demonstrate the potential of this technique to visualize structure at the microscopic level deep in biological tissue in situ. PMID:24466482

Primary Orbital Chondromyxoid Fibroma: A Rare Case.

PubMed

Mullen, Martin G; Somogyi, Marie; Maxwell, Sean P; Prabhu, Vikram; Yoo, David K

A 56-year-old male with history of chronic sinusitis was found to have a 3 cm left orbital lesion on CT. Subsequent MRI demonstrated a multilobulated enhancing soft tissue lesion at the superotemporal region of the left orbit. Initial biopsy was reported as a low-grade sarcoma. On further evaluation, a consensus was made that the lesion was likely a benign mixed mesenchymal type tumor but should nonetheless be surgically removed. Left lateral orbitotomy was performed which revealed a tumor originating in the lateral orbital bone with segments eroding through the wall of the orbit. Intraoperative frozen sections revealed myoepitheliod tissue with locally aggressive features and the tumor was completely removed. The final histopathologic analysis of the tissue was consistent with a chondromyxoid fibroma. Chondomyxoid fibroma is a rare entity in the orbital bones and is more commonly seen in long bones.

The influence of composition and location on the toughness of human atherosclerotic femoral plaque tissue.

PubMed

Cunnane, E M; Barrett, H E; Kavanagh, E G; Mongrain, R; Walsh, M T

2016-02-01

The toughness of femoral atherosclerotic tissue is of pivotal importance to understanding the mechanism of luminal expansion during cutting balloon angioplasty (CBA) in the peripheral vessels. Furthermore, the ability to relate this parameter to plaque composition, pathological inclusions and location within the femoral vessels would allow for the improvement of existing CBA technology and for the stratification of patient treatment based on the predicted fracture response of the plaque tissue to CBA. Such information may lead to a reduction in clinically observed complications, an improvement in trial results and an increased adoption of the CBA technique to reduce vessel trauma and further endovascular treatment uptake. This study characterises the toughness of atherosclerotic plaque extracted from the femoral arteries of ten patients using a lubricated guillotine cutting test to determine the critical energy release rate. This information is related to the location that the plaque section was removed from within the femoral vessels and the composition of the plaque tissue, determined using Fourier Transform InfraRed spectroscopy, to establish the influence of location and composition on the toughness of the plaque tissue. Scanning electron microscopy (SEM) is employed to examine the fracture surfaces of the sections to determine the contribution of tissue morphology to toughness. Toughness results exhibit large inter and intra patient and location variance with values ranging far above and below the toughness of healthy porcine arterial tissue (Range: 1330-3035 for location and 140-4560J/m(2) for patients). No significant difference in mean toughness is observed between patients or location. However, the composition parameter representing the calcified tissue content of the plaque correlates significantly with sample toughness (r=0.949, p<0.001). SEM reveals the presence of large calcified regions in the toughest sections that are absent from the least tough sections. Regression analysis highlights the potential of employing the calcified tissue content of the plaque as a preoperative tool for predicting the fracture response of a target lesion to CBA (R(2)=0.885, p<0.001). This study addresses a gap in current knowledge regarding the influence of plaque location, composition and morphology on the toughness of human femoral plaque tissue. Such information is of great importance to the continued improvement of endovascular treatments, particularly cutting balloon angioplasty (CBA), which require experimentally derived data as a framework for assessing clinical cases and advancing medical devices. This study identifies that femoral plaque tissue exhibits large inter and intra patient and location variance regarding tissue toughness. Increasing calcified plaque content is demonstrated to correlate significantly with increasing toughness. This highlights the potential for predicting target lesion toughness which may lead to an increased adoption of the CBA technique and also further the uptake of endovascular treatment. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A mechano-biological model of multi-tissue evolution in bone

NASA Astrophysics Data System (ADS)

Frame, Jamie; Rohan, Pierre-Yves; Corté, Laurent; Allena, Rachele

2017-12-01

Successfully simulating tissue evolution in bone is of significant importance in predicting various biological processes such as bone remodeling, fracture healing and osseointegration of implants. Each of these processes involves in different ways the permanent or transient formation of different tissue types, namely bone, cartilage and fibrous tissues. The tissue evolution in specific circumstances such as bone remodeling and fracturing healing is currently able to be modeled. Nevertheless, it remains challenging to predict which tissue types and organization can develop without any a priori assumptions. In particular, the role of mechano-biological coupling in this selective tissue evolution has not been clearly elucidated. In this work, a multi-tissue model has been created which simultaneously describes the evolution of bone, cartilage and fibrous tissues. The coupling of the biological and mechanical factors involved in tissue formation has been modeled by defining two different tissue states: an immature state corresponding to the early stages of tissue growth and representing cell clusters in a weakly neo-formed Extra Cellular Matrix (ECM), and a mature state corresponding to well-formed connective tissues. This has allowed for the cellular processes of migration, proliferation and apoptosis to be described simultaneously with the changing ECM properties through strain driven diffusion, growth, maturation and resorption terms. A series of finite element simulations were carried out on idealized cantilever bending geometries. Starting from a tissue composition replicating a mid-diaphysis section of a long bone, a steady-state tissue formation was reached over a statically loaded period of 10,000 h (60 weeks). The results demonstrated that bone formation occurred in regions which are optimally physiologically strained. In two additional 1000 h bending simulations both cartilaginous and fibrous tissues were shown to form under specific geometrical and loading cases and cartilage was shown to lead to the formation of bone in a beam replicating a fracture healing initial tissue distribution. This finding is encouraging in that it is corroborated by similar experimental observations of cartilage leading bone formation during the fracture healing process. The results of this work demonstrate that a multi-tissue mechano-biological model of tissue evolution has the potential for predictive analysis in the design and implementations of implants, describing fracture healing and bone remodeling processes.

The spatial resolution of silicon-based electron detectors in beta-autoradiography.

PubMed

Cabello, Jorge; Wells, Kevin

2010-03-21

Thin tissue autoradiography is an imaging modality where ex-vivo tissue sections are placed in direct contact with autoradiographic film. These tissue sections contain a radiolabelled ligand bound to a specific biomolecule under study. This radioligand emits beta - or beta+ particles ionizing silver halide crystals in the film. High spatial resolution autoradiograms are obtained using low energy radioisotopes, such as (3)H where an intrinsic 0.1-1 microm spatial resolution can be achieved. Several digital alternatives have been presented over the past few years to replace conventional film but their spatial resolution has yet to equal film, although silicon-based imaging technologies have demonstrated higher sensitivity compared to conventional film. It will be shown in this work how pixel size is a critical parameter for achieving high spatial resolution for low energy uncollimated beta imaging. In this work we also examine the confounding factors impeding silicon-based technologies with respect to spatial resolution. The study considers charge diffusion in silicon and detector noise, and this is applied to a range of radioisotopes typically used in autoradiography. Finally an optimal detector geometry to obtain the best possible spatial resolution for a specific technology and a specific radioisotope is suggested.

Using oligonucleotide aptamer probes for immunostaining of formalin-fixed and paraffin-embedded tissues

PubMed Central

Zeng, Zihua; Zhang, Peng; Zhao, Nianxi; Sheehan, Andrea M; Tung, Ching-Hsuan; Chang, Chung-Che; Zu, Youli

2011-01-01

For tissue immunostaining, antibodies are currently the only clinically validated and commercially available probes. Aptamers, which belong to a class of small molecule ligands composed of short single-stranded oligonucleotides, have emerged as probes over the last several decades; however, their potential clinical value has not yet been fully explored. Using cultured cells and an RNA-based CD30 aptamer, we recently demonstrated that the synthetic aptamer is useful as a specific probe for flow cytometric detection of CD30-expressing lymphoma cells. In this study, we further validated the use of this aptamer probe for immunostaining of formalin-fixed and paraffin-embedded lymphoma tissues. Using CD30 antibody as a standard control, we demonstrated that the synthetic CD30 aptamer specifically recognized and immunostained tumor cells of classical Hodgkin lymphoma and anaplastic large cell lymphoma, but did not react with background cells within tumor sites. Notably, the CD30 aptamer probe optimally immunostained lymphoma cells with lower temperature antigen retrieval (37 vs 96°C for antibody) and shorter probing reaction times (20 vs 90 min for antibody) than typical antibody immunostaining protocols. In addition, the CD30 aptamer probe showed no nonspecific background staining of cell debris in necrotic tissue and exhibited no cross-reaction to tissues that do not express CD30, as confirmed by a standard CD30 antibody staining. Therefore, our findings indicate that the synthetic oligonucleotide CD30 aptamer can be used as a probe for immunostaining of fixed tissue sections for disease diagnosis. PMID:20693984

Automated high-throughput assessment of prostate biopsy tissue using infrared spectroscopic chemical imaging

NASA Astrophysics Data System (ADS)

Bassan, Paul; Sachdeva, Ashwin; Shanks, Jonathan H.; Brown, Mick D.; Clarke, Noel W.; Gardner, Peter

2014-03-01

Fourier transform infrared (FT-IR) chemical imaging has been demonstrated as a promising technique to complement histopathological assessment of biomedical tissue samples. Current histopathology practice involves preparing thin tissue sections and staining them using hematoxylin and eosin (H&E) after which a histopathologist manually assess the tissue architecture under a visible microscope. Studies have shown that there is disagreement between operators viewing the same tissue suggesting that a complementary technique for verification could improve the robustness of the evaluation, and improve patient care. FT-IR chemical imaging allows the spatial distribution of chemistry to be rapidly imaged at a high (diffraction-limited) spatial resolution where each pixel represents an area of 5.5 × 5.5 μm2 and contains a full infrared spectrum providing a chemical fingerprint which studies have shown contains the diagnostic potential to discriminate between different cell-types, and even the benign or malignant state of prostatic epithelial cells. We report a label-free (i.e. no chemical de-waxing, or staining) method of imaging large pieces of prostate tissue (typically 1 cm × 2 cm) in tens of minutes (at a rate of 0.704 × 0.704 mm2 every 14.5 s) yielding images containing millions of spectra. Due to refractive index matching between sample and surrounding paraffin, minimal signal processing is required to recover spectra with their natural profile as opposed to harsh baseline correction methods, paving the way for future quantitative analysis of biochemical signatures. The quality of the spectral information is demonstrated by building and testing an automated cell-type classifier based upon spectral features.

Optical scattering coefficient estimated by optical coherence tomography correlates with collagen content in ovarian tissue

NASA Astrophysics Data System (ADS)

Yang, Yi; Wang, Tianheng; Biswal, Nrusingh C.; Wang, Xiaohong; Sanders, Melinda; Brewer, Molly; Zhu, Quing

2011-09-01

Optical scattering coefficient from ex vivo unfixed normal and malignant ovarian tissue was quantitatively extracted by fitting optical coherence tomography (OCT) A-line signals to a single scattering model. 1097 average A-line measurements at a wavelength of 1310 nm were performed at 108 sites obtained from 18 ovaries. The average scattering coefficient obtained from the normal tissue group consisted of 833 measurements from 88 sites was 2.41 mm-1 (+/-0.59), while the average coefficient obtained from the malignant tissue group consisted of 264 measurements from 20 sites was 1.55 mm-1 (+/-0.46). The malignant ovarian tissue showed significant lower scattering than the normal group (p < 0.001). The amount of collagen within OCT imaging depth was analyzed from the tissue histological section stained with Sirius Red. The average collagen area fraction (CAF) obtained from the normal tissue group was 48.4% (+/-12.3%), while the average CAF obtained from the malignant tissue group was 11.4% (+/-4.7%). A statistical significance of the collagen content was found between the two groups (p < 0.001). These results demonstrated that quantitative measurements of optical scattering coefficient from OCT images could be a potential powerful method for ovarian cancer detection.

Reconstruction of the forehead acoustic properties in an Indo-Pacific humpback dolphin (Sousa chinensis), with investigation on the responses of soft tissue sound velocity to temperature.

PubMed

Song, Zhongchang; Zhang, Yu; Berggren, Per; Wei, Chong

2017-02-01

Computed tomography (CT) imaging and ultrasound experimental measurements were combined to reconstruct the acoustic properties (density, velocity, and impedance) of the head from a deceased Indo-Pacific humpback dolphin (Sousa chinensis). The authors extracted 42 soft forehead tissue samples to estimate the sound velocity and density properties at room temperature, 25.0  °C. Hounsfield Units (HUs) of the samples were read from CT scans. Linear relationships between the tissues' HUs and velocity, and HUs and density were revealed through regression analyses. The distributions of the head acoustic properties at axial, coronal, and sagittal cross sections were reconstructed, suggesting that the forehead soft tissues were characterized by low-velocity in the melon, high-velocity in the muscle and connective tissues. Further, the sound velocities of melon, muscle, and connective tissue pieces were measured under different temperatures to investigate tissues' velocity response to temperature. The results demonstrated nonlinear relationships between tissues' sound velocity and temperature. This study represents a first attempt to provide general information on acoustic properties of this species. The results could provide meaningful information for understanding the species' bioacoustic characteristics and for further investigation on sound beam formation of the dolphin.

Compact diode laser source for multiphoton biological imaging

PubMed Central

Niederriter, Robert D.; Ozbay, Baris N.; Futia, Gregory L.; Gibson, Emily A.; Gopinath, Juliet T.

2016-01-01

We demonstrate a compact, pulsed diode laser source suitable for multiphoton microscopy of biological samples. The center wavelength is 976 nm, near the peak of the two-photon cross section of common fluorescent markers such as genetically encoded green and yellow fluorescent proteins. The laser repetition rate is electrically tunable between 66.67 kHz and 10 MHz, with 2.3 ps pulse duration and peak powers >1 kW. The laser components are fiber-coupled and scalable to a compact package. We demonstrate >600 μm depth penetration in brain tissue, limited by laser power. PMID:28101420

Microscope mode secondary ion mass spectrometry imaging with a Timepix detector.

PubMed

Kiss, Andras; Jungmann, Julia H; Smith, Donald F; Heeren, Ron M A

2013-01-01

In-vacuum active pixel detectors enable high sensitivity, highly parallel time- and space-resolved detection of ions from complex surfaces. For the first time, a Timepix detector assembly was combined with a secondary ion mass spectrometer for microscope mode secondary ion mass spectrometry (SIMS) imaging. Time resolved images from various benchmark samples demonstrate the imaging capabilities of the detector system. The main advantages of the active pixel detector are the higher signal-to-noise ratio and parallel acquisition of arrival time and position. Microscope mode SIMS imaging of biomolecules is demonstrated from tissue sections with the Timepix detector.

Investigating MALDI MSI parameters (Part 2) - On the use of a mechanically shuttered trigger system for improved laser energy stability.

PubMed

Steven, Rory T; Dexter, Alex; Bunch, Josephine

2016-07-15

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is now widely used to desorb, ionize and detect molecules from complex samples and tissue sections. The detected ion intensity within MALDI MS and MSI is intimately linked to the laser energy per pulse incident upon the sample during analysis. Laser energy/power stability can be significantly affected by the manner in which the laser is operated. High-repetition rate diode-pumped solid-state (DPSS) lasers are being increasingly adopted to enable high-throughput MALDI MSI analysis. Within this work two different laser-triggering setups are used to demonstrate the effect of laser energy instabilities due to spiking and thermal control phenomena and a setup with a shutter to remove these effects. The effect of non-equilibrium laser operation on MALDI MSI data versus the more stable laser pulse energy of the shutter-triggered system is demonstrated in thin films of α-cyano-4-hydroxycinnamic acid (CHCA) and for imaging of murine brain tissue sections. Significant unwanted variations in absolute and relative detected ion intensity are shown where energy variation is introduced by these phenomena, which return to equilibrium within the setup employed here over timescales relevant to MALDI MS analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue.

PubMed

Yoshitake, Tadayuki; Giacomelli, Michael G; Cahill, Lucas C; Schmolze, Daniel B; Vardeh, Hilde; Faulkner-Jones, Beverly E; Connolly, James L; Fujimoto, James G

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

PubMed Central

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-01-01

Abstract. Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue. PMID:28032121

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

NASA Astrophysics Data System (ADS)

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Histology and ultrastructure of picosecond laser intrastromal photorefractive keratectomy (ISPRK)

NASA Astrophysics Data System (ADS)

Krueger, Ronald R.; Quantock, Andrew J.; Ito, Mitsutoshi; Assil, Kerry K.; Schanzlin, David J.

1995-05-01

Picosecond intrastromal ablation is currently under investigation as a new minimally invasive way of correcting refractive error. When the laser pulses are placed in an expanding spiral pattern along a lamellar plane, the technique is called intrastromal photorefractive keratectomy (ISPRK). We performed ISPRK on six human eye bank eyes. Thirty picosecond pulses at 1000 Hz and 20 - 25 (mu) J per pulse were separated by 15 microns. A total of 3 layers were placed in the anterior stroma separated by 15 microns. The eyes were then preserved and sectioned for light, scanning and transmission electron microscopy. Light and scanning electron microscopy reveals that picosecond intrastromal ablation using an ISPRK pattern demonstrates multiple, coalescing intrastromal cavities oriented parallel to the corneal surface. These cavities possess a smooth appearing inner wall. Using transmission electron microscopy, we noticed tissue loss surrounding some cavities with collagen fibril termination and thinning of collagen lamella. Other cavities we formed by separation of lamella with little evidence of tissue loss. A pseudomembrane lines the edge of some cavities. Although underlying tissue disruption was occasionally seen along the border of a cavity in no case was there any evidence of thermal damage or tissue necrosis. Ablation and loss of tissue in ISPRK results in nonthermal microscopic corneal thinning around some cavities whereas others demonstrate only lamellar separation. Alternative patterns and energy parameters should be investigated to bring this technology to its full potential in refractive surgery.

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

PubMed

Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

2015-07-01

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

Quantitative bioimaging of p-boronophenylalanine in thin liver tissue sections as a tool for treatment planning in boron neutron capture therapy.

PubMed

Reifschneider, Olga; Schütz, Christian L; Brochhausen, Christoph; Hampel, Gabriele; Ross, Tobias; Sperling, Michael; Karst, Uwe

2015-03-01

An analytical method using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was developed and applied to assess enrichment of 10B-containing p-boronophenylalanine-fructose (BPA-f) and its pharmacokinetic distribution in human tissues after application for boron neutron capture therapy (BNCT). High spatial resolution (50 μm) and limits of detection in the low parts-per-billion range were achieved using a Nd:YAG laser of 213 nm wavelength. External calibration by means of 10B-enriched standards based on whole blood proved to yield precise quantification results. Using this calibration method, quantification of 10B in cancerous and healthy tissue was carried out. Additionally, the distribution of 11B was investigated, providing 10B enrichment in the investigated tissues. Quantitative imaging of 10B by means of LA-ICP-MS was demonstrated as a new option to characterise the efficacy of boron compounds for BNCT.

Importance of tissue preparation methods in FTIR micro-spectroscopical analysis of biological tissues: 'traps for new users'.

PubMed

Zohdi, Vladislava; Whelan, Donna R; Wood, Bayden R; Pearson, James T; Bambery, Keith R; Black, M Jane

2015-01-01

Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled prior to FTIR micro-spectroscopic imaging requires special consideration, particularly with regards to methods for preservation of the samples. We have performed FTIR micro-spectroscopy on rodent heart and liver tissue sections (two spectroscopically very different biological tissues) that were prepared by desiccation drying, ethanol substitution and formalin fixation and have compared the resulting spectra with that of fully hydrated freshly excised tissues. We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions. In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol. Indeed, we demonstrate that preserving samples through desiccation drying, ethanol substitution or formalin fixation significantly alters the biochemical information detected using spectroscopic methods when compared to spectra of fresh hydrated tissue. It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.

Tissue adhesion to bioactive glass-coated silicone tubing in a rat model of peritoneal dialysis catheters and catheter tunnels.

PubMed

Ross, Edward A; Batich, Christopher D; Clapp, William L; Sallustio, Judith E; Lee, Nadeen C

2003-02-01

Silicone peritoneal dialysis catheters do not develop tissue ingrowth, lack a mechanical barrier to periluminal bacterial migration and need cuffs for anchorage. We hypothesized that a bioactive glass coating composed of silicon, calcium, sodium and phosphorous oxides would cause a beneficial tissue reaction causing catheter adhesion, and tested this in a rat model. A hexane solvent-based method of coating silicone tubes with Bioglass powder was used, which maintained flexibility, and then the ultrastructure was confirmed with scanning electron microscopy (EM). Segments 2.5 cm were implanted subcutaneously in 8 Sprague-Dawley rats, with uncoated tubes as a contralateral control, and histology was done at 2, 4 and 6 weeks, including special stains and EM. The uncoated segments grossly had no adherence to surrounding tissue, and were physically separate from a thin fibrous capsule of approximately 50 micro width. Trichrome stains demonstrated the capsule was rich in collagen. There was minimal adjacent tissue reaction. In contrast, the coated tubes were palpably fixed to the soft tissues, and sections demonstrated an adjacent prominent layer of macrophages and multinucleated giant cells. Small numbers of lymphocytes were noted. This cellular reaction increased over the 6-week implant duration, and was also associated with neovascularization of the tissue adjacent to the segments (33 vessels in coated vs. 20 in controls per x 200 field, P < 0.0001). Many refractile silicone particles and prominent multinucleated giant cells were present, with small numbers of lymphocytes and macrophages. Stains showed scattered discontinuous calcific deposits. These findings are consistent with reports that the Bioglass(R) silicon oxide leads to the formation of a layer of hydroxyapatite, which binds to collagen and induces a tissue cellular reaction. In summary, bioactive glass coatings can improve the tissue retention of silicone tubing by promoting adhesion by collagen and cell proliferation, and are promising for future studies of peritoneal dialysis catheters.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

PubMed Central

Saitoh, Sei; Ohno, Nobuhiko; Saitoh, Yurika; Terada, Nobuo; Shimo, Satoshi; Aida, Kaoru; Fujii, Hideki; Kobayashi, Tetsuro; Ohno, Shinichi

2018-01-01

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets. PMID:29622846

Cytoskeletal remodeling of connective tissue fibroblasts in response to static stretch is dependent on matrix material properties

PubMed Central

Abbott, Rosalyn D; Koptiuch, Cathryn; Iatridis, James C; Howe, Alan K; Badger, Gary J; Langevin, Helene M

2012-01-01

In areolar “loose” connective tissue, fibroblasts remodel their cytoskeleton within minutes in response to static stretch resulting in increased cell body cross-sectional area that relaxes the tissue to a lower state of resting tension. It remains unknown whether the loosely arranged collagen matrix, characteristic of areolar connective tissue, is required for this cytoskeletal response to occur. The purpose of this study was to evaluate cytoskeletal remodeling of fibroblasts in and dissociated from areolar and dense connective tissue in response to 2 hours of static stretch in both native tissue and collagen gels of varying crosslinking. Rheometric testing indicated that the areolar connective tissue had a lower dynamic modulus and was more viscous than the dense connective tissue. In response to stretch, cells within the more compliant areolar connective tissue adopted a large “sheet-like” morphology that was in contrast to the smaller dendritic morphology in the dense connective tissue. By adjusting the in vitro collagen crosslinking, and the resulting dynamic modulus, it was demonstrated that cells dissociated from dense connective tissue are capable of responding when seeded into a compliant matrix, while cells dissociated from areolar connective tissue can lose their ability to respond when their matrix becomes stiffer. This set of experiments indicated stretch-induced fibroblast expansion was dependent on the distinct matrix material properties of areolar connective tissues as opposed to the cells’ tissue of origin. These results also suggest that disease and pathological processes with increased crosslinks, such as diabetes and fibrosis, could impair fibroblast responsiveness in connective tissues. PMID:22552950

Early fixation of cobalt-chromium based alloy surgical implants to bone using a tissue-engineering approach.

PubMed

Ogawa, Munehiro; Tohma, Yasuaki; Ohgushi, Hajime; Takakura, Yoshinori; Tanaka, Yasuhito

2012-01-01

To establish the methods of demonstrating early fixation of metal implants to bone, one side of a Cobalt-Chromium (CoCr) based alloy implant surface was seeded with rabbit marrow mesenchymal cells and the other side was left unseeded. The mesenchymal cells were further cultured in the presence of ascorbic acid, β-glycerophosphate and dexamethasone, resulting in the appearance of osteoblasts and bone matrix on the implant surface. Thus, we succeeded in generating tissue-engineered bone on one side of the CoCr implant. The CoCr implants were then implanted in rabbit bone defects. Three weeks after the implantation, evaluations of mechanical test, undecalcified histological section and electron microscope analysis were performed. Histological and electron microscope images of the tissue engineered surface exhibited abundant new bone formation. However, newly formed bone tissue was difficult to detect on the side without cell seeding. In the mechanical test, the mean values of pull-out forces were 77.15 N and 44.94 N for the tissue-engineered and non-cell-seeded surfaces, respectively. These findings indicate early bone fixation of the tissue-engineered CoCr surface just three weeks after implantation.

Early Fixation of Cobalt-Chromium Based Alloy Surgical Implants to Bone Using a Tissue-engineering Approach

PubMed Central

Ogawa, Munehiro; Tohma, Yasuaki; Ohgushi, Hajime; Takakura, Yoshinori; Tanaka, Yasuhito

2012-01-01

To establish the methods of demonstrating early fixation of metal implants to bone, one side of a Cobalt-Chromium (CoCr) based alloy implant surface was seeded with rabbit marrow mesenchymal cells and the other side was left unseeded. The mesenchymal cells were further cultured in the presence of ascorbic acid, β-glycerophosphate and dexamethasone, resulting in the appearance of osteoblasts and bone matrix on the implant surface. Thus, we succeeded in generating tissue-engineered bone on one side of the CoCr implant. The CoCr implants were then implanted in rabbit bone defects. Three weeks after the implantation, evaluations of mechanical test, undecalcified histological section and electron microscope analysis were performed. Histological and electron microscope images of the tissue engineered surface exhibited abundant new bone formation. However, newly formed bone tissue was difficult to detect on the side without cell seeding. In the mechanical test, the mean values of pull-out forces were 77.15 N and 44.94 N for the tissue-engineered and non-cell-seeded surfaces, respectively. These findings indicate early bone fixation of the tissue-engineered CoCr surface just three weeks after implantation. PMID:22754313

Red fluorescent protein responsible for pigmentation in trematode-infected Porites compressa tissues.

PubMed

Palmer, Caroline V; Roth, Melissa S; Gates, Ruth D

2009-02-01

Reports of coral disease have increased dramatically over the last decade; however, the biological mechanisms that corals utilize to limit infection and resist disease remain poorly understood. Compromised coral tissues often display non-normal pigmentation that potentially represents an inflammation-like response, although these pigments remain uncharacterized. Using spectral emission analysis and cryo-histological and electrophoretic techniques, we investigated the pink pigmentation associated with trematodiasis, infection with Podocotyloides stenometre larval trematode, in Porites compressa. Spectral emission analysis reveals that macroscopic areas of pink pigmentation fluoresce under blue light excitation (450 nm) and produce a broad emission peak at 590 nm (+/-6) with a 60-nm full width at half maximum. Electrophoretic protein separation of pigmented tissue extract confirms the red fluorescence to be a protein rather than a low-molecular-weight compound. Histological sections demonstrate green fluorescence in healthy coral tissue and red fluorescence in the trematodiasis-compromised tissue. The red fluorescent protein (FP) is limited to the epidermis, is not associated with cells or granules, and appears unstructured. These data collectively suggest that the red FP is produced and localized in tissue infected by larval trematodes and plays a role in the immune response in corals.

Automated tumor analysis for molecular profiling in lung cancer

PubMed Central

Boyd, Clinton; James, Jacqueline A.; Loughrey, Maurice B.; Hougton, Joseph P.; Boyle, David P.; Kelly, Paul; Maxwell, Perry; McCleary, David; Diamond, James; McArt, Darragh G.; Tunstall, Jonathon; Bankhead, Peter; Salto-Tellez, Manuel

2015-01-01

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics. PMID:26317646

Effects of n-acetylcysteine in a rat model of ischemia and reperfusion injury.

PubMed

Cuzzocrea, S; Mazzon, E; Costantino, G; Serraino, I; De Sarro, A; Caputi, A P

2000-08-18

Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of n-acetylcysteine (NAC), a free radical scavenger, in rats subjected to SAO shock. Treatment of rats with NAC (applied at 20 mg/kg, 5 min prior to reperfusion, followed by an infusion of 20 mg/kg/h) attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. NAC also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine in the plasma of the SAO-shocked rats after reperfusion. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) synthetase (PARS) revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and PARS were markedly reduced in tissue sections obtained from SAO-shocked rats which had received NAC. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localised in the vascular endothelial cells. Ileum tissue section obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. NAC treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). NAC treatment improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and significantly improved survival. Taken together, our results clearly demonstrate that NAC treatment exert a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

Laser Microdissection and Atmospheric Pressure Chemical Ionization Mass Spectrometry Coupled for Multimodal Imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lorenz, Matthias; Ovchinnikova, Olga S; Kertesz, Vilmos

2013-01-01

This paper describes the coupling of ambient laser ablation surface sampling, accomplished using a laser capture microdissection system, with atmospheric pressure chemical ionization mass spectrometry for high spatial resolution multimodal imaging. A commercial laser capture microdissection system was placed in close proximity to a modified ion source of a mass spectrometer designed to allow for sampling of laser ablated material via a transfer tube directly into the ionization region. Rhodamine 6G dye of red sharpie ink in a laser etched pattern as well as cholesterol and phosphatidylcholine in a cerebellum mouse brain thin tissue section were identified and imaged frommore » full scan mass spectra. A minimal spot diameter of 8 m was achieved using the 10X microscope cutting objective with a lateral oversampling pixel resolution of about 3.7 m. Distinguishing between features approximately 13 m apart in a cerebellum mouse brain thin tissue section was demonstrated in a multimodal fashion including co-registered optical and mass spectral chemical images.« less

Terahertz Imaging of Three-Dimensional Dehydrated Breast Cancer Tumors

NASA Astrophysics Data System (ADS)

Bowman, Tyler; Wu, Yuhao; Gauch, John; Campbell, Lucas K.; El-Shenawee, Magda

2017-06-01

This work presents the application of terahertz imaging to three-dimensional formalin-fixed, paraffin-embedded human breast cancer tumors. The results demonstrate the capability of terahertz for in-depth scanning to produce cross section images without the need to slice the tumor. Samples of tumors excised from women diagnosed with infiltrating ductal carcinoma and lobular carcinoma are investigated using a pulsed terahertz time domain imaging system. A time of flight estimation is used to obtain vertical and horizontal cross section images of tumor tissues embedded in paraffin block. Strong agreement is shown comparing the terahertz images obtained by electronically scanning the tumor in-depth in comparison with histopathology images. The detection of cancer tissue inside the block is found to be accurate to depths over 1 mm. Image processing techniques are applied to provide improved contrast and automation of the obtained terahertz images. In particular, unsharp masking and edge detection methods are found to be most effective for three-dimensional block imaging.

Multi-institutional comparison of whole slide digital imaging and optical microscopy for interpretation of hematoxylin-eosin-stained breast tissue sections.

PubMed

Krishnamurthy, Savitri; Mathews, Kurt; McClure, Steven; Murray, Melissa; Gilcrease, Michael; Albarracin, Constance; Spinosa, John; Chang, Bernard; Ho, James; Holt, John; Cohen, Arthur; Giri, Dilip; Garg, Karuna; Bassett, Roland L; Liang, Kevin

2013-12-01

Whole slide imaging (WSI) is now used for educational purposes, for consultation, and for archiving and quantitation of immunostains. However, it is not routinely used for the primary diagnosis of hematoxylin-eosin-stained tissue sections. To compare WSI using the Aperio digital pathology system (Aperio Technologies, Inc, Vista, California) with optical microscopy (OM) for the interpretation of hematoxylin-eosin-stained tissue sections of breast lesions. The study was conducted at 3 clinical sites; 3 breast pathologists interpreted 150 hematoxylin-eosin-stained slides at each site, 3 times each by WSI and 3 times each by OM. For WSI, slides were scanned using an Aperio ScanScope and interpreted on a computer monitor using Aperio ImageScope software and Aperio Spectrum data management software. Pathologic interpretations were recorded using the College of American Pathologists breast checklist. WSI diagnoses were compared with OM diagnoses for accuracy, precision (interpathologist variation), and reproducibility (intrapathologist variation). Results were considered accurate only if the interpretation matched exactly between WSI and OM. The proportion of accurate results reported by each pathologist was expressed as a percentage for the comparison of the 2 platforms. The accuracy of WSI for classifying lesions as not carcinoma or as noninvasive (ductal or lobular) or invasive (ductal, lobular, or other) carcinoma was 90.5%. The accuracy of OM was 92.1%. The precision and reproducibility of WSI and OM were determined on the basis of pairwise comparisons (3 comparisons for each slide, resulting in 36 possible comparisons). The overall precision of WSI was 90.5% in comparison with 92.1% for OM; reproducibility of WSI was 91.6% in comparison with 94.5% for OM, respectively. In this study, we demonstrated that WSI and OM have similar accuracy, precision, and reproducibility for interpreting hematoxylin-eosin-stained breast tissue sections. Further clinical studies using routine surgical pathology specimens would be useful to confirm these findings and facilitate the incorporation of WSI into diagnostic practice.

Towards High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry Coupled to Shear Force Microscopy

NASA Astrophysics Data System (ADS)

Nguyen, Son N.; Sontag, Ryan L.; Carson, James P.; Corley, Richard A.; Ansong, Charles; Laskin, Julia

2018-02-01

Constant mode ambient mass spectrometry imaging (MSI) of tissue sections with high lateral resolution of better than 10 μm was performed by combining shear force microscopy with nanospray desorption electrospray ionization (nano-DESI). Shear force microscopy enabled precise control of the distance between the sample and nano-DESI probe during MSI experiments and provided information on sample topography. Proof-of-concept experiments were performed using lung and brain tissue sections representing spongy and dense tissues, respectively. Topography images obtained using shear force microscopy were comparable to the results obtained using contact profilometry over the same region of the tissue section. Variations in tissue height were found to be dependent on the tissue type and were in the range of 0-5 μm for lung tissue and 0-3 μm for brain tissue sections. Ion images of phospholipids obtained in this study are in good agreement with literature data. Normalization of nano-DESI MSI images to the signal of the internal standard added to the extraction solvent allowed us to construct high-resolution ion images free of matrix effects.

Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC–ESI-MS–MS

DOE PAGES

Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R.; ...

2015-06-18

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland.more » AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.« less

Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC–ESI-MS–MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R.

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland.more » AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.« less

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses.

PubMed

Glaros, Trevor G; Blancett, Candace D; Bell, Todd M; Natesan, Mohan; Ulrich, Robert G

2015-01-01

The bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers. Reasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei. Our results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

Demonstration of a focused ion-beam cross-sectioning technique for ultrastructural examination of resin-dentin interfaces.

PubMed

Van Meerbeek, B; Conn, L J; Duke, E S; Schraub, D; Ghafghaichi, F

1995-03-01

focused ion-beam (FIB) etching, commonly used as a cross-sectioning technique for failure analysis of semiconductor devices, has recently been applied to biological tissues to expose their ultrastructure for examination. It was the aim of this investigation to determine the practical utility of FIB to cross-section resin-dentin interfaces in order to morphologically evaluate the completeness of resin penetration into the exposed collagen scaffold at the resin-dentin bond interface. Two representative commercially available dentin adhesive systems were bonded to mid-coronal dentin. After appropriate fixation and dehydration of the resin-bonded dentin samples, a scanned focused ion-beam of a few tens of nano-meters in diameter was used to cross=section the resin-dentin interface. Examination of the interfacial ultrastructure was accomplished using a field-emission SEM. Results indicate possible artifact production at the cross-sectioned interface, hiding its actual ultrastructure, probably due to a heat-effect with possible recrystallization. Further studies of FIB are needed to optimize its usefulness for resin-dentin interface examinations and other biological tissue applications. Complete resin saturation of the demineralized dentin surface-layer has been claimed to be the key factor for a long-lasting resin-dentin bond. A "clean" artifact-free micro-cross-sectioning technique may provide indisputable ultra-structural information about the depth of resin penetration into the demineralized zone. Such a test would be useful in the development of dentin adhesive systems.

Vandetanib-eluting Radiopaque Beads: In vivo Pharmacokinetics, Safety and Toxicity Evaluation following Swine Liver Embolization.

PubMed

Denys, Alban; Czuczman, Peter; Grey, David; Bascal, Zainab; Whomsley, Rhys; Kilpatrick, Hugh; Lewis, Andrew L

2017-01-01

To evaluate the plasma and tissue pharmacokinetics, safety and toxicity following intra-arterial hepatic artery administration of Vandetanib (VTB)-eluting Radiopaque Beads (VERB) in healthy swine. In a first phase, healthy swine were treated with hepatic intra-arterial administration of VERB at target dose loading strengths of 36 mg/mL (VERB36), 72 mg/mL (VERB72) and 120 mg/mL (VERB120). Blood and tissue samples were taken and analysed for VTB and metabolites to determine pharmacokinetic parameters for the different dose forms over 30 days. In a second phase, animals were treated with unloaded radiopaque beads or high dose VTB loaded beads (VERB100, 100 mg/mL). Tissue samples from embolized and non-embolized areas of the liver were evaluated at necropsy (30 and 90 days) for determination of VTB and metabolite levels and tissue pathology. Imaging was performed prior to sacrifice using multi-detector computed tomography (MDCT) and imaging findings correlated with pathological changes in the tissue and location of the radiopaque beads. The peak plasma levels of VTB (C max ) released from the various doses of VERB ranged between 6.19-17.3 ng/mL indicating a low systemic burst release. The plasma profile of VTB was consistent with a distribution phase up to 6 h after administration followed by elimination with a half-life of 20-23 h. The AUC of VTB and its major metabolite N-desmethyl vandetanib (NDM VTB) was approximately linear with the dose strength of VERB. VTB plasma levels were at or below limits of detection two weeks after administration. In liver samples, VTB and NDM VTB were present in treated sections at 30 days after administration at levels above the in vitro IC 50 for biological effectiveness. At 90 days both analytes were still present in treated liver but were near or below the limit of quantification in untreated liver sections, demonstrating sustained release from the VERB. Comparison of the reduction of the liver lobe size and associated tissue changes suggested a more effective embolization with VERB compared to the beads without drug. Hepatic intra-arterial administration of VERB results in a low systemic exposure and enables sustained delivery of VTB to target tissues following embolization. Changes in the liver tissue are consistent with an effective embolization and this study has demonstrated that VERB100 is well tolerated with no obvious systemic toxicity.

Research on terahertz properties of rat brain tissue sections during dehydration

NASA Astrophysics Data System (ADS)

Cui, Gangqiang; Liang, Jianfeng; Zhao, Hongwei; Zhao, Xianghui; Chang, Chao

2018-01-01

Biological tissue sections are always kept in a system purged with dry nitrogen for the measurement of terahertz spectrum. However, the injected nitrogen will cause dehydration of tissue sections, which will affect the accuracy of spectrum measurement. In this paper, terahertz time-domain spectrometer is used to measure the terahertz spectra of rat brain tissue sections during dehydration. The changes of terahertz properties, including terahertz transmittance, refractive index and extinction coefficient during dehydration are also analyzed. The amplitudes of terahertz time-domain spectra increase gradually during the dehydration process. Besides, the terahertz properties show obvious changes during the dehydration process. All the results indicate that the injected dry nitrogen has a significant effect on the terahertz spectra and properties of tissue sections. This study contributes to further research and application of terahertz technology in biomedical field.

Development of acute hydrocephalus does not change brain tissue mechanical properties in adult rats, but in juvenile rats

PubMed Central

Pong, Alice C.; Jugé, Lauriane; Bilston, Lynne E.; Cheng, Shaokoon

2017-01-01

Introduction Regional changes in brain stiffness were previously demonstrated in an experimental obstructive hydrocephalus juvenile rat model. The open cranial sutures in the juvenile rats have influenced brain compression and mechanical properties during hydrocephalus development and the extent by which closed cranial sutures in adult hydrocephalic rat models affect brain stiffness in-vivo remains unclear. The aims of this study were to determine changes in brain tissue mechanical properties and brain structure size during hydrocephalus development in adult rat with fixed cranial volume and how these changes were related to brain tissue deformation. Methods Hydrocephalus was induced in 9 female ten weeks old Sprague-Dawley rats by injecting 60 μL of a kaolin suspension (25%) into the cisterna magna under anaesthesia. 6 sham-injected age-matched female SD rats were used as controls. MR imaging (9.4T, Bruker) was performed 1 day before and then at 3 days post injection. T2-weighted anatomical MR images were collected to quantify ventricle and brain tissue cross-sectional areas. MR elastography (800 Hz) was used to measure the brain stiffness (G*, shear modulus). Results Brain tissue in the adult hydrocephalic rats was more compressed than the juvenile hydrocephalic rats because the skulls of the adult hydrocephalic rats were unable to expand like the juvenile rats. In the adult hydrocephalic rats, the cortical gray matter thickness and the caudate-putamen cross-sectional area decreased (Spearman, P < 0.001 for both) but there were no significant changes in cranial cross-sectional area (Spearman, P = 0.35), cortical gray matter stiffness (Spearman, P = 0.24) and caudate-putamen (Spearman, P = 0.11) stiffness. No significant changes in the size of brain structures were observed in the controls. Conclusions This study showed that although brain tissue in the adult hydrocephalic rats was severely compressed, their brain tissue stiffness did not change significantly. These results are in contrast with our previous findings in juvenile hydrocephalic rats which had significantly less brain compression (as the brain circumference was able to stretch with the cranium due to the open skull sutures) and had a significant increase in caudate putamen stiffness. These results suggest that change in brain mechanical properties in hydrocephalus is complex and is not solely dependent on brain tissue deformation. Further studies on the interactions between brain tissue stiffness, deformation, tissue oedema and neural damage are necessary before MRE can be used as a tool to track changes in brain biomechanics in hydrocephalus. PMID:28837671

Development of acute hydrocephalus does not change brain tissue mechanical properties in adult rats, but in juvenile rats.

PubMed

Pong, Alice C; Jugé, Lauriane; Bilston, Lynne E; Cheng, Shaokoon

2017-01-01

Regional changes in brain stiffness were previously demonstrated in an experimental obstructive hydrocephalus juvenile rat model. The open cranial sutures in the juvenile rats have influenced brain compression and mechanical properties during hydrocephalus development and the extent by which closed cranial sutures in adult hydrocephalic rat models affect brain stiffness in-vivo remains unclear. The aims of this study were to determine changes in brain tissue mechanical properties and brain structure size during hydrocephalus development in adult rat with fixed cranial volume and how these changes were related to brain tissue deformation. Hydrocephalus was induced in 9 female ten weeks old Sprague-Dawley rats by injecting 60 μL of a kaolin suspension (25%) into the cisterna magna under anaesthesia. 6 sham-injected age-matched female SD rats were used as controls. MR imaging (9.4T, Bruker) was performed 1 day before and then at 3 days post injection. T2-weighted anatomical MR images were collected to quantify ventricle and brain tissue cross-sectional areas. MR elastography (800 Hz) was used to measure the brain stiffness (G*, shear modulus). Brain tissue in the adult hydrocephalic rats was more compressed than the juvenile hydrocephalic rats because the skulls of the adult hydrocephalic rats were unable to expand like the juvenile rats. In the adult hydrocephalic rats, the cortical gray matter thickness and the caudate-putamen cross-sectional area decreased (Spearman, P < 0.001 for both) but there were no significant changes in cranial cross-sectional area (Spearman, P = 0.35), cortical gray matter stiffness (Spearman, P = 0.24) and caudate-putamen (Spearman, P = 0.11) stiffness. No significant changes in the size of brain structures were observed in the controls. This study showed that although brain tissue in the adult hydrocephalic rats was severely compressed, their brain tissue stiffness did not change significantly. These results are in contrast with our previous findings in juvenile hydrocephalic rats which had significantly less brain compression (as the brain circumference was able to stretch with the cranium due to the open skull sutures) and had a significant increase in caudate putamen stiffness. These results suggest that change in brain mechanical properties in hydrocephalus is complex and is not solely dependent on brain tissue deformation. Further studies on the interactions between brain tissue stiffness, deformation, tissue oedema and neural damage are necessary before MRE can be used as a tool to track changes in brain biomechanics in hydrocephalus.

Joint learning of ultrasonic backscattering statistical physics and signal confidence primal for characterizing atherosclerotic plaques using intravascular ultrasound.

PubMed

Sheet, Debdoot; Karamalis, Athanasios; Eslami, Abouzar; Noël, Peter; Chatterjee, Jyotirmoy; Ray, Ajoy K; Laine, Andrew F; Carlier, Stephane G; Navab, Nassir; Katouzian, Amin

2014-01-01

Intravascular Ultrasound (IVUS) is a predominant imaging modality in interventional cardiology. It provides real-time cross-sectional images of arteries and assists clinicians to infer about atherosclerotic plaques composition. These plaques are heterogeneous in nature and constitute fibrous tissue, lipid deposits and calcifications. Each of these tissues backscatter ultrasonic pulses and are associated with a characteristic intensity in B-mode IVUS image. However, clinicians are challenged when colocated heterogeneous tissue backscatter mixed signals appearing as non-unique intensity patterns in B-mode IVUS image. Tissue characterization algorithms have been developed to assist clinicians to identify such heterogeneous tissues and assess plaque vulnerability. In this paper, we propose a novel technique coined as Stochastic Driven Histology (SDH) that is able to provide information about co-located heterogeneous tissues. It employs learning of tissue specific ultrasonic backscattering statistical physics and signal confidence primal from labeled data for predicting heterogeneous tissue composition in plaques. We employ a random forest for the purpose of learning such a primal using sparsely labeled and noisy samples. In clinical deployment, the posterior prediction of different lesions constituting the plaque is estimated. Folded cross-validation experiments have been performed with 53 plaques indicating high concurrence with traditional tissue histology. On the wider horizon, this framework enables learning of tissue-energy interaction statistical physics and can be leveraged for promising clinical applications requiring tissue characterization beyond the application demonstrated in this paper. Copyright © 2013 Elsevier B.V. All rights reserved.

Multispectral fluorescence imaging of human ovarian and Fallopian tube tissue for early stage cancer detection

NASA Astrophysics Data System (ADS)

Tate, Tyler; Baggett, Brenda; Rice, Photini; Watson, Jennifer; Orsinger, Gabe; Nymeyer, Ariel C.; Welge, Weston A.; Keenan, Molly; Saboda, Kathylynn; Roe, Denise J.; Hatch, Kenneth; Chambers, Setsuko; Black, John; Utzinger, Urs; Barton, Jennifer

2015-03-01

With early detection, five year survival rates for ovarian cancer are over 90%, yet no effective early screening method exists. Emerging consensus suggests that perhaps over 50% of the most lethal form of the disease, high grade serous ovarian cancer, originates in the Fallopian tube. Cancer changes molecular concentrations of various endogenous fluorophores. Using specific excitation wavelengths and emissions bands on a Multispectral Fluorescence Imaging (MFI) system, spatial and spectral data over a wide field of view can be collected from endogenous fluorophores. Wavelength specific reflectance images provide additional information to normalize for tissue geometry and blood absorption. Ratiometric combination of the images may create high contrast between neighboring normal and abnormal tissue. Twenty-six women undergoing oophorectomy or debulking surgery consented the use of surgical discard tissue samples for MFI imaging. Forty-nine pieces of ovarian tissue and thirty-two pieces of Fallopian tube tissue were collected and imaged with excitation wavelengths between 280 nm and 550 nm. After imaging, each tissue sample was fixed, sectioned and HE stained for pathological evaluation. Comparison of mean intensity values between normal, benign, and cancerous tissue demonstrate a general trend of increased fluorescence of benign tissue and decreased fluorescence of cancerous tissue when compared to normal tissue. The predictive capabilities of the mean intensity measurements are tested using multinomial logistic regression and quadratic discriminant analysis. Adaption of the system for in vivo Fallopian tube and ovary endoscopic imaging is possible and is briefly described.

Ultrahigh-resolution endoscopic optical coherence tomography

NASA Astrophysics Data System (ADS)

Chen, Yu; Herz, Paul R.; Hsiung, Pei-Lin; Aguirre, Aaron D.; Mashimo, Hiroshi; Desai, Saleem; Pedrosa, Macos; Koski, Amanda; Schmitt, Joseph M.; Fujimoto, James G.

2005-01-01

Early detection of gastrointestinal cancer is essential for the patient treatment and medical care. Endoscopically guided biopsy is currently the gold standard for the diagnosis of early esophageal cancer, but can suffer from high false negative rates due to sampling errors. Optical coherence tomography (OCT) is an emerging medical imaging technology which can generate high resolution, cross-sectional images of tissue in situ and in real time, without the removal of tissue specimen. Although endoscopic OCT has been used successfully to identify certain pathologies in the gastrointestinal tract, the resolution of current endoscopic OCT systems has been limited to 10 - 15 m for clinical procedures. In this study, in vivo imaging of the gastrointestinal tract is demonstrated at a three-fold higher resolution (< 5 m), using a portable, broadband, Cr4+:Forsterite laser as the optical light source. Images acquired from the esophagus, gastro-esophageal junction and colon on animal model display tissue microstructures and architectural details at high resolution, and the features observed in the OCT images are well-matched with histology. The clinical feasibility study is conducted through delivering OCT imaging catheter using standard endoscope. OCT images of normal esophagus, Barrett's esophagus, and esophageal cancers are demonstrated with distinct features. The ability of high resolution endoscopic OCT to image tissue morphology at an unprecedented resolution in vivo would facilitate the development of OCT as a potential imaging modality for early detection of neoplastic changes.

Intraoperative Evaluation of Breast Tumor Margins with Optical Coherence Tomography

PubMed Central

Nguyen, Freddy T.; Zysk, Adam M.; Chaney, Eric J.; Kotynek, Jan G.; Oliphant, Uretz J.; Bellafiore, Frank J.; Rowland, Kendrith M.; Johnson, Patricia A.; Boppart, Stephen A.

2009-01-01

As breast cancer screening rates increase, smaller and more numerous lesions are being identified earlier, leading to more breast-conserving surgical procedures. Achieving a clean surgical margin represents a technical challenge with important clinical implications. Optical coherence tomography (OCT) is introduced as an intraoperative high-resolution imaging technique that assesses surgical breast tumor margins by providing real-time microscopic images up to 2 mm beneath the tissue surface. In a study of 37 patients split between training and study groups, OCT images covering 1 cm2 regions were acquired from surgical margins of lumpectomy specimens, registered with ink, and correlated with corresponding histological sections. A 17 patient training set used to establish standard imaging protocols and OCT evaluation criteria demonstrated that areas of higher scattering tissue with a heterogeneous pattern were indicative of tumor cells and tumor tissue, in contrast to lower scattering adipocytes found in normal breast tissue. The remaining 20 patients were enrolled into the feasibility study. Of these lumpectomy specimens, 11 were identified with a positive or close surgical margin and 9 were identified with a negative margin under OCT. Based on histological findings, 9 true positives, 9 true negatives, 2 false positives, and 0 false negatives were found, yielding a sensitivity of 100% and specificity of 82%. These results demonstrate the potential of OCT as a real-time method for intraoperative margin assessment in breast conserving surgeries. PMID:19910294

Reference spectra from squamous epithelium and connective tissue allow whole section proteomics analysis.

PubMed

Roesch-Ely, Mariana; Schnölzer, Martina; Nees, Matthias; Plinkert, Peter K; Bosch, Franz X

2010-01-01

We reasoned that micro-dissection of tumour cells for protein expression studies should be omitted since tumour-stroma interactions are an important part of the biology of solid tumours. To study such interactions in head and neck squamous cell carcinoma (HNSCC) development, we generated reference protein spectra for normal squamous epithelium and connective tissue by SELDI-TOF-MS. Calgranulins A and B, Annexin1 and Histone H4 were found to be strongly enriched in the epithelium. The alpha-defensins 1-3 and the haemoglobin subunits were identified in the connective tissue. Tumour-distant epithelia, representing early pre-malignant lesions, showed up-regulated expression of the stromal alpha-defensins, whereas the epithelial Annexin 1 was down-regulated. Thus, tumour microenvironment interactions occur very early in the carcinogenic process. These data demonstrate that omitting micro-dissection is actually beneficial for studying changes in protein expression during development and progression of solid tumours.

[Detection of Toxoplasma gondii DNA in human lymph node tissue by in situ hybridization].

PubMed

Liu, C; Ke, O; Tan, D; Zhang, Z

1998-01-01

To detect the presence of Toxoplasma gondii in lymph node tissue in patients with Toxoplasma infection. T. gondii (RH strain) specific DNA fragment clones were obtained by using PCR and gene recombination technique. The DNA fragments used as hybridization probes were labelled with digoxigenin by random primer method. The technique of in situ hybridization (ISH) was used to detect T. g DNA in the lymph node sections. Four out of 120 samples T. g DNA were found positive, one with Hodgkin's disease (HD) (1/32), one with non-Hodgkin's lymphoma (NHL) (1/41) and 2 with chronic lymphadenitis (CL) (2/47). The total positive rate was 3.3%. It was demonstrated that this highly specific probe could detect 10 pg of the total RH strain T. g DNA. ISH was applicable in detecting pathogens in the lymph node tissues of individuals with Toxoplasma infection.

Metabolic changes in psoriatic skin under topical corticosteroid treatment.

PubMed

Sitter, Beathe; Johnsson, Margareta Karin; Halgunset, Jostein; Bathen, Tone Frost

2013-08-14

MR spectroscopy of intact biopsies can provide a metabolic snapshot of the investigated tissue. The aim of the present study was to explore the metabolic pattern of uninvolved skin, psoriatic skin and corticosteroid treated psoriatic skin. The three types of skin biopsy samples were excised from patients with psoriasis (N = 10). Lesions were evaluated clinically, and tissue biopsies were excised and analyzed by one-dimensional 1H MR spectroscopy. Relative levels were calculated for nine tissue metabolites. Subsequently, relative amounts of epidermis, dermis and subcutaneous tissue were scored by histopathological evaluation of HES stained sections. Seven out of 10 patients experienced at least 40% reduction in clinical score after corticosteroid treatment. Tissue biopsies from psoriatic skin contained lower levels of the metabolites myo-inositol and glucose, and higher levels of choline and taurine compared to uninvolved skin. In corticosteroid treated psoriatic skin, tissue levels of glucose, myo-inositol, GPC and glycine were increased, whereas choline was reduced, in patients with good therapeutic effect. These tissue levels are becoming more similar to metabolite levels in uninvolved skin. This MR method demonstrates that metabolism in psoriatic skin becomes similar to that of uninvolved skin after effective corticosteroid treatment. MR profiling of skin lesions reflect metabolic alterations related to pathogenesis and treatment effects.

An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.

PubMed

Amini, Parisa; Ettlin, Julia; Opitz, Lennart; Clementi, Elena; Malbon, Alexandra; Markkanen, Enni

2017-08-23

Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult. We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step. We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.

21 CFR 1271.150 - Current good tissue practice requirements.

Code of Federal Regulations, 2011 CFR

2011-04-01

....150 Section 1271.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.150... implemented for reproductive HCT/Ps described in § 1271.10 and regulated solely under section 361 of the...

21 CFR 1271.150 - Current good tissue practice requirements.

Code of Federal Regulations, 2012 CFR

2012-04-01

....150 Section 1271.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.150... implemented for reproductive HCT/Ps described in § 1271.10 and regulated solely under section 361 of the...

21 CFR 1271.150 - Current good tissue practice requirements.

Code of Federal Regulations, 2013 CFR

2013-04-01

....150 Section 1271.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.150... implemented for reproductive HCT/Ps described in § 1271.10 and regulated solely under section 361 of the...

21 CFR 1271.150 - Current good tissue practice requirements.

Code of Federal Regulations, 2014 CFR

2014-04-01

....150 Section 1271.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.150... implemented for reproductive HCT/Ps described in § 1271.10 and regulated solely under section 361 of the...

Use of cryostat sections from snap-frozen nervous tissue for combining stereological estimates with histological, cellular, or molecular analyses on adjacent sections.

PubMed

Schmitz, C; Dafotakis, M; Heinsen, H; Mugrauer, K; Niesel, A; Popken, G J; Stephan, M; Van de Berg, W D; von Hörsten, S; Korr, H

2000-10-01

Adequate tissue preparation is essential for both modern stereological and immunohistochemical investigations. However, combining these methodologies in a single study presents a number of obstacles pertaining to optimal histological preparation. Tissue shrinkage and loss of nuclei/nucleoli from the unprotected section surfaces of unembedded tissue used for immunohistochemistry may be problematic with regard to adequate stereological design. In this study, frozen cryostat sections from hippocampal and cerebellar regions of two rat strains and cerebellar and cerebral regions from a human brain were analyzed to determine the potential impact of these factors on estimates of neuron number obtained using the optical disector. Neuronal nuclei and nucleoli were clearly present in thin sections of snap-frozen rat (3 microm) and human (6 microm) tissue, indicating that neuronal nuclei/nucleoli are not unavoidably lost from unprotected section surfaces of unembedded tissue. In order to quantify the potential impact of any nuclear loss, optical fractionator estimates of rat hippocampal pyramidal cells in areas CA1-3 and cerebellar granule and Purkinje cells were made using minimal (1 microm) upper guard zones. Estimates did not differ from data reported previously in the literature. This data indicates that cryostat sections of snap-frozen nervous tissue may successfully be used for estimating total neuronal numbers using optical disectors.

Three-photon imaging of ovarian cancer

NASA Astrophysics Data System (ADS)

Barton, Jennifer K.; Amirsolaimani, Babak; Rice, Photini; Hatch, Kenneth; Kieu, Khanh

2016-02-01

Optical imaging methods have the potential to detect ovarian cancer at an early, curable stage. Optical imaging has the disadvantage that high resolution techniques require access to the tissue of interest, but miniature endoscopes that traverse the natural orifice of the reproductive tract, or access the ovaries and fallopian tubes through a small incision in the vagina wall, can provide a minimally-invasive solution. We have imaged both rodent and human ovaries and fallopian tubes with a variety of endoscope-compatible modalities. The recent development of fiber-coupled femtosecond lasers will enable endoscopic multiphoton microscopy (MPM). We demonstrated two- and three-photon excited fluorescence (2PEF, 3PEF), and second- and third-harmonic generation microscopy (SHG, THG) in human ovarian and fallopian tube tissue. A study was undertaken to understand the mechanisms of contrast in these images. Six patients (normal, cystadenoma, and ovarian adenocarcinoma) provided ovarian and fallopian tube biopsies. The tissue was imaged with three-dimensional optical coherence tomography, multiphoton microscopy, and frozen for histological sectioning. Tissue sections were stained with hematoxylin and eosin, Masson's trichrome, and Sudan black. Approximately 1 μm resolution images were obtained with an excitation source at 1550 nm. 2PEF signal was absent. SHG signal was mainly from collagen. 3PEF and THG signal came from a variety of sources, including a strong signal from fatty connective tissue and red blood cells. Adenocarcinoma was characterized by loss of SHG signal, whereas cystic abnormalities showed strong SHG. There was limited overlap of two- and three- photon signals, suggesting that three-photon imaging can provide additional information for early diagnosis of ovarian cancer.

Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae.

PubMed

Chovanec, P; Hovorka, O; Novák, K

2008-01-01

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.

Volumetric laser endomicroscopy in Barrett's esophagus: a feasibility study on histological correlation.

PubMed

Swager, A; Boerwinkel, D F; de Bruin, D M; Weusten, B L; Faber, D J; Meijer, S L; van Leeuwen, T G; Curvers, W L; Bergman, J J

2016-08-01

Volumetric laser endomicroscopy (VLE) is a novel balloon-based optical coherence tomography (OCT) imaging technique that may improve detection of early neoplasia in Barrett's esophagus (BE). Most OCT studies lack a direct correlation between histology and OCT images. The aim is to investigate the optimal approach for achieving one-to-one correlation of ex-vivo VLE images of endoscopic resection (ER) specimens with histology. BE patients with and without early neoplasia underwent ER after delineating areas with electrocoagulation markers (ECM). After ER, specimens underwent additional ex-vivo marking with several different markers (ink, pin, Gold Probe) followed by ex-vivo VLE scanning. ER specimens were carefully sectioned into tissue blocks guided by the markers. Histology and VLE slides were considered a match if ≥ 2 markers were visible on both modalities and mucosal patterns aside from these markers matched on both histology and VLE. From 16 ER specimens 120 tissue blocks were sectioned of which 23 contained multiple markers. Fourteen histology-VLE matches were identified. ECMs and ink markers proved to be the most effective combination for matching. The last 6/16 ER specimens yielded 9/14 matches, demonstrating a learning curve due to methodological improvements in marker placement and tissue block sectioning. One-to-one correlation of VLE and histology is complex but feasible. The groundwork laid in this study will provide high-quality histology-VLE correlations that will allow further research on VLE features of early neoplasia in BE. © 2015 International Society for Diseases of the Esophagus.

A computational framework to detect normal and tuberculosis infected lung from H and E-stained whole slide images

NASA Astrophysics Data System (ADS)

Niazi, M. Khalid Khan; Beamer, Gillian; Gurcan, Metin N.

2017-03-01

Accurate detection and quantification of normal lung tissue in the context of Mycobacterium tuberculosis infection is of interest from a biological perspective. The automatic detection and quantification of normal lung will allow the biologists to focus more intensely on regions of interest within normal and infected tissues. We present a computational framework to extract individual tissue sections from whole slide images having multiple tissue sections. It automatically detects the background, red blood cells and handwritten digits to bring efficiency as well as accuracy in quantification of tissue sections. For efficiency, we model our framework with logical and morphological operations as they can be performed in linear time. We further divide these individual tissue sections into normal and infected areas using deep neural network. The computational framework was trained on 60 whole slide images. The proposed computational framework resulted in an overall accuracy of 99.2% when extracting individual tissue sections from 120 whole slide images in the test dataset. The framework resulted in a relatively higher accuracy (99.7%) while classifying individual lung sections into normal and infected areas. Our preliminary findings suggest that the proposed framework has good agreement with biologists on how define normal and infected lung areas.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens of cartilage tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Constructs grown on Mir (A) tended to become more spherical, whereas those grown on Earth (B) maintained their initial disc shape. These findings might be related to differences in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1g (Earth's gravity). In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion, increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. A and B are full cross sections of constructs from Mir and Earth groups shown at 10-power. C and D are representative areas at the construct surfaces enlarged to 200-power. They are stained red with safranin-O. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Photo credit: Proceedings of the National Academy of Sciences.

Structural locus of transmucosal albumin efflux in canine ileum. A fluorescent study.

PubMed

Granger, D N; Cook, B H; Taylor, A E

1976-12-01

This study demonstrates the effects of elevated intestinal venous pressure on the intestinal tissue spaces and the histological locus of the transmucosal albumin flux under such conditions. The authors were able to localize albumin in the tissues using an Evans blue-albumin fluorescence technique. This technique makes use of the fluorescence properties and albumin affinity of Evans blue dye (T-1824). Evans blue dye has a high affinity for albumin and emits a red-orange fluorescence at a wavelength of 720 nm. Evans blue was mixed with a solution of bovine serum albumin at concentrations that yield negligible amounts of free dye. Control ileal samples were obtained in order to visualize the natural tissue morphology and fluorescence. The Evans blue-albumin solution was injected and tissue samples were obtained 15 and 60 min postinjection, then venous outflow was occluded and after 15 and 60 min the tissues were sampled. Each sample was immediately frozen, freeze dried, embedded in paraffin, and 7-mu sections were made. The Evans blue-albumin was demonstrated histologically with a fluorescence microscope. No leakage sites were apparent at normal venous pressures. However, after elevation of venous pressure, Evans blue-albumin was observed in the interepithelial and/or intraepithelial spaces of villus tips, but no Evans blue-albumin was observed either between or within the epithelial cells of the crypts, or within the tubular crypt lumina. These results indicate that at elevated venous pressures, the transmucosal albumin flux occurs exclusively at the villus tip region, suggesting a great vulnerability of the cells found in this region to elevations in tissue pressure as compared to the crypt epithelial cells.

The use of morphological characteristics and texture analysis in the identification of tissue composition in prostatic neoplasia.

PubMed

Diamond, James; Anderson, Neil H; Bartels, Peter H; Montironi, Rodolfo; Hamilton, Peter W

2004-09-01

Quantitative examination of prostate histology offers clues in the diagnostic classification of lesions and in the prediction of response to treatment and prognosis. To facilitate the collection of quantitative data, the development of machine vision systems is necessary. This study explored the use of imaging for identifying tissue abnormalities in prostate histology. Medium-power histological scenes were recorded from whole-mount radical prostatectomy sections at x 40 objective magnification and assessed by a pathologist as exhibiting stroma, normal tissue (nonneoplastic epithelial component), or prostatic carcinoma (PCa). A machine vision system was developed that divided the scenes into subregions of 100 x 100 pixels and subjected each to image-processing techniques. Analysis of morphological characteristics allowed the identification of normal tissue. Analysis of image texture demonstrated that Haralick feature 4 was the most suitable for discriminating stroma from PCa. Using these morphological and texture measurements, it was possible to define a classification scheme for each subregion. The machine vision system is designed to integrate these classification rules and generate digital maps of tissue composition from the classification of subregions; 79.3% of subregions were correctly classified. Established classification rates have demonstrated the validity of the methodology on small scenes; a logical extension was to apply the methodology to whole slide images via scanning technology. The machine vision system is capable of classifying these images. The machine vision system developed in this project facilitates the exploration of morphological and texture characteristics in quantifying tissue composition. It also illustrates the potential of quantitative methods to provide highly discriminatory information in the automated identification of prostatic lesions using computer vision.

Histologic assessment of mesh fixation following laser-assisted tissue soldering in a lapine model.

PubMed

Lanzafame, Raymond J; Brondon, Philip; Stadler, Istvan; DeVore, Dale P; Soltz, Robert; Soltz, Barbara A

2005-08-01

Wound histology and mesh bioincorporation following intraperitoneal fixation using laser-assisted soldering was evaluated. 2.8-3.2 kg NZW rabbits underwent laparotomy. Controls had 2x2 cm segments of Mersilene stapled to peritoneum. Group 2 segments were affixed with 55% collagen solder onlay by fiber-coupled diode laser (1.43 +/- 10 micro, 2.5 W CW, 4 mm spot, 60 degrees C set temperature). Group 4 had Mersilene inlaid into melted solder. Group 3 had solder-embedded Vicryl mesh affixed. Animals were euthanized at 0, 2, 4, 6 weeks. Fixed sections were assessed for integrity, inflammation, and fibrosis using H & E, Masson's Trichrome and Evans Van Gieson staining. Histology demonstrated cell types, local mesh reaction, and progressive evidence of solder reabsorption mimicking normal healing and bioincorporation. Mersilene groups demonstrated normal arrangement of collagen-rich layers around mesh. Collagen-based tissue soldering permits normal wound healing and may mitigate use of staples. Further development of this strategy is warranted. (c) 2005 Wiley-Liss, Inc.

Grinding and polishing instead of sectioning for the tissue samples with a graft: Implications for light and electron microscopy.

PubMed

Mukhamadiyarov, Rinat A; Sevostyanova, Victoria V; Shishkova, Daria K; Nokhrin, Andrey V; Sidorova, Olga D; Kutikhin, Anton G

2016-06-01

A broad use of the graft replacement requires a detailed investigation of the host-graft interaction, including both histological examination and electron microscopy. A high quality sectioning of the host tissue with a graft seems to be complicated; in addition, it is difficult to examine the same tissue area by both of the mentioned microscopy techniques. To solve these problems, we developed a new technique of epoxy resin embedding with the further grinding, polishing, and staining. Graft-containing tissues prepared by grinding and polishing preserved their structure; however, sectioning frequently required the explantation of the graft and led to tissue disintegration. Moreover, stained samples prepared by grinding and polishing may then be assessed by both light microscopy and backscattered scanning electron microscopy. Therefore, grinding and polishing outperform sectioning when applied to the tissues with a graft. Copyright © 2016 Elsevier Ltd. All rights reserved.

Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation

PubMed Central

Einaga, Naoki; Yoshida, Akio; Noda, Hiroko; Suemitsu, Masaaki; Nakayama, Yuki; Sakurada, Akihisa; Kawaji, Yoshiko; Yamaguchi, Hiromi; Sasaki, Yasushi; Tokino, Takashi; Esumi, Mariko

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied. PMID:28498833

Improved resolution by mounting of tissue sections for laser microdissection.

PubMed

van Dijk, M C R F; Rombout, P D M; Dijkman, H B P M; Ruiter, D J; Bernsen, M R

2003-08-01

Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor. To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated. Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue. The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser. The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.

Improved resolution by mounting of tissue sections for laser microdissection

PubMed Central

van Dijk, M C R F; Rombout, P D M; Dijkman, H B P M; Ruiter, D J; Bernsen, M R

2003-01-01

Background: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor. Aims: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated. Methods: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10–2000 cells isolated by microdissection from mounted and unmounted tissue. Results: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser. Conclusions: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted. PMID:12890747

CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker

PubMed Central

Molano, Monica; Tabrizi, Sepehr N.; Garland, Suzanne M.; Roberts, Jennifer M.; Machalek, Dorothy A.; Phillips, Samuel; Chandler, David; Hillman, Richard J.; Grulich, Andrew E.; Jin, Fengyi; Poynten, I. Mary; Templeton, David J.; Cornall, Alyssa M.

2016-01-01

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL. PMID:27529629

CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker.

PubMed

Molano, Monica; Tabrizi, Sepehr N; Garland, Suzanne M; Roberts, Jennifer M; Machalek, Dorothy A; Phillips, Samuel; Chandler, David; Hillman, Richard J; Grulich, Andrew E; Jin, Fengyi; Poynten, I Mary; Templeton, David J; Cornall, Alyssa M

2016-01-01

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.

Concentrations of cadmium and selected essential elements in malignant large intestine tissue

PubMed Central

Dziki, Adam; Kilanowicz, Anna; Sapota, Andrzej; Duda-Szymańska, Joanna; Daragó, Adam

2015-01-01

Introduction Colorectal cancer is one of the most common cancers worldwide. Incidence rates of large intestine cancer indicate a role of environmental and occupational factors. The role of essential elements and their interaction with toxic metals can contribute to the explanation of a complex mechanism by which large intestine cancer develops. Bearing this in mind, determining the levels of essential and toxic elements in tissues (organs), as well as in body fluids, seems to shed light on their role in the mode of action in malignant disease. Aim Determination of the levels of cadmium, zinc, copper, selenium, calcium, magnesium, and iron in large intestine malignant tissue. Material and methods Two intraoperative intestine sections were investigated: one from the malignant tissue and the other one from the normal tissue, collected from each person with diagnosed large intestine cancer. Cadmium, zinc, copper, calcium, magnesium, and iron levels were determined with atomic absorption spectrometry, and selenium levels by spectrofluorimetric method. Results The levels of copper, selenium, and magnesium were higher in the malignant than in normal tissues. In addition, the zinc/copper and calcium/magnesium relationship was altered in malignant tissue, where correlations were lower compared to non-malignant tissue. Conclusions The results seems to demonstrate disturbed homeostasis of some essential elements. However, it is hard to confirm their involvement in the aetiology of colorectal cancer. PMID:27110307

Multiview hyperspectral topography of tissue structural and functional characteristics

NASA Astrophysics Data System (ADS)

Zhang, Shiwu; Liu, Peng; Huang, Jiwei; Xu, Ronald

2012-12-01

Accurate and in vivo characterization of structural, functional, and molecular characteristics of biological tissue will facilitate quantitative diagnosis, therapeutic guidance, and outcome assessment in many clinical applications, such as wound healing, cancer surgery, and organ transplantation. However, many clinical imaging systems have limitations and fail to provide noninvasive, real time, and quantitative assessment of biological tissue in an operation room. To overcome these limitations, we developed and tested a multiview hyperspectral imaging system. The multiview hyperspectral imaging system integrated the multiview and the hyperspectral imaging techniques in a single portable unit. Four plane mirrors are cohered together as a multiview reflective mirror set with a rectangular cross section. The multiview reflective mirror set was placed between a hyperspectral camera and the measured biological tissue. For a single image acquisition task, a hyperspectral data cube with five views was obtained. The five-view hyperspectral image consisted of a main objective image and four reflective images. Three-dimensional topography of the scene was achieved by correlating the matching pixels between the objective image and the reflective images. Three-dimensional mapping of tissue oxygenation was achieved using a hyperspectral oxygenation algorithm. The multiview hyperspectral imaging technique is currently under quantitative validation in a wound model, a tissue-simulating blood phantom, and an in vivo biological tissue model. The preliminary results have demonstrated the technical feasibility of using multiview hyperspectral imaging for three-dimensional topography of tissue functional properties.

3D imaging of cleared human skin biopsies using light-sheet microscopy: A new way to visualize in-depth skin structure.

PubMed

Abadie, S; Jardet, C; Colombelli, J; Chaput, B; David, A; Grolleau, J-L; Bedos, P; Lobjois, V; Descargues, P; Rouquette, J

2018-05-01

Human skin is composed of the superimposition of tissue layers of various thicknesses and components. Histological staining of skin sections is the benchmark approach to analyse the organization and integrity of human skin biopsies; however, this approach does not allow 3D tissue visualization. Alternatively, confocal or two-photon microscopy is an effective approach to perform fluorescent-based 3D imaging. However, owing to light scattering, these methods display limited light penetration in depth. The objectives of this study were therefore to combine optical clearing and light-sheet fluorescence microscopy (LSFM) to perform in-depth optical sectioning of 5 mm-thick human skin biopsies and generate 3D images of entire human skin biopsies. A benzyl alcohol and benzyl benzoate solution was used to successfully optically clear entire formalin fixed human skin biopsies, making them transparent. In-depth optical sectioning was performed with LSFM on the basis of tissue-autofluorescence observations. 3D image analysis of optical sections generated with LSFM was performed by using the Amira ® software. This new approach allowed us to observe in situ the different layers and compartments of human skin, such as the stratum corneum, the dermis and epidermal appendages. With this approach, we easily performed 3D reconstruction to visualise an entire human skin biopsy. Finally, we demonstrated that this method is useful to visualise and quantify histological anomalies, such as epidermal hyperplasia. The combination of optical clearing and LSFM has new applications in dermatology and dermatological research by allowing 3D visualization and analysis of whole human skin biopsies. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Towards High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry Coupled to Shear Force Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Son N.; Sontag, Ryan L.; Carson, James P.

Constant mode ambient mass spectrometry imaging (MSI) of tissue sections with high lateral resolution of better than 10 µm was performed by combining shear force microscopy with nanospray desorption electrospray ionization (nano-DESI). Shear force microscopy enabled precise control of the distance between the sample and nano-DESI probe during MSI experiments and provided information on sample topography. Proof-of-concept experiments were performed using lung and brain tissue sections representing spongy and dense tissues, respectively. Topography images obtained using shear force microscopy were comparable to the results obtained using contact profilometry over the same region of the tissue section. Variations in tissue heightmore » were found to be dependent on the tissue type and were in the range of 0-5 µm for lung tissue and 0-3 µm for brain tissue sections. Ion images of phospholipids obtained in this study are in good agreement with literature data. Normalization of nano-DESI MSI images to the signal of the internal standard added to the extraction solvent allowed us to construct high-resolution ion images free of matrix effects.« less

Diagnostic utility of melanin production by fungi: study on tissue sections and culture smears with Masson-Fontana stain.

PubMed

Sundaram, Challa; Shantveer, G Uppin; Umabala, Pamidi; Lakshmi, Vemu

2014-01-01

Dematiaceous fungi appear brown in tissue section due to melanin in their cell walls. When the brown color is not seen on routine H and E and culture is not available, differentiation of dematiaceous fungi from other fungi is difficult on morphology alone. To study if melanin production by dematiaceous fungi can help differentiate them from other types of fungi. Fifty tissue sections of various fungal infections and 13 smears from cultures of different species of fungi were stained with Masson Fontana stain to assess melanin production. The tissue sections included biopsies from 26 culture-proven fungi and 24 biopsies of filamentous fungi diagnosed on morphology alone with no culture confirmation. All culture-proven dematiaceous fungi and Zygomycetes showed strong positivity in sections and culture smears. Aspergillus sp showed variable positivity and intensity. Cryptococcus neoformans showed strong positivity in tissue sections and culture smears. Tissue sections of septate filamentous fungi (9/15), Zygomycetes (4/5), and fungi with both hyphal and yeast morphology (4/4) showed positivity for melanin. The septate filamentous fungi negative for melanin were from biopsy samples of fungal sinusitis including both allergic and invasive fungal sinusitis and colonizing fungal balls. Melanin is produced by both dematiaceous and non-dematiaceous fungi. Masson-Fontana stain cannot reliably differentiate dematiaceous fungi from other filamentous fungi like Aspergillus sp; however, absence of melanin in the hyphae may be used to rule out dematiaceous fungi from other filamentous fungi. In the differential diagnosis of yeast fungi, Cryptococcus sp can be differentiated from Candida sp by Masson-Fontana stain in tissue sections.

Model development and experimental validation for analyzing initial transients of irradiation of tissues during thermal therapy using short pulse lasers.

PubMed

Ganguly, Mohit; Miller, Stephanie; Mitra, Kunal

2015-11-01

Short pulse lasers with pulse durations in the range of nanoseconds and shorter are effective in the targeted delivery of heat energy for precise tissue heating and ablation. This photothermal therapy is useful where the removal of cancerous tissue sections is required. The objective of this paper is to use finite element modeling to demonstrate the differences in the thermal response of skin tissue to short-pulse and continuous wave laser irradiation in the initial stages of the irradiation. Models have been developed to validate the temperature distribution and heat affected zone during laser irradiation of excised rat skin samples and live anesthetized mouse tissue. Excised rat skin samples and live anesthetized mice were subjected to Nd:YAG pulsed laser (1,064 nm, 500 ns) irradiation of varying powers. A thermal camera was used to measure the rise in surface temperature as a result of the laser irradiation. Histological analyses of the heat affected zone created in the tissue samples due to the temperature rise were performed. The thermal interaction of the laser with the tissue was quantified by measuring the thermal dose delivered by the laser. Finite element geometries of three-dimensional tissue sections for continuum and vascular models were developed using COMSOL Multiphysics. Blood flow was incorporated into the vascular model to mimic the presence of discrete blood vessels and contrasted with the continuum model without blood perfusion. The temperature rises predicted by the continuum and the vascular models agreed with the temperature rises observed at the surface of the excised rat tissue samples and live anesthetized mice due to laser irradiation respectively. The vascular model developed was able to predict the cooling produced by the blood vessels in the region where the vessels were present. The temperature rise in the continuum model due to pulsed laser irradiation was higher than that due to continuous wave (CW) laser irradiation in the initial stages of the irradiation. The temperature rise due to pulsed and CW laser irradiation converged as the time of irradiation increased. A similar trend was observed when comparing the thermal dose for pulsed and CW laser irradiation in the vascular model. Finite element models (continuum and vascular) were developed that can be used to predict temperature rise and quantify the thermal dose resulting from laser irradiation of excised rat skin samples and live anesthetized mouse tissue. The vascular model incorporating blood perfusion effects predicted temperature rise better in the live animal tissue. The models developed demonstrated that pulsed lasers caused greater temperature rise and delivered a greater thermal dose than CW lasers of equal average power, especially during the initial transients of irradiation. This analysis will be beneficial for thermal therapy applications where maximum delivery of thermal dose over a short period of time is important. © 2015 Wiley Periodicals, Inc.

Multimodal ophthalmic imaging using spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography

NASA Astrophysics Data System (ADS)

El-Haddad, Mohamed T.; Malone, Joseph D.; Li, Jianwei D.; Bozic, Ivan; Arquitola, Amber M.; Joos, Karen M.; Patel, Shriji N.; Tao, Yuankai K.

2017-08-01

Ophthalmic surgery involves manipulation of delicate, layered tissue structures on milli- to micrometer scales. Traditional surgical microscopes provide an inherently two-dimensional view of the surgical field with limited depth perception which precludes accurate depth-resolved visualization of these tissue layers, and limits the development of novel surgical techniques. We demonstrate multimodal swept-source spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography (SS-SESLO-OCT) to address current limitations of image-guided ophthalmic microsurgery. SS-SESLO-OCT provides inherently co-registered en face and cross-sectional field-of-views (FOVs) at a line rate of 400 kHz and >2 GPix/s throughput. We show in vivo imaging of the anterior segment and retinal fundus of a healthy volunteer, and preliminary results of multi-volumetric mosaicking for ultrawide-field retinal imaging with 90° FOV. Additionally, a scan-head was rapid-prototyped with a modular architecture which enabled integration of SS-SESLO-OCT with traditional surgical microscope and slit-lamp imaging optics. Ex vivo surgical maneuvers were simulated in cadaveric porcine eyes. The system throughput enabled volumetric acquisition at 10 volumes-per-second (vps) and allowed visualization of surgical dynamics in corneal sweeps, compressions, and dissections, and retinal sweeps, compressions, and elevations. SESLO en face images enabled simple real-time co-registration with the surgical microscope FOV, and OCT cross-sections provided depth-resolved visualization of instrument-tissue interactions. Finally, we demonstrate novel augmented-reality integration with the surgical view using segmentation overlays to aid surgical guidance. SS-SESLO-OCT may benefit clinical diagnostics by enabling aiming, registration, and mosaicking; and intraoperative imaging by allowing for real-time surgical feedback, instrument tracking, and overlays of computationally extracted biomarkers of disease.

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis

PubMed Central

YANG, ZHIZHOU; SUN, ZHAORUI; LIU, HONGMEI; REN, YI; SHAO, DANBING; ZHANG, WEI; LIN, JINFENG; WOLFRAM, JOY; WANG, FENG; NIE, SHINAN

2015-01-01

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson’s trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury. PMID:25815693

Diode laser prostatectomy (VLAP): initial canine evaluation

NASA Astrophysics Data System (ADS)

Kopchok, George E.; Verbin, Chris; Ayres, Bruce; Peng, Shi-Kaung; White, Rodney A.

1995-05-01

This study evaluated the acute and chronic effects of diode laser (960 nm) prostatectomy using a Prolase II fiber in a canine model (n equals 5). The laser fiber consists of a 1000 um quartz fiber which reflects a cone of laser energy, at 45 degree(s) to the axis of the fiber, into the prostatic urethra (Visual Laser Ablation of Prostate). Perineal access was used to guide a 15.5 Fr cystoscope to the level of the prostate. Under visual guidance and continual saline irrigation, 60 watts of laser power was delivered for 60 seconds at 3, 9, and 12 o'clock and 30 seconds at the 6 o'clock (posterior) positions for a total energy fluence of 12,600 J. One prostate received an additional 60 second exposure at 3 and 9 o'clock for a total fluence of 19,800 J. The prostates were evaluated at one day (n equals 1) and 8 weeks (n equals 4). The histopathology of laser effects at one day show areas of necrosis with loss of glandular structures and stromal edema. Surrounding this area was a zone of degenerative glandular structures extending up to 17.5 mm (cross sectional diameter). The histopathology of the 8 week laser treated animals demonstrated dilated prostatic urethras with maximum cross- sectional diameter of 23.4 mm (mean equals 18.5 +/- 3.9 mm). This study demonstrates the effectiveness of diode laser energy for prostatic tissue coagulation and eventual sloughing. The results also demonstrate the safety of diode laser energy, with similar tissue response as seen with Nd:YAG laser, for laser prostatectomy.

Study on nasopharyngeal cancer tissue using surface-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Ge, Xiaosong; Lin, Xueliang; Xu, Zhihong; Wei, Guoqiang; Huang, Wei; Lin, Duo

2016-10-01

Surface-enhanced Raman spectroscopy (SERS) can provide detailed molecular structure and composition information, and has demonstrated great potential in biomedical filed. This spectroscopy technology has become one of the most important optical techniques in the early diagnosis of cancer. Nasopharyngeal cancer (NPC) is a malignant neoplasm arising in the nasopharyngeal epithelial lining, which has relatively high incidence and death rate in Southeast Asia and southern China. This paper reviews the current progress of SERS in the field of cancer diagnostics, including gastric cancer, colorectal cancer, cervical cancer and nasopharyngeal cancer. In addition to above researches, we recently develop a novel NPC detection method based on tissue section using SERS, and obtain primary results. The proposed method has promising potential for the detection of nasopharyngeal carcinoma.

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

Endomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast wide-field imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated.

Electrochemical removal of metallic implants from Technovit 9100 New embedded hard and soft tissues prior to histological sectioning.

PubMed

Willbold, Elmar; Reebmann, Mattias; Jeffries, Richard; Witte, Frank

2013-11-01

Solid metallic implants in soft or hard tissues are serious challenges for histological processing. However, metallic implants are more frequently used in e.g. cardiovascular or orthopaedic therapies. Before clinical use, these devices need to be tested thoroughly in a biological environment and histological analysis of their biocompatibility is a major requirement. To allow the histological analysis of metallic implants in tissues especially in calcified hard tissues, we describe a method for embedding these tissues in the resin Technovit 9100 New and removing the metallic implants by electrochemical dissolution. With the combination of these two processes, we are able to achieve 5 μm thick sections from soft or hard tissues with a superior preservation of tissue architecture and especially the implant-tissue interface. These sections can be stained by classical stainings, immunohistochemical and enzymehistochemical as well as DNA-based staining methods.

A high-resolution optical imaging system for obtaining the serial transverse section images of biologic tissue

NASA Astrophysics Data System (ADS)

Wu, Li; Zhang, Bin; Wu, Ping; Liu, Qian; Gong, Hui

2007-05-01

A high-resolution optical imaging system was designed and developed to obtain the serial transverse section images of the biologic tissue, such as the mouse brain, in which new knife-edge imaging technology, high-speed and high-sensitive line-scan CCD and linear air bearing stages were adopted and incorporated with an OLYMPUS microscope. The section images on the tip of the knife-edge were synchronously captured by the reflection imaging in the microscope while cutting the biologic tissue. The biologic tissue can be sectioned at interval of 250 nm with the same resolution of the transverse section images obtained in x and y plane. And the cutting job can be automatically finished based on the control program wrote specially in advance, so we save the mass labor of the registration of the vast images data. In addition, by using this system a larger sample can be cut than conventional ultramicrotome so as to avoid the loss of the tissue structure information because of splitting the tissue sample to meet the size request of the ultramicrotome.

A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.

PubMed

Koh, James; Hogue, Joyce A; Sosa, Julie A

2016-01-01

The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo therapeutic testing. This highly adaptable system provides a unique platform for live-cell ex vivo provocative testing of human tumor responsiveness to a range of physiological agonists or candidate therapeutic compounds.

Tactile mapping system: a novel imaging technology for surface topography and elasticity of tissues or organs.

PubMed

Oie, Tomonori; Suzuki, Hisato; Fukuda, Toru; Murayama, Yoshinobu; Omata, Sadao; Kanda, Keiichi; Nakayama, Yasuhide

2009-11-01

: We demonstrated that the tactile mapping system (TMS) has a high degree of spatial precision in the distribution mapping of surface elasticity of tissues or organs. : Samples used were a circumferential section of a small-caliber porcine artery (diameter: ∼3 mm) and an elasticity test pattern with a line and space configuration for the distribution mapping of elasticity, prepared by regional micropatterning of a 14-μm thick gelatin hydrogel coating on a polyurethane sheet. Surface topography and elasticity in normal saline were simultaneously investigated by TMS using a probe with a diameter of 5 or 12 μm, a spatial interval of 1 to 5 μm, and an indentation depth of 4 μm. : In the test pattern, a spatial resolution in TMS of <5 μm was acquired under water with a minimal probe diameter and spatial interval of the probe movement. TMS was used for the distribution mapping of surface elasticity in a flat, circumferential section (thickness: ∼0.5 mm) of a porcine artery, and the concentric layers of the vascular wall, including the collagen-rich and elastin-rich layers, could be clearly differentiated in terms of surface elasticity at the spatial resolution of <2 μm. : TMS is a simple and inexpensive technique for the distribution mapping of the surface elasticity in vascular tissues at the spatial resolution <2 μm. TMS has the ability to analyze a complex structure of the tissue samples under normal saline.

UV-laser-based microscopic dissection of tree rings - a novel sampling tool for δ(13) C and δ(18) O studies.

PubMed

Schollaen, Karina; Heinrich, Ingo; Helle, Gerhard

2014-02-01

UV-laser-based microscopic systems were utilized to dissect and sample organic tissue for stable isotope measurements from thin wood cross-sections. We tested UV-laser-based microscopic tissue dissection in practice for high-resolution isotopic analyses (δ(13) C/δ(18) O) on thin cross-sections from different tree species. The method allows serial isolation of tissue of any shape and from millimetre down to micrometre scales. On-screen pre-defined areas of interest were automatically dissected and collected for mass spectrometric analysis. Three examples of high-resolution isotopic analyses revealed that: in comparison to δ(13) C of xylem cells, woody ray parenchyma of deciduous trees have the same year-to-year variability, but reveal offsets that are opposite in sign depending on whether wholewood or cellulose is considered; high-resolution tree-ring δ(18) O profiles of Indonesian teak reflect monsoonal rainfall patterns and are sensitive to rainfall extremes caused by ENSO; and seasonal moisture signals in intra-tree-ring δ(18) O of white pine are weighted by nonlinear intra-annual growth dynamics. The applications demonstrate that the use of UV-laser-based microscopic dissection allows for sampling plant tissue at ultrahigh resolution and unprecedented precision. This new technique facilitates sampling for stable isotope analysis of anatomical plant traits like combined tree eco-physiological, wood anatomical and dendroclimatological studies. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Adenosine A2A Receptor Blockade or Deletion Diminishes Fibrocyte Accumulation in the Skin in a Murine Model of Scleroderma, Bleomycin-induced Fibrosis

PubMed Central

Katebi, Majid; Fernandez, Patricia; Chan, Edwin S. L.; Cronstein, Bruce N.

2015-01-01

Peripheral blood fibrocytes are a newly identified circulating leukocyte subpopulation that migrates into injured tissue where it may display fibroblast-like properties and participate in wound healing and fibrosis of skin and other organs. Previous studies in our lab demonstrated that A2A receptor-deficient and A2A antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis, thus the aim of this study was to determine whether the adenosine A2A receptor regulates recruitment of fibrocytes to the dermis in this bleomycin-induced model of dermal fibrosis. Sections of skin from normal mice and bleomycin-treated wild type, A2A knockout and A2A antagonist-treated mice were stained for Procollagen α2 Type I and CD34 and the double stained cells, fibrocytes, were counted in the tissue sections. There were more fibrocytes in the dermis of bleomycin-treated mice than normal mice and the increase was abrogated by deletion or blockade of adenosine A2A receptors. Because fibrocytes play a central role in tissue fibrosis these results suggest that diminished adenosine A2A receptor-mediated recruitment of fibrocytes into tissue may play a role in the pathogenesis of fibrosing diseases of the skin. Moreover, these results provide further evidence that adenosine A2A receptors may represent a new target for the treatment of such fibrosing diseases as scleroderma or nephrogenic fibrosing dermopathy. PMID:18709547

78 FR 3437 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-16

... tissue sections for diagnosing cancer by using the differences in spatial positioning of certain genes... and cancerous human breast tissue section images that can be used for large experiments. Additionally.... Supervised learning framework for screening nuclei in tissue [[Page 3438

[Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots].

PubMed

Rafalovskaia-Orlovskaia, E P; Gorgidze, L A; Gladkikh, A A; Tauger, S M; Vorob'ev, I A

2012-01-01

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.

HIV increased the release of dickkopf-1 (DKK1) protein from human astrocytes by a Cx43 hemichannel-dependent mechanism

PubMed Central

Orellana, Juan Andres; Sáez, Juan Carlos; Bennett, Michael Vander Lann; Berman, Joan Weinberger; Morgello, Susan; Eugenin, Eliseo Alberto

2015-01-01

Human immunodeficiency virus-1 (HIV) is a public health issue and a major complication of the disease is NeuroAIDS. In vivo, microglia/macrophages are the main cells infected. However, a low but significant number of HIV infected astrocytes also has been detected, but their role in the pathogenesis of NeuroAIDS is not well understood. Our previous data indicates that gap junction channels amplify toxicity from few HIV infected into uninfected astrocytes. Now, we demonstrated that HIV infection of astrocytes results in opening of connexin43 hemichannels (Cx43 HCs). HIV induced opening of Cx43 HCs resulted in dysregulated secretion of dickkopf-1 protein (DKK1, a soluble wnt pathway inhibitor). Treatment of mixed cultures of neurons and astrocytes with DKK1, in the absence of HIV infection, resulted in collapse of neuronal processes. HIV infection of mixed cultures of human neurons and astrocytes also resulted in collapse of neuronal processes by a DKK1 dependent mechanism. In addition, dysregulated DKK1 expression in astrocytes was observed in human brain tissue sections of individuals with HIV encephalitis as compared to tissue sections from uninfected individuals. Thus, we demonstrated that HIV infection of astrocytes induces dysregulation of DKK1 by a HC-dependent mechanism that contributes to the brain pathogenesis observed in HIV infected individuals. PMID:24134157

Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

NASA Astrophysics Data System (ADS)

Xu, Zhang; Reilley, Michael; Li, Run; Xu, Min

2017-06-01

We report chemometric wide-field fluorescence microscopy for imaging the spatial distribution and concentration of endogenous fluorophores in thin tissue sections. Nonnegative factorization aided by spatial diversity is used to learn both the spectral signature and the spatial distribution of endogenous fluorophores from microscopic fluorescence color images obtained under broadband excitation and detection. The absolute concentration map of individual fluorophores is derived by comparing the fluorescence from "pure" fluorophores under the identical imaging condition following the identification of the fluorescence species by its spectral signature. This method is then demonstrated by characterizing the concentration map of endogenous fluorophores (including tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide) for lung tissue specimens. The absolute concentrations of these fluorophores are all found to decrease significantly from normal, perilesional, to cancerous (squamous cell carcinoma) tissue. Discriminating tissue types using the absolute fluorophore concentration is found to be significantly more accurate than that achievable with the relative fluorescence strength. Quantification of fluorophores in terms of the absolute concentration map is also advantageous in eliminating the uncertainties due to system responses or measurement details, yielding more biologically relevant data, and simplifying the assessment of competing imaging approaches.

A finite element model to study the effect of tissue anisotropy on ex vivo arterial shear wave elastography measurements

NASA Astrophysics Data System (ADS)

Shcherbakova, D. A.; Debusschere, N.; Caenen, A.; Iannaccone, F.; Pernot, M.; Swillens, A.; Segers, P.

2017-07-01

Shear wave elastography (SWE) is an ultrasound (US) diagnostic method for measuring the stiffness of soft tissues based on generated shear waves (SWs). SWE has been applied to bulk tissues, but in arteries it is still under investigation. Previously performed studies in arteries or arterial phantoms demonstrated the potential of SWE to measure arterial wall stiffness—a relevant marker in prediction of cardiovascular diseases. This study is focused on numerical modelling of SWs in ex vivo equine aortic tissue, yet based on experimental SWE measurements with the tissue dynamically loaded while rotating the US probe to investigate the sensitivity of SWE to the anisotropic structure. A good match with experimental shear wave group speed results was obtained. SWs were sensitive to the orthotropy and nonlinearity of the material. The model also allowed to study the nature of the SWs by performing 2D FFT-based and analytical phase analyses. A good match between numerical group velocities derived using the time-of-flight algorithm and derived from the dispersion curves was found in the cross-sectional and axial arterial views. The complexity of solving analytical equations for nonlinear orthotropic stressed plates was discussed.

A novel slice preparation to study medullary oromotor and autonomic circuits in vitro

PubMed Central

Nasse, Jason S.

2014-01-01

Background The medulla is capable of controlling and modulating ingestive behavior and gastrointestinal function. These two functions, which are critical to maintaining homeostasis, are governed by an interconnected group of nuclei dispersed throughout the medulla. As such, in vitro experiments to study the neurophysiologic details of these connections have been limited by spatial constraints of conventional slice preparations. New method This study demonstrates a novel method of sectioning the medulla so that sensory, integrative, and motor nuclei that innervate the gastrointestinal tract and the oral cavity remain intact. Results: Immunohistochemical staining against choline-acetyl-transferase and dopamine-β-hydroxylase demonstrated that within a 450 μm block of tissue we are able to capture sensory, integrative and motor nuclei that are critical to oromotor and gastrointestinal function. Within slice tracing shows that axonal projections from the NST to the reticular formation and from the reticular formation to the hypoglossal motor nucleus (mXII) persist. Live-cell calcium imaging of the slice demonstrates that stimulation of either the rostral or caudal NST activates neurons throughout the NST, as well as the reticular formation and mXII. Comparison with existing methods This new method of sectioning captures a majority of the nuclei that are active when ingesting a meal. Tradition planes of section, i.e. coronal, horizontal or sagittal, contain only a limited portion of the substrate. Conclusions Our results demonstrate that both anatomical and physiologic connections of oral and visceral sensory nuclei that project to integrative and motor nuclei remain intact with this new plane of section. PMID:25196216

A novel slice preparation to study medullary oromotor and autonomic circuits in vitro.

PubMed

Nasse, Jason S

2014-11-30

The medulla is capable of controlling and modulating ingestive behavior and gastrointestinal function. These two functions, which are critical to maintaining homeostasis, are governed by an interconnected group of nuclei dispersed throughout the medulla. As such, in vitro experiments to study the neurophysiologic details of these connections have been limited by spatial constraints of conventional slice preparations. This study demonstrates a novel method of sectioning the medulla so that sensory, integrative, and motor nuclei that innervate the gastrointestinal tract and the oral cavity remain intact. Immunohistochemical staining against choline-acetyl-transferase and dopamine-β-hydroxylase demonstrated that within a 450 μm block of tissue we are able to capture sensory, integrative and motor nuclei that are critical to oromotor and gastrointestinal function. Within slice tracing shows that axonal projections from the NST to the reticular formation and from the reticular formation to the hypoglossal motor nucleus (mXII) persist. Live-cell calcium imaging of the slice demonstrates that stimulation of either the rostral or caudal NST activates neurons throughout the NST, as well as the reticular formation and mXII. This new method of sectioning captures a majority of the nuclei that are active when ingesting a meal. Tradition planes of section, i.e. coronal, horizontal or sagittal, contain only a limited portion of the substrate. Our results demonstrate that both anatomical and physiologic connections of oral and visceral sensory nuclei that project to integrative and motor nuclei remain intact with this new plane of section. Published by Elsevier B.V.

Spatial cluster analysis of nanoscopically mapped serotonin receptors for classification of fixed brain tissue

NASA Astrophysics Data System (ADS)

Sams, Michael; Silye, Rene; Göhring, Janett; Muresan, Leila; Schilcher, Kurt; Jacak, Jaroslaw

2014-01-01

We present a cluster spatial analysis method using nanoscopic dSTORM images to determine changes in protein cluster distributions within brain tissue. Such methods are suitable to investigate human brain tissue and will help to achieve a deeper understanding of brain disease along with aiding drug development. Human brain tissue samples are usually treated postmortem via standard fixation protocols, which are established in clinical laboratories. Therefore, our localization microscopy-based method was adapted to characterize protein density and protein cluster localization in samples fixed using different protocols followed by common fluorescent immunohistochemistry techniques. The localization microscopy allows nanoscopic mapping of serotonin 5-HT1A receptor groups within a two-dimensional image of a brain tissue slice. These nanoscopically mapped proteins can be confined to clusters by applying the proposed statistical spatial analysis. Selected features of such clusters were subsequently used to characterize and classify the tissue. Samples were obtained from different types of patients, fixed with different preparation methods, and finally stored in a human tissue bank. To verify the proposed method, samples of a cryopreserved healthy brain have been compared with epitope-retrieved and paraffin-fixed tissues. Furthermore, samples of healthy brain tissues were compared with data obtained from patients suffering from mental illnesses (e.g., major depressive disorder). Our work demonstrates the applicability of localization microscopy and image analysis methods for comparison and classification of human brain tissues at a nanoscopic level. Furthermore, the presented workflow marks a unique technological advance in the characterization of protein distributions in brain tissue sections.

Negating Tissue Contracture Improves Volume Maintenance and Longevity of In Vivo Engineered Tissues.

PubMed

Lytle, Ian F; Kozlow, Jeffrey H; Zhang, Wen X; Buffington, Deborah A; Humes, H David; Brown, David L

2015-10-01

Engineering large, complex tissues in vivo requires robust vascularization to optimize survival, growth, and function. Previously, the authors used a "chamber" model that promotes intense angiogenesis in vivo as a platform for functional three-dimensional muscle and renal engineering. A silicone membrane used to define the structure and to contain the constructs is successful in the short term. However, over time, generated tissues contract and decrease in size in a manner similar to capsular contracture seen around many commonly used surgical implants. The authors hypothesized that modification of the chamber structure or internal surface would promote tissue adherence and maintain construct volume. Three chamber configurations were tested against volume maintenance. Previously studied, smooth silicone surfaces were compared to chambers modified for improved tissue adherence, with multiple transmembrane perforations or lined with a commercially available textured surface. Tissues were allowed to mature long term in a rat model, before analysis. On explantation, average tissue masses were 49, 102, and 122 mg; average volumes were 74, 158 and 176 μl; and average cross-sectional areas were 1.6, 6.7, and 8.7 mm for the smooth, perforated, and textured groups, respectively. Both perforated and textured designs demonstrated significantly greater measures than the smooth-surfaced constructs in all respects. By modifying the design of chambers supporting vascularized, three-dimensional, in vivo tissue engineering constructs, generated tissue mass, volume, and area can be maintained over a long time course. Successful progress in the scale-up of construct size should follow, leading to improved potential for development of increasingly complex engineered tissues.

Quantitative shear-wave optical coherence elastography with a programmable phased array ultrasound as the wave source.

PubMed

Song, Shaozhen; Le, Nhan Minh; Huang, Zhihong; Shen, Tueng; Wang, Ruikang K

2015-11-01

The purpose of this study is to implement a beam-steering ultrasound as the wave source for shear-wave optical coherence elastography (SW-OCE) to achieve an extended range of elastic imaging of the tissue sample. We introduce a linear phased array ultrasound transducer (LPAUT) as the remote and programmable wave source and a phase-sensitive optical coherence tomography (OCT) as the sensitive shear-wave detector. The LPAUT is programmed to launch acoustic radiation force impulses (ARFI) focused at desired locations within the range of OCT imaging, upon which the elasticity map of the entire OCT B-scan cross section is recovered by spatial compounding of the elastic maps derived from each launch of AFRIs. We also propose a directional filter to separate the shear-wave propagation at different directions in order to reduce the effect of tissue heterogeneity on the shear-wave propagation within tissue. The feasibility of this proposed approach is then demonstrated by determining the stiffness of tissue-mimicking phantoms with agarose concentrations of 0.5% and 1% and also by imaging the Young's modulus of retinal and choroidal tissues within a porcine eye ball ex vivo. The approach opens up opportunities to combine medical ultrasound imaging and SW-OCE for high-resolution localized quantitative assessment of tissue biomechanical property.

In vivo microwave-based thermoacoustic tomography of rats (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lin, Li; Zhou, Yong; Wang, Lihong V.

2016-03-01

Microwave-based thermoacoustic tomography (TAT), based on the measurement of ultrasonic waves induced by microwave pulses, can reveal tissue dielectric properties that may be closely related to the physiological and pathological status of the tissues. Using microwaves as the excitation source improved imaging depth because of their deep penetration into biological tissues. We demonstrate, for the first time, in vivo microwave-based thermoacoustic imaging in rats. The transducer is rotated around the rat in a full circle, providing a full two-dimensional view. Instead of a flat ultrasonic transducer, we used a virtual line detector based on a cylindrically focused transducer. A 3 GHz microwave source with 0.6 µs pulse width and an electromagnetically shielded transducer with 2.25 MHz central frequency provided clear cross-sectional images of the rat's body. The high imaging contrast, based on the tissue's rate of absorption, and the ultrasonically defined spatial resolution combine to reveal the spine, kidney, muscle, and other deeply seated anatomical features in the rat's abdominal cavity. This non-invasive and non-ionizing imaging modality achieved an imaging depth beyond 6 cm in the rat's tissue. Cancer diagnosis based on information about tissue properties from microwave band TAT can potentially be more accurate than has previously been achievable.

Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

PubMed Central

Sarkar, Purbasha; Bosneaga, Elena; Yap, Edgar G.; Das, Jyotirmoy; Tsai, Wen-Ting; Cabal, Angelo; Neuhaus, Erica; Maji, Dolonchampa; Kumar, Shailabh; Joo, Michael; Yakovlev, Sergey; Csencsits, Roseann; Yu, Zeyun; Bajaj, Chandrajit; Downing, Kenneth H.; Auer, Manfred

2014-01-01

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls. PMID:25207917

Self-spectral calibration for spectral domain optical coherence tomography

NASA Astrophysics Data System (ADS)

Zhang, Xianling; Gao, Wanrong; Bian, Haiyi; Chen, Chaoliang; Liao, Jiuling

2013-06-01

A different real-time self-wavelength calibration method for spectral domain optical coherence tomography is presented in which interference spectra measured from two arbitrary points on the tissue surface are used for calibration. The method takes advantages of two favorable conditions of optical coherence tomography (OCT) signal. First, the signal back-scattered from the tissue surface is generally much stronger than that from positions in the tissue interior, so the spectral component of the surface interference could be extracted from the measured spectrum. Second, the tissue surface is not a plane and a phase difference exists between the light reflected from two different points on the surface. Compared with the zero-crossing automatic method, the introduced method has the advantage of removing the error due to dispersion mismatch or the common phase error. The method is tested experimentally to demonstrate the improved signal-to-noise ratio, higher axial resolution, and slower sensitivity degradation with depth when compared to the use of the zero-crossing method and applied to two-dimensional cross-sectional images of human finger skin.

Effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in the digestive system of tilapia (Oreochromis niloticus).

PubMed

Dai, Wei; Du, Huahua; Fu, Linglin; Jin, Chengguan; Xu, Zirong; Liu, Huitao

2009-02-01

With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-microg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia.

In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics

PubMed Central

Grundberg, Ida; Kiflemariam, Sara; Mignardi, Marco; Imgenberg-Kreuz, Juliana; Edlund, Karolina; Micke, Patrick; Sundström, Magnus; Sjöblom, Tobias

2013-01-01

Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy. PMID:24280411

Distribution of Particles in the Z-axis of Tissue Sections: Relevance for Counting Methods.

PubMed

von Bartheld, Christopher S

2012-01-01

The distribution of particles in the z-axis of thick tissue sections has gained considerable attention, primarily because of implications for the accuracy of modern stereological counting methods. Three major types of artifacts can affect these sections: loss of particles from the surfaces of tissue sections (lost caps), homogeneous collapse in the z-axis, and differential deformation in the z-axis. Initially it was assumed that thick sections were not compromised by differential shrinkage or compression (differential uniform deformation). Studies in the last decade showed that such artifacts are common and that they depend on embedding media and sectioning devices. Paraffin, glycolmethacrylate and vibratome sections are affected by this artifact, but not celloidin sections or cryostat-derived cryosections. Differential distribution of particles in the z-axis is likely due to compression of the surface areas (margins) during sectioning, resulting in differential particle densities in the core and margin of tissue sections. This deformation of tissue sections can be rapidly assessed by measuring the position of particles in the z-axis. The analysis is complicated by potential secondary effects on section surfaces through loss of particles, the so-called "lost caps" phenomenon. Secondary effects necessitate the use of guard spaces, while their use in case of primary effects (compression due to sectioning) would enhance the artifact's impact on bias. Symmetric versus asymmetric patterns of z-axis distortion can give clues to distinguish primary and secondary effects. Studies that use the optical disector need to take these parameters into account to minimize biases.

Silica Encapsulated Gold Nanoparticles as SERS Labels for the Detection of Lymphoma B-Cells in Tissue Sections

NASA Astrophysics Data System (ADS)

Al-Faouri, Tamara

The surface of silica encapsulated gold nanoparticles with trans-1,2-bis (4-pyridyl) ethylene Raman active dye were utilized as SERS labels to target CD20 surface protein on lymphoma B-cells in human tissue sections with CLL or FL. SERS labels were functionalized with various antibody linkers including carboxylic, aldehyde, and heterobifunctional PEG chains with an NHS end, to permit them to bind to tissue section samples. NP samples and tissue sections were characterized through UV-Vis spectroscopy, TEM, XPS, Zeta potential measurements, Dark Field microscopy, Raman spectroscopy, NMR, and AFM. The number of SERS labels present on a tissue sample was estimated using dark field images and a particle counting software. It was found that the heterobifunctional PEG chains linker provided the most specific binding of SERS labels with an estimated NP count of 1.33x106 NPs on the whole tissue and produced the highest Raman scatter intensity of approximately 48600 counts.

Fluorescent Staining of Tea Pathogenic Fungi in Tea Leaves Using Fluorescein-labeled Lectin

NASA Astrophysics Data System (ADS)

Yamada, Kengo; Yoshida, Katsuyuki; Sonoda, Ryoichi

Fluorochrome-labeled lectin, fluorescein conjugated wheat germ agglutinin (F-WGA) was applied to stain tea pathogenic fungi in tea leaf tissue. Infected leaves were fixed and decolorized with a mixture of ethanol and acetic acid, and cleared with 10% KOH for whole mount before staining with F-WGA. Hyphae of Pestalotiopsis longiseta, Pseudocercospora ocellata, Botrytis cinerea and Colletotrichum theae-sinensis fluoresced brightly in whole mount and sectioned samples of infected leaf tissue. In browned tissue, hyphae did not fluoresce frequently in whole mount sample. Autofluorescence of leaf tissue was strong in browned tissue of sections, it was removed by 10% KOH treatment before staining. Penetration hyphae of C. theae-sinensis in cell wall of trichome and hyphae in basal part of trichome did not fluoresced frequently. In whole mount samples of tea leaf infected with Exobasidium vexans and E. reticulatum, hymenia appeared on leaf surface fluoresced, but hyphae in leaf tissue did not fluoresce. In sectioned samples, hyphae fluoresced brightly when sections were treated with 10% KOH before staining.

Low vacuum scanning electron microscopy for paraffin sections utilizing the differential stainability of cells and tissues with platinum blue.

PubMed

Inaga, Sumire; Hirashima, Sayuri; Tanaka, Keiichi; Katsumoto, Tetsuo; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

2009-07-01

The present study introduces a novel method for the direct observation of histological paraffin sections by low vacuum scanning electron microscopy (LVSEM) with platinum blue (Pt-blue) treatment. Pt-blue was applied not only as a backscattered electron (BSE) signal enhancer but also as a histologically specific stain. In this method, paraffin sections of the rat tongue prepared for conventional light microscopy (LM) were stained on glass slides with a Pt-blue staining solution (pH 9) and observed in a LVSEM using BSE detector. Under LVSEM, overviews of whole sections as well as three-dimensional detailed observations of individual cells and tissues could be easily made at magnifications from x40 to x10,000. Each kind of cell and tissue observed in the section could be clearly distinguished due to the different yields of BSE signals, which depended on the surface structures and different affinities to Pt-blue. Thus, we roughly classified cellular and tissue components into three groups according to the staining intensity of Pt-blue observed by LM and LVSEM: 1) a strongly stained (deep blue by LM and brightest by LVSEM) group which included epithelial tissue, endothelium and mast cells; 2) a moderately stained (light blue and bright) group which included muscular tissue and nervous tissue; 3) an unstained or weakly stained (colorless and dark) group which included elastic fibers and collagen fibers. We expect that this method will prove useful for the three-dimensional direct observation of histological paraffin sections of various tissues by LVSEM with higher resolutions than LM.

Functional anatomy of the nasal bones and adjacent structures. Consequences for nasal surgery.

PubMed

Popko, M; Verlinde-Schellekens, S A M W; Huizing, E H; Bleys, R L A W

2018-03-01

The periosteum of the nasal bones, the periosteal-perichondrial nasal envelope, and the cartilaginous support of the bony vault were studied in serial coronal sections of four human cadaver noses. To differentiate between the various tissue components, the sections were stained according to Mallory-Cason and Verhoeff-Van Gieson stain. The results demonstrated: 1. the presence of clearly distinguishable layers of the periosteum covering the nasal bones; 2. the presence of a continuous periosteal-perichondrial covering of the bony and cartilaginous nasal vaults; 3. the way the cartilaginous support of the bony vault is constructed. The findings described in the present study may have clinical relevance in nasal surgery.

Expression and clinical significance of PIWIL2 in hilar cholangiocarcinoma tissues and cell lines.

PubMed

Chen, Y J; Xiong, X F; Wen, S Q; Tian, L; Cheng, W L; Qi, Y Q

2015-06-26

The objective of this study was to explore the relationship between PIWI-like protein 2 (PIWIL2) and clinicopathological charac-teristics and prognosis after radical resection. To accomplish this, we analyzed PIWIL2 expression in hilar cholangiocarcinoma tissues and cell lines. PIWIL2 expression was detected by immunohistochemistry in 41 hilar cholangiocarcinoma samples and 10 control tissues. Western blotting and immunocytofluorescence were used to investigate PIWIL2 expression in the cholangiocarcinoma cell line QBC939 and the bile duct epithelial cell line HIBEpic. Univariate and multivariate surviv-al analyses were performed using the Kaplan-Meier method for hilar cholangiocarcinoma patients who underwent radical resection. PIWIL2 expression was significantly higher in the hilar cholangiocarcinoma tissues and QBC939 cells than in control tissues and HIBEpic cells, respectively (P < 0.05). Poorly and moderately differentiated cholan-giocarcinoma tissues had significantly higher PIWIL2 expression than well-differentiated tissues (P < 0.05). Univariate analysis demonstrated that high PIWIL2 expression was associated with shorter survival time after radical resection (P < 0.05). Multivariate analysis showed that PI-WIL2 expression was an independent prognostic factor after radical re-section of hilar cholangiocarcinoma (P < 0.05). PIWIL2 expression was also associated with tumor-node-metastasis stage and differentiation. PIWIL2 was an independent prognostic factor after radical resection of hilar cholangiocarcinoma.

Comparative evaluation of diagnostic tests for the detection of Mycobacterium avium subsp. paratuberculosis in the tissues of sheep affected with distinct pathology of paratuberculosis.

PubMed

Sonawane, Ganesh G; Tripathi, Bhupendra N

2016-12-01

Paratuberculosis or Johne's disease is a chronic infectious granulomatous enteritis, mainly of cattle, sheep, goats, and other domestic and wild animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). Currently, MAP has been recognized as an important animal pathogen with significant zoonotic and public health concerns. The early detection of infected animals using suitable diagnostic methods helps in developing control and preventive strategies for the herd. Therefore, the present study was aimed to determine the comparative efficacy of certain diagnostic methods used in the identification and confirmation of MAP in the ovine tissues with distinct pathology of paratuberculosis. The ileum and mesenteric lymph node (MLN) tissues were collected from 38 sheep infected with paratuberculosis from organized farms of Rajasthan. These animals were further classified as paucibacillary (PB; n=15) or multibacillary (MB; n=23) on the basis of histopathological findings and mycobacterial loads. The ileum and MLN tissues of these animals were subjected to IS900 and 251 gene polymerase chain reaction (PCR) and bacterial culture. The tissue sections from MB, PB, and uninfected control sheep groups were stained using indirect immunoperoxidase technique (IPT) and Ziehl-Neelsen (ZN) method. On bacterial culture examination of the ileum and MLN tissues using Herrold's egg yolk medium, MAP was isolated in 14 (60.9%) MB and 5 (33.3%) PB sheep. Of 38 sheep, IS900 PCR detected 21 (55.2%) positive for MAP, of which 19 (82.6%) were MB and 2 (13.3%) were PB sheep. Similarly, 251 gene PCR detected 25 (65.7%) sheep positive for MAP infection, of which 21 (91.3%) were MB and 4 (27%) were PB sheep. Thus, 251 gene PCR was found superior to IS900 PCR in the detection of MAP from the tissues. In PB sheep, IPT and ZN tests were positive in 87.5% and 50% of ileum sections and 70% and 37.5% in MLN sections, respectively. In MB sheep, IPT and ZN tests detected all animals as positive for MAP organisms or antigen and had equal sensitivity in the detection of MAP. The overall sensitivity of IPT was found superior (95%) to ZN staining (80%) in the demonstration of acid-fast bacteria or its antigen in the tissues. The sensitivity of all the tests in the detection of MAP was lower in PB sheep than in MB sheep. Bacterial culture detected MAP in only 50% of sheep and was found less sensitive than other tests used in the present study. Comparing the overall sensitivity of both the PCR assays, 251 gene PCR was found superior to IS900 gene PCR. The sensitivity of IPT was found superior (95%) to the ZN staining (80%) in the demonstration of acid-fast bacteria in the tissues. In the present study, IPT was found superior in the detection of MAP in PB and MB form of ovine paratuberculosis. This test can be used in the confirmation of post mortem diagnosis and research of paratuberculosis along with histopathology. However, 251 gene PCR assay was found easier to perform than IPT and could be used as paratuberculosis screening test in endemic sheep farms for blood and fecal samples. Copyright © 2016.

Detection of the multiphoton signals in stained tissue using nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Zeng, Yaping; Xu, Jian; Kang, Deyong; Lin, Jiangbo; Chen, Jianxin

2016-10-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging, has become a powerful, important tool for tissue imaging at the molecular level. Recently, MPM is also used to image hematoxylin and eosin (H and E)-stained sections in cancer diagnostics. However, several studies have showed that the MPM images of tissue stained with H and E are significantly different from unstained tissue sections. Our aim was to detect of the multiphoton signals in stained tissue by using MPM. In this paper, MPM was used to image histological sections of esophageal invasive carcinoma tissues stained with H, E, H and E and fresh tissue. To detect of the multiphoton signals in stained tissue, the emission spectroscopic of tissue stained with H, E, H and E were obtained. For comparison, the fresh tissues were also investigated. Our results showed that the tissue stained with H, E, H and E could be detected by their TPEF signals. While the tissue stained with H and fresh tissue could be detected by their TPEF and SHG signals. In this work, we detect of the multiphoton signals in stained tissue. These findings will be useful for choosing suitable staining method so to improve the quality of MPM imaging in the future.

Preclinical evaluation of nuclear morphometry and tissue topology for breast carcinoma detection and margin assessment

PubMed Central

Nyirenda, Ndeke; Farkas, Daniel L.

2010-01-01

Prevention and early detection of breast cancer are the major prophylactic measures taken to reduce the breast cancer related mortality and morbidity. Clinical management of breast cancer largely relies on the efficacy of the breast-conserving surgeries and the subsequent radiation therapy. A key problem that limits the success of these surgeries is the lack of accurate, real-time knowledge about the positive tumor margins in the surgically excised tumors in the operating room. This leads to tumor recurrence and, hence, the need for repeated surgeries. Current intraoperative techniques such as frozen section pathology or touch imprint cytology severely suffer from poor sampling and non-optimal detection sensitivity. Even though histopathology analysis can provide information on positive tumor margins post-operatively (~2–3 days), this information is of no immediate utility in the operating rooms. In this article, we propose a novel image analysis method for tumor margin assessment based on nuclear morphometry and tissue topology and demonstrate its high sensitivity/specificity in preclinical animal model of breast carcinoma. The method relies on imaging nuclear-specific fluorescence in the excised surgical specimen and on extracting nuclear morphometric parameters (size, number, and area fraction) from the spatial distribution of the observed fluorescence in the tissue. We also report the utility of tissue topology in tumor margin assessment by measuring the fractal dimension in the same set of images. By a systematic analysis of multiple breast tissues specimens, we show here that the proposed method is not only accurate (~97% sensitivity and 96% specificity) in thin sections, but also in three-dimensional (3D) thick tissues that mimic the realistic lumpectomy specimens. Our data clearly precludes the utility of nuclear size as a reliable diagnostic criterion for tumor margin assessment. On the other hand, nuclear area fraction addresses this issue very effectively since it is a combination of both nuclear size and count in any given region of the analyzed image, and thus yields high sensitivity and specificity (~97%) in tumor detection. This is further substantiated by an independent parameter, fractal dimension, based on the tissue topology. Although the basic definition of cancer as an uncontrolled cell growth entails a high nuclear density in tumor regions, a simple but systematic exploration of nuclear distribution in thick tissues by nuclear morphometry and tissue topology as performed in this study has never been carried out, to the best of our knowledge. We discuss the practical aspects of implementing this imaging approach in automated tissue sampling scenario where the accuracy of tumor margin assessment can be significantly increased by scanning the entire surgical specimen rather than sampling only a few sections as in current histopathology analysis. PMID:20446030

Cell-based and biomaterial approaches to connective tissue repair

NASA Astrophysics Data System (ADS)

Stalling, Simone Suzette

Connective tissue injuries of skin, tendon and ligament, heal by a reparative process in adults, filling the wound site with fibrotic, disorganized scar tissue that poorly reflects normal tissue architecture or function. Conversely, fetal skin and tendon have been shown to heal scarlessly. Complete regeneration is not intrinsically ubiquitous to all fetal tissues; fetal diaphragmatic and gastrointestinal injuries form scars. In vivo studies suggest that the presence of fetal fibroblasts is essential for scarless healing. In the orthopaedic setting, adult anterior cruciate ligament (ACL) heals poorly; however, little is known about the regenerative capacity of fetal ACL or fetal ACL fibroblasts. We characterized in vitro wound healing properties of fetal and adult ACL fibroblasts demonstrating that fetal ACL fibroblasts migrate faster and elaborate greater quantities of type I collagen, suggesting the healing potential of the fetal ACL may not be intrinsically poor. Similar to fetal ACL fibroblasts, fetal dermal fibroblasts also exhibit robust cellular properties. We investigated the age-dependent effects of dermal fibroblasts on tendon-to-bone healing in rat supraspinatus tendon injuries, a reparative injury model. We hypothesized delivery of fetal dermal fibroblasts would increase tissue organization and mechanical properties in comparison to adult dermal fibroblasts. However, at 1 and 8 weeks, the presence of dermal fibroblasts, either adult or fetal, had no significant effect on tissue histology or mechanical properties. There was a decreasing trend in cross-sectional area of repaired tendons treated with fetal dermal fibroblasts in comparison to adult, but this finding was not significant in comparison to controls. Finally, we synthesized a novel polysaccharide, methacrylated methylcellulose (MA-MC), and fabricated hydrogels using a well-established photopolymerization technique. We characterized the physical and mechanical properties of MA-MC hydrogels in vitro as well as in a subcutaneous mouse model. Stable MA-MC hydrogels, of varying weight percentages, demonstrated tunable swelling and mechanical properties in the absence of cytotoxic degradation products. In vivo, 6wt% MA-MC hydrogels maintained their shape and mechanical integrity while eliciting a minimal inflammatory response; highly desirable properties for soft tissue reconstruction. These cellulose-based photopolymerizable hydrogels can be further optimized for drug delivery and tissue engineering applications to enhance wound repair.

A simple method for maintaining relative positions of separate tissue elements during processing for electron microscopy.

PubMed

Stirling, C A

1978-09-01

Molten (328 K) 20% gelatin is used as a 'glue' to hold together separate tissue elements or tissue elements that may be separated when cutting small blocks of tissue for plastic embedding. Standard aldehyde and osmium fixation, dehydration and epoxy embedding are compatible with this as is semi-thin sectioning for light microscopy or thin sectioning for electron microscopy.

Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery.

PubMed

Bennàssar, A; Vilata, A; Puig, S; Malvehy, J

2014-02-01

Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P < 0·001). The results demonstrate the feasibility of confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours. © 2013 British Association of Dermatologists.

Quantitative analysis of transcranial and intraparenchymal light penetration in human cadaver brain tissue.

PubMed

Tedford, Clark E; DeLapp, Scott; Jacques, Steven; Anders, Juanita

2015-04-01

Photobiomodulation (PBM) also known as low-level light therapy has been used successfully for the treatment of injury and disease of the nervous system. The use of PBM to treat injury and diseases of the brain requires an in-depth understanding of light propagation through tissues including scalp, skull, meninges, and brain. This study investigated the light penetration gradients in the human cadaver brain using a Transcranial Laser System with a 30 mm diameter beam of 808 nm wavelength light. In addition, the wavelength-dependence of light scatter and absorbance in intraparenchymal brain tissue using 660, 808, and 940 nm wavelengths was investigated. Intact human cadaver heads (n = 8) were obtained for measurement of light propagation through the scalp/skull/meninges and into brain tissue. The cadaver heads were sectioned in either the transverse or mid-sagittal. The sectioned head was mounted into a cranial fixture with an 808 nm wavelength laser system illuminating the head from beneath with either pulsed-wave (PW) or continuous-wave (CW) laser light. A linear array of nine isotropic optical fibers on a 5 mm pitch was inserted into the brain tissue along the optical axis of the beam. Light collected from each fiber was delivered to a multichannel power meter. As the array was lowered into the tissue, the power from each probe was recorded at 5 mm increments until the inner aspect of the dura mater was reached. Intraparenchymal light penetration measurements were made by delivering a series of wavelengths (660, 808, and 940 nm) through a separate optical fiber within the array, which was offset from the array line by 5 mm. Local light penetration was determined and compared across the selected wavelengths. Unfixed cadaver brains provide good anatomical localization and reliable measurements of light scatter and penetration in the CNS tissues. Transcranial application of 808 nm wavelength light penetrated the scalp, skull, meninges, and brain to a depth of approximately 40 mm with an effective attenuation coefficient for the system of 2.22 cm(-1) . No differences were observed in the results between the PW and CW laser light. The intraparenchymal studies demonstrated less absorption and scattering for the 808 nm wavelength light compared to the 660 or 940 nm wavelengths. Transcranial light measurements of unfixed human cadaver brains allowed for determinations of light penetration variables. While unfixed human cadaver studies do not reflect all the conditions seen in the living condition, comparisons of light scatter and penetration and estimates of fluence levels can be used to establish further clinical dosing. The 808 nm wavelength light demonstrated superior CNS tissue penetration. © 2015 Wiley Periodicals, Inc.

Volumetric structured illumination microscopy enabled by tunable focus lens (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hinsdale, Taylor; Malik, Bilal; Olsovsky, Cory; Jo, Javier A.; Maitland, Kristen C.

2016-03-01

We present a volumetric imaging method for biological tissue that is free of mechanically scanning components. The optical sectioning in the system is obtained by structured illumination microscopy (SIM) with the depth of focus being varied by the use of an electronic tunable-focus lens (ETL). The performance of the axial scanning mechanism was evaluated and characterized in conjunction with SIM to ensure volumetric images could be recorded and reconstructed without significant losses in optical section thickness and lateral resolution over the full desired scan range. It was demonstrated that sub-cellular image resolutions were obtainable in both microsphere films and in ex vivo oral mucosa, spanning multiple cell layers, without significant losses in image quality. The mechanism proposed here has the ability to be integrated into any wide-field microscopy system to convert it into a three-dimensional imaging platform without the need for axial scanning of the sample or imaging optics. The ability to axially scan independent of mechanical movement also provides the opportunity for the development of endoscopic systems which can create volumetric images of tissue in vivo.

Automated Liquid Microjunction Surface Sampling-HPLC-MS/MS Analysis of Drugs and Metabolites in Whole-Body Thin Tissue Sections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kertesz, Vilmos; Van Berkel, Gary J

A fully automated liquid extraction-based surface sampling system utilizing a commercially available autosampler coupled to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection is reported. Discrete spots selected for droplet-based sampling and automated sample queue generation for both the autosampler and MS were enabled by using in-house developed software. In addition, co-registration of spatially resolved sampling position and HPLC-MS information to generate heatmaps of compounds monitored for subsequent data analysis was also available in the software. The system was evaluated with whole-body thin tissue sections from propranolol dosed rat. The hands-free operation of the system was demonstrated by creating heatmapsmore » of the parent drug and its hydroxypropranolol glucuronide metabolites with 1 mm resolution in the areas of interest. The sample throughput was approximately 5 min/sample defined by the time needed for chromatographic separation. The spatial distributions of both the drug and its metabolites were consistent with previous studies employing other liquid extraction-based surface sampling methodologies.« less

Nondestructive tissue analysis for ex vivo and in vivo cancer diagnosis using a handheld mass spectrometry system

PubMed Central

Zhang, Jialing; Rector, John; Lin, John Q.; Young, Jonathan H.; Sans, Marta; Katta, Nitesh; Giese, Noah; Yu, Wendong; Nagi, Chandandeep; Suliburk, James; Liu, Jinsong; Bensussan, Alena; DeHoog, Rachel J.; Garza, Kyana Y.; Ludolph, Benjamin; Sorace, Anna G.; Syed, Anum; Zahedivash, Aydin; Milner, Thomas E.; Eberlin, Livia S.

2018-01-01

Conventional methods for histopathologic tissue diagnosis are labor- and time-intensive and can delay decision-making during diagnostic and therapeutic procedures. We report the development of an automated and biocompatible handheld mass spectrometry device for rapid and nondestructive diagnosis of human cancer tissues. The device, named MasSpec Pen, enables controlled and automated delivery of a discrete water droplet to a tissue surface for efficient extraction of biomolecules. We used the MasSpec Pen for ex vivo molecular analysis of 20 human cancer thin tissue sections and 253 human patient tissue samples including normal and cancerous tissues from breast, lung, thyroid, and ovary. The mass spectra obtained presented rich molecular profiles characterized by a variety of potential cancer biomarkers identified as metabolites, lipids, and proteins. Statistical classifiers built from the histologically validated molecular database allowed cancer prediction with high sensitivity (96.4%), specificity (96.2%), and overall accuracy (96.3%), as well as prediction of benign and malignant thyroid tumors and different histologic subtypes of lung cancer. Notably, our classifier allowed accurate diagnosis of cancer in marginal tumor regions presenting mixed histologic composition. Last, we demonstrate that the MasSpec Pen is suited for in vivo cancer diagnosis during surgery performed in tumor-bearing mouse models, without causing any observable tissue harm or stress to the animal. Our results provide evidence that the MasSpec Pen could potentially be used as a clinical and intraoperative technology for ex vivo and in vivo cancer diagnosis. PMID:28878011

Rotator cuff tear measurement by arthropneumotomography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kilcoyne, R.F.; Matsen, F.A. III

1983-02-01

Five years of experience with a method of shoulder arthrography using upright tomography in cases of suspected or known rotator cuff tears has demonstrated its effectiveness. The value of the procedure lies in its ability to demonstrate the size of the cuff tear and the thickness of the remaining cuff tissue. This information provides the surgeon with a preoperative estimate of the difficulty of the repair and the prognosis for a good functional recovery. In 33 cases, there was good correlation between the upright thin-section tomogram findings and the surgical results. The tomograms provided better information about the size ofmore » the tear and the quality of the remaining cuff than did plain arthrograms.« less

Chromium mapping in male mice reproductive glands exposed to CrCl 3 using proton and X-ray synchrotron radiation microbeams

NASA Astrophysics Data System (ADS)

Ortega, R.; Devès, G.; Bonnin-Mosbah, M.; Salomé, M.; Susini, J.; Anderson, L. M.; Kasprzak, K. S.

2001-07-01

Preconception exposure to certain chemicals may increase risk of tumors in offspring, especially with regard to occupational metals such as chromium. However, the mechanism of chromium trans-generation carcinogenicity remains unknown. Using scanning proton X-ray microanalysis we have been able to detect chromium in testicular tissue sections from mice treated by intraperitoneal injection of 1 mmol/kg CrCl 3. Chromium concentration was about 5 μg/g dry mass in average, but higher concentrations were found within the limiting membrane of the testes, the tunica albuginea. In addition, synchrotron radiation X-ray fluorescence measurements, with microscopic resolution, clearly demonstrated the presence of chromium in the tunica albuginea but also within isolated cells from the interstitial connective tissue.

The biomechanics of leg ulceration.

PubMed Central

Chant, A.

1999-01-01

Research performed in the late 1960s, using 24Na, suggested that the perfusion of skin and subcutaneous tissues is critically dependent on the relationship between capillary (Pc) and tissue pressures (Pt). Perfusion changes differed significantly between controls and patients with venous disease and the differences could be interpreted as evidence that Pt remained high in venous diseased patients. From this starting point, a biomechanical theory for the aetiology of venous ulceration was developed and tested by measuring skin elasticity, limb cross-sectional area and laser Doppler flux. The results confirm that, modelled as a two-compartment system (vascular and interstitial fluid), forces can be demonstrated sufficient to cause intermittent capillary closure and subsequent reperfusion injury. These forces are maximal in the gaiter area, the site of most leg ulcers. Images Figure 2 Figure 4 PMID:10364960

A novel protocol for generating intact, whole-head spider cephalothorax tissue sections.

PubMed

Long, Skye M

2018-04-01

The diversity of spider behavior and sensory systems provides an excellent opportunity for comparative studies of the relationship between the brain and behavior. However, the morphology of spiders poses a challenge for histologists since the spider cephalothorax contains heterogeneous tissues and has both tough external and internal sclerotized regions. Unlike the heads of insects, the cephalothorax is highly pressurized, which can cause tissues to shift during processing and can reduce tissue cohesion in thick sections. This work describes a novel protocol for producing thick whole-head sections for morphological study by softening the exoskeleton and stabilizing friable tissue, without freezing or dehydration. It also presents an effective whole-head DiI staining method that uses minimal dehydration and highlights neural structures.

Microgravity

NASA Image and Video Library

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens of cartilage tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Constructs grown on Mir (A) tended to become more spherical, whereas those grown on Earth (B) maintained their initial disc shape. These findings might be related to differences in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1g (Earth's gravity). In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion, increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. A and B are full cross sections of constructs from Mir and Earth groups shown at 10-power. C and D are representative areas at the construct surfaces enlarged to 200-power. They are stained red with safranin-O. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Photo credit: Proceedings of the National Academy of Sciences.

Cholecystokinin receptors on gallbladder muscle and pancreatic acinar cells: a comparative study

DOE Office of Scientific and Technical Information (OSTI.GOV)

von Schrenck, T.; Moran, T.H.; Heinz-Erian, P.

1988-10-01

To compare receptors for cholecystokinin (CCK) in pancreas and gallbladder, we measured binding of 125I-Bolton-Hunter-labeled CCK-8 (125I-BH-CCK-8) to tissue sections from guinea pig gallbladder and pancreas under identical conditions. In both tissues, binding had similar time-, temperature-, and pH dependence, was reversible, saturable and inhibited only by CCK related peptides or CCK receptor antagonists. Autoradiography localized 125I-BH-CCK-8 binding to the smooth muscle layer in the gallbladder. Binding of 125I-BH-CCK-8 to gallbladder sections was inhibited by various agonists with the following potencies (IC50):CCK-8 (0.4 nM) greater than des(SO3)CCK-8 (0.07 microM) greater than gastrin-17-I (1.7 +/- 0.3 microM) and by various receptormore » antagonists with the following potencies: L364,718 (1.5 nM) greater than CR 1409 (0.19 microM) greater than asperlicin = CBZ-CCK-(27-32)-NH2 (1 microM) greater than Bt2cGMP (120 microM). Similar potencies were found for the agonists and antagonists for pancreas sections. Inhibition of binding of 125I-BH-CCK-8 by 11 different analogues of proglumide gave similar potencies for both pancreas and gallbladder. The potencies of agonists in stimulating and antagonists in inhibiting CCK-stimulated contraction or amylase release correlated closely with their abilities to inhibit 125I-BH-CCK-8 binding to gallbladder or pancreas sections or acini, respectively. The present results demonstrate and characterize a method that can be used to compare the CCK receptors in guinea pig gallbladder and pancreas under identical conditions. Moreover, this study demonstrates that gallbladder and pancreatic CCK receptors have similar affinities for the various agonists and antagonists tested and, therefore, provides no evidence that they represent different subtypes of CCK receptors that can be distinguished pharmacologically.« less

Penile histomorphometrical evaluation in hypertensive rats treated with sildenafil or enalapril alone or in combination: a comparison with normotensive and untreated hypertensive rats.

PubMed

Felix-Patrício, Bruno; Medeiros, Jorge L; De Souza, Diogo B; Costa, Waldemar S; Sampaio, Francisco J B

2015-01-01

Erectile dysfunction (ED) is frequently associated to hypertension and antihypertensive drugs; however, the penile morphological aspects on these situations are poorly known. Evaluate the penile morphology of untreated hypertensive rats and rats treated with enalapril or sildenafil alone or in combination to verify the hypothesis that morphological alterations promoted by hypertension on corpus cavernosum could be ameliorated by the use of angiotensin-converting enzyme inhibitors and/or phosphodiesterase type 5 inhibitors. Fifty male rats were assigned into five groups: normotensive rats, untreated spontaneously hypertensive rats (SHRs), and SHR treated with enalapril or sildenafil alone or in combination. Blood pressure was measured weekly. At the conclusion of the study, the rats were euthanized, and their penises were collected for histomorphometrical analysis. The cross-sectional areas of the penis, tunica albuginea, and corpus cavernosum were measured. The density of the corpus cavernosum structures was quantified. Both groups of SHR rats treated with enalapril became normotensive. Untreated SHR showed no difference in penile and cavernosal cross-sectional area compared with normotensive rats; however, those rats treated with enalapril or sildenafil alone demonstrated an increase in these parameters. Rats receiving combination therapy showed no cross-sectional area differences compared with normotensive rats. Cavernosal connective tissue density was increased, while the sinusoidal spaces were diminished in untreated SHR. All treatments were effective in maintaining connective tissue density in comparison with normotensive animals. Cavernosal smooth muscle density was similar in all groups, with the exception of the combination therapy group, which demonstrated a reduction in smooth muscle. Hypertension promoted structural alterations in the corpus cavernosum that may be related to ED. Enalapril- and sildenafil-treated animals had preservation of normal corpus cavernosum structure and an increase in penile and cavernosal cross-sectional area. The combination of these drugs showed less benefit than individual use. © 2014 International Society for Sexual Medicine.

Raman spectroscopic analysis of human skin tissue sections ex-vivo: evaluation of the effects of tissue processing and dewaxing

NASA Astrophysics Data System (ADS)

Ali, Syed M.; Bonnier, Franck; Tfayli, Ali; Lambkin, Helen; Flynn, Kathleen; McDonagh, Vincent; Healy, Claragh; Clive Lee, T.; Lyng, Fiona M.; Byrne, Hugh J.

2013-06-01

Raman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections and the effects of tissue processing. Both hand and thigh sections of human cadavers were analyzed in their unprocessed and formalin-fixed, paraffin-processed (FFPP), and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum (SC), intermediate underlying epithelium, and dermal layers for sections from both anatomical sites. The SC is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that of the dermis is dominated by the characteristics of collagen. For a given anatomical site, little difference in layer structure and biochemistry is observed between samples from different cadavers. However, the hand and thigh sections are consistently differentiated for all cadavers, largely based on lipidic profiles. In dewaxed FFPP samples, while the SC, intermediate, and dermal layers are clearly differentiated by KMCA of Raman maps of tissue sections, the lipidic contributions to the spectra are significantly reduced, with the result that respective skin layers from different anatomical sites become indistinguishable. While efficient at removing the fixing wax, the tissue processing also efficiently removes the structurally similar lipidic components of the skin layers. In studies of dermatological processes in which lipids play an important role, such as wound healing, dewaxed samples are therefore not appropriate. Removal of the lipids does however accentuate the spectral features of the cellular and protein components, which may be more appropriate for retrospective analysis of disease progression and biochemical analysis using tissue banks.

Decreased Nephrin and GLEPP-1, But Increased VEGF, Flt-1, and Nitrotyrosine, Expressions in Kidney Tissue Sections From Women With Preeclampsia

PubMed Central

Zhao, Shuang; Gu, Xin; Groome, Lynn J.; Wang, Yuping

2011-01-01

Renal injury is a common pathophysiological feature in women with preeclampsia as evidenced by increased protein leakage (proteinuria) and glomerular injury (glomerular endotheliosis). Recently, podocyturia was found in preeclampsia, suggesting podocyte shedding occurs in this pregnancy disorder. However, podocyte function in preeclampsia is poorly understood. In this study, the authors have examined podocyte-specific protein expressions for nephrin, glomerular epithelial protein 1 (GLEPP-1), and ezrin in kidney biopsy tissue sections from women with preeclampsia. Expressions for vascular endothelial growth factor (VEGF) and its receptor Flt-1 and oxidative stress marker nitrotyrosine and antioxidant CuZn-superoxide dismutase (CuZn-SOD) were also examined. Kidney tissue sections from nonhypertensive and chronic hypertensive participants were stained as controls. The findings were (1) nephrin and GLEPP-1 were mainly expressed in glomerular podocytes; (2) ezrin was expressed in both glomerular podocytes and tubular epithelial cells; (3) compared to tissue sections from nonhypertensive and chronic hypertensive participants, nephrin and GLEPP-1 expressions were much reduced in tissue sections from preeclampsia and ezrin expression was reduced in podocytes; (4) enhanced VEGF, Flt-1, and nitrotyrosine, but reduced CuZn-SOD, expressions were observed in both glomerular podocytes and endothelial cells in tissue sections from preeclampsia; and (5) the expression pattern for nephrin, GLEPP-1, ezrin, VEGF, Flt-1, and CuZn-SOD were similar between tissue sections from nonhypertensive and chronic hypertensive participants. Although the authors could not conclude from this biopsy study whether the podocyte injury is the cause or effect of the preeclampsia phenotype, the data provide compelling evidence that podocyte injury accompanied by altered angiogenesis process and increased oxidative stress occurs in kidney of patients with preeclampsia. PMID:19528353

A comparison of digitized frozen section and smear preparations for intraoperative neurotelepathology.

PubMed

Gould, Peter V; Saikali, Stephan

2012-01-01

Intraoperative consultations in neuropathology are often assessed by smear preparations rather than by frozen sections. Both techniques are standard practice for light microscopic examination on site, but there is little data comparing these techniques in a telepathology setting. Thirty cases of brain tumours submitted for intraoperative consultation at our institution between July and December 2010 were identified in which both frozen section and tissue smear preparations were available for digitization at 20× magnification. Slides were digitized using a Hamamatsu Nanozoomer 2.0 HT whole slide scanner, and resulting digital images were visualized at 1680 × 1050 pixel resolution with NDP. view software. The original intraoperative diagnosis was concordant with the sign out diagnosis in 29/30 cases; one tumeur was initially interpreted as a high grade glioma but proved to be a lymphoma at sign out. Digitized frozen section slides were sufficient for diagnosis at 10× magnification in 27/30 cases. Digitized tissue smears were sufficient for diagnosis at 10× magnification in 28/30 cases. In two cases tumour was present on the tissue smear but not the frozen section (one case of recurrent astrocytoma, one case of meningeal carcinomatosis). In one case of lymphoma, tumour was present on frozen section only. These discrepancies were attributed to tissue sampling rather than image quality. Examination of digitized slides at higher magnfication (20×) permitted confirmation of mitoses and Rosenthal fibers on tissue smear preparations, but did not change the primary diagnosis. Intra-slide variations in tissue thickness on smear preparations led to variable loss of focus in digitized images, but did not affect image quality in thinner areas of the smear or impede diagnosis. Digitized tissue smears are suitable for intraoperative neurotelepathology and provide comparable information to digitized frozen sections at medium power magnification.

Optical coherence tomography monitoring of vocal fold femtosecond laser microsurgery

NASA Astrophysics Data System (ADS)

Wisweh, Henning; Merkel, Ulrich; Hüller, Ann-Kristin; Lüerßen, Kathrin; Lubatschowski, Holger

2007-07-01

Surgery of benign pathological alterations of the vocal folds results in permanent disphonia if the bounderies of the vocal fold layers are disregarded. Precise cutting with a femtosecond laser (fs-laser) combined with simultanous imaging of the layered structure enables accurate resections with respect to the layer boundaries. Earlier works demonstrated the capability of optical coherence tomography (OCT) for utilization on vocal folds. The layered structure can be imaged with a spatial resolution of 10-20μm up to a depth of 1.5mm. The performance of fs-laser cutting was analyzed on extracted porcine vocal folds with OCT monitoring. Histopathological sections of the same processed samples could be well correlated with the OCT images. With adequate laser parameters thermal effects induced only negligable damage to the processed tissue. The dimensions of the thermal necrosis were determined to be smaller than 1μm. OCT contolled fs-laser cutting of porcine vocal fold tissue in the μm range with minimal tissue damage is presented.

Mapping Antiretroviral Drugs in Tissue by IR-MALDESI MSI Coupled to the Q Exactive and Comparison with LC-MS/MS SRM Assay

NASA Astrophysics Data System (ADS)

Barry, Jeremy A.; Robichaud, Guillaume; Bokhart, Mark T.; Thompson, Corbin; Sykes, Craig; Kashuba, Angela D. M.; Muddiman, David C.

2014-12-01

This work describes the coupling of the IR-MALDESI imaging source with the Q Exactive mass spectrometer. IR-MALDESI MSI was used to elucidate the spatial distribution of several HIV drugs in cervical tissues that had been incubated in either a low or high concentration. Serial sections of those analyzed by IR-MALDESI MSI were homogenized and analyzed by LC-MS/MS to quantify the amount of each drug present in the tissue. By comparing the two techniques, an agreement between the average intensities from the imaging experiment and the absolute quantities for each drug was observed. This correlation between these two techniques serves as a prerequisite to quantitative IR-MALDESI MSI. In addition, a targeted MS2 imaging experiment was also conducted to demonstrate the capabilities of the Q Exactive and to highlight the added selectivity that can be obtained with SRM or MRM imaging experiments.

Real-time three-dimensional ultrasound-assisted axillary plexus block defines soft tissue planes.

PubMed

Clendenen, Steven R; Riutort, Kevin; Ladlie, Beth L; Robards, Christopher; Franco, Carlo D; Greengrass, Roy A

2009-04-01

Two-dimensional (2D) ultrasound is commonly used for regional block of the axillary brachial plexus. In this technical case report, we described a real-time three-dimensional (3D) ultrasound-guided axillary block. The difference between 2D and 3D ultrasound is similar to the difference between plain radiograph and computer tomography. Unlike 2D ultrasound that captures a planar image, 3D ultrasound technology acquires a 3D volume of information that enables multiple planes of view by manipulating the image without movement of the ultrasound probe. Observation of the brachial plexus in cross-section demonstrated distinct linear hyperechoic tissue structures (loose connective tissue) that initially inhibited the flow of the local anesthesia. After completion of the injection, we were able to visualize the influence of arterial pulsation on the spread of the local anesthesia. Possible advantages of this novel technology over current 2D methods are wider image volume and the capability to manipulate the planes of the image without moving the probe.

Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria.

PubMed

Thomas, Pious; Sekhar, Aparna Chandra

2014-01-01

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50-100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed 'Brownian motion'-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as 'Cytobacts' and 'Peribacts', respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host.

Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria

PubMed Central

Thomas, Pious; Sekhar, Aparna Chandra

2014-01-01

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50–100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed ‘Brownian motion’-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as ‘Cytobacts’ and ‘Peribacts’, respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host. PMID:24790123

Various clinical application of phase contrast X-ray

NASA Astrophysics Data System (ADS)

Oh, Chilhwan; Park, Sangyong; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Je, Jungho

2008-02-01

In biomedical application study using phase contrast X-ray, both sample thickness or density and absorption difference are very important factors in aspects of contrast enhancement. We present experimental evidence that synchrotron hard X-ray are suitable for radiological imaging of biological samples down to the cellular level. We investigated the potential of refractive index radiology using un-monochromatized synchrotron hard X-rays for the imaging of cell and tissue in various diseases. Material had been adopted various medical field, such as apoE knockout mouse in cardiologic field, specimen from renal and prostatic carcinoma patient in urology, basal cell epithelioma in dermatology, brain tissue from autosy sample of pakinson's disease, artificially induced artilrtis tissue from rabbits and extracted tooth from patients of crack tooth syndrome. Formalin and paraffin fixed tissue blocks were cut in 3 mm thickness for the X-ray radiographic imaging. From adjacent areas, 4 μm thickness sections were also prepared for hematoxylin-eosin staining. Radiographic images of dissected tissues were obtained using the hard X-rays from the 7B2 beamline of the Pohang Light Source (PLS). The technique used for the study was the phase contrast images were compared with the optical microscopic images of corresponding histological slides. Radiographic images of various diseased tissues showed clear histological details of organelles in normal tissues. Most of cancerous lesions were well differentiated from adjacent normal tissues and detailed histological features of each tumor were clearly identified. Also normal microstructures were identifiable by the phase contrast imaging. Tissue in cancer or other disease showed clearly different findings from those of surrounding normal tissue. For the first time we successfully demonstrated that synchrotron hard X-rays can be used for radiological imaging of relatively thick tissue samples with great histological details.

3D on-chip microscopy of optically cleared tissue

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2018-02-01

Traditional pathology relies on tissue biopsy, micro-sectioning, immunohistochemistry and microscopic imaging, which are relatively expensive and labor-intensive, and therefore are less accessible in resource-limited areas. Low-cost tissue clearing techniques, such as the simplified CLARITY method (SCM), are promising to potentially reduce the cost of disease diagnosis by providing 3D imaging and phenotyping of thicker tissue samples with simpler preparation steps. However, the mainstream imaging approach for cleared tissue, fluorescence microscopy, suffers from high-cost, photobleaching and signal fading. As an alternative approach to fluorescence, here we demonstrate 3D imaging of SCMcleared tissue using on-chip holography, which is based on pixel-super-resolution and multi-height phase recovery algorithms to digitally compute the sample's amplitude and phase images at various z-slices/depths through the sample. The tissue clearing procedures and the lens-free imaging system were jointly optimized to find the best illumination wavelength, tissue thickness, staining solution pH, and the number of hologram heights to maximize the imaged tissue volume, minimize the amount of acquired data, while maintaining a high contrast-to-noise ratio for the imaged cells. After this optimization, we achieved 3D imaging of a 200-μm thick cleared mouse brain tissue over a field-of-view of <20mm2 , and the resulting 3D z-stack agrees well with the images acquired with a scanning lens-based microscope (20× 0.75NA). Moreover, the lens-free microscope achieves an order-of-magnitude better data efficiency compared to its lens-based counterparts for volumetric imaging of samples. The presented low-cost and high-throughput lens-free tissue imaging technique enabled by CLARITY can be used in various biomedical applications in low-resource-settings.

IR-MALDESI MASS SPECTROMETRY IMAGING OF BIOLOGICAL TISSUE SECTIONS USING ICE AS A MATRIX

PubMed Central

Robichaud, Guillaume; Barry, Jeremy A.; Muddiman, David C.

2014-01-01

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Mass Spectrometry imaging of biological tissue sections using a layer of deposited ice as an energy absorbing matrix was investigated. Dynamics of plume ablation were first explored using a nanosecond exposure shadowgraphy system designed to simultaneously collect pictures of the plume with a camera and collect the FT-ICR mass spectrum corresponding to that same ablation event. Ablation of fresh tissue analyzed with and without using ice as a matrix were both compared using this technique. Effect of spot-to-spot distance, number of laser shots per pixel and tissue condition (matrix) on ion abundance was also investigated for 50 µm thick tissue sections. Finally, the statistical method called design of experiments was used to compare source parameters and determine the optimal conditions for IR-MALDESI of tissue sections using deposited ice as a matrix. With a better understanding of the fundamentals of ablation dynamics and a systematic approach to explore the experimental space, it was possible to improve ion abundance by nearly one order of magnitude. PMID:24385399

Raman Spectroscopy Study of Prostatic Adenocarcinoma Bulk Tissues

NASA Astrophysics Data System (ADS)

Devpura, S.; Dai, H.; Thakur, J. S.; Naik, R.; Cao, A.; Pandya, A.; Auner, G. W.; Sarkar, F.; Sakr, W.; Naik, V.

2009-03-01

Prostate cancer is one of the most common types of cancer among men. The mortality rate for this disease can be dramatically reduced if it can be diagnosed in its early stages. Raman spectroscopy is one of the optical techniques which can provide fingerprints of a disease in terms of its molecular composition which changes due to the onset of disease. The aim of this project is to investigate the differences in the Raman spectra to identify benign epithelium (BE), prostatic intraepithelial neoplasia (PIN) and adenocarcinoma of various Gleason grades in archived bulk tissues embedded in paraffin wax. For each tissue, two adjacent tissue sections were cut and dewaxed, where one of the sections was stained using haematoxylin and eosin for histological examination and the other unstained adjacent section was used for Raman spectroscopic studies. We have collected Raman spectra from 10 prostatic adenocarcinoma dewaxed tissue sections using Raman microscope (785 nm excitation laser). The data were analyzed using statistical methods of principal component analysis and discriminant function analysis to classify the tissue regions. The results indicate that Raman Spectroscopy can differentiate between BE, PIN and Cancer regions.

Monitoring Dopamine ex Vivo during Electrical Stimulation Using Liquid-Microjunction Surface Sampling.

PubMed

Gill, Emily L; Marks, Megan; Yost, Richard A; Vedam-Mai, Vinata; Garrett, Timothy J

2017-12-19

Liquid-microjunction surface sampling (LMJ-SS) is an ambient ionization technique based on the continuous flow of solvent using an in situ microextraction device in which solvent moves through the probe, drawing in the analytes in preparation for ionization using an electrospray ionization source. However, unlike traditional mass spectrometry (MS) techniques, it operates under ambient pressure and requires no sample preparation, thereby making it ideal for rapid sampling of thicker tissue sections for electrophysiological and other neuroscientific research studies. Studies interrogating neural synapses, or a specific neural circuit, typically employ thick, ex vivo tissue sections maintained under near-physiological conditions to preserve tissue viability and maintain the neural networks. Deep brain stimulation (DBS) is a surgical procedure used to treat the neurological symptoms that are associated with certain neurodegenerative and neuropsychiatric diseases. Parkinson's disease (PD) is a neurological disorder which is commonly treated with DBS therapy. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta portion of the brain. Here, we demonstrate that the LMJ-SS methodology can provide a platform for ex vivo analysis of the brain during electrical stimulation, such as DBS. We employ LMJ-SS in the ex vivo analysis of mouse brain tissue for monitoring dopamine during electrical stimulation of the striatum region. The mouse brain tissue was sectioned fresh post sacrifice and maintained in artificial cerebrospinal fluid to create near-physiological conditions before direct sampling using LMJ-SS. A selection of metabolites, including time-sensitive metabolites involved in energy regulation in the brain, were identified using standards, and the mass spectral database mzCloud was used to assess the feasibility of the methodology. Thereafter, the intensity of m/z 154 corresponding to protonated dopamine was monitored before and after electrical stimulation of the striatum region, showing an increase in signal directly following a stimulation event. Dopamine is the key neurotransmitter implicated in PD, and although electrochemical detectors have shown such increases in dopamine post-DBS, this is the first study to do so using MS methodologies.

Antibody incubation at 37°C improves fluorescent immunolabeling in free-floating thick tissue sections.

PubMed

Xiao, Xia; Feng, Ya-Ping; Du, Bin; Sun, Han-Ru; Ding, You-Quan; Qi, Jian-Guo

2017-03-01

Fluorescent immunolabeling and imaging in free-floating thick (50-60 μm) tissue sections is relatively simple in practice and enables design-based non-biased stereology, or 3-D reconstruction and analysis. This method is widely used for 3-D in situ quantitative biology in many areas of biological research. However, the labeling quality and efficiency of standard protocols for fluorescent immunolabeling of these tissue sections are not always satisfactory. Here, we systematically evaluate the effects of raising the conventional antibody incubation temperatures (4°C or 21°C) to mammalian body temperature (37°C) in these protocols. Our modification significantly enhances the quality (labeling sensitivity, specificity, and homogeneity) and efficiency (antibody concentration and antibody incubation duration) of fluorescent immunolabeling of free-floating thick tissue sections.

Immunohistochemical distribution of chromogranin A in medicolegal autopsy materials.

PubMed

Yoshida, Chiemi; Ishikawa, Takaki; Michiue, Tomomi; Zhao, Dong; Komatsu, Ayumi; Quan, Li; Maeda, Hitoshi

2009-04-01

Chromogranin A (CgA) was recently reported as a marker of various stress responses. The aim of this study was to investigate the immunohistochemical distribution of CgA in human tissues in medicolegal autopsy cases as a basis for postmortem investigation of stress responses. The autopsy cases (n=30, within 48 h postmortem) comprised cases of mechanical asphyxia (n=15: strangulation, n=8; hanging, n=7) and acute myocardial infarction/ischemia (AMI, n=15). Routinely formalin-fixed paraffin-embedded tissue sections, including those of the hypothalamus, pituitary gland, cardiac muscle, lungs, liver, kidneys, spleen, skeletal muscle, skin, thyroid gland, submandibular gland, pancreas, and adrenal gland, were stained with polyclonal anti-human CgA antibodies and CgA positivity was quantitatively examined. Localization of CgA immunopositivity was clearly demonstrated in specific cell components in all tissue sections. CgA was mainly observed in the anterior lobe of the pituitary, adrenal medulla, neurons and some gliocytes in the hypothalamus, submandibular gland, follicular epithelial cells and connective tissue in the thyroid gland and pancreatic islet cells. CgA immunopositivity showed no significant difference between mechanical asphyxia and AMI cases. Positivity was slightly higher in adenohypophysis, adrenal medullar, and pancreatic islet cells (approximately 50-80%) than in the thyroid and submandibular glands (approximately 30-60%); however, a large case difference was observed in hypothalamic CgA immunopositivity (0-100%). These findings suggest that hypothalamic CgA immunopositivity can be used as a marker for investigating individual differences in stress responses during the death process. Further investigation of other causes of death is needed.

Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

EPA Science Inventory

Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

EPA Science Inventory

Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

THE INTRARENAL DISTRIBUTION OF TRITIATED PARA-AMINOHIPPURIC ACID DETERMINED BY A MODIFIED TECHNIQUE OF SECTION FREEZE-DRY RADIOAUTOGRAPHY

PubMed Central

Bordier, Betrand; Ornstein, Leonard; Wedeen, Richard P.

1970-01-01

Section freeze-dry radioautography has been used to examine the intrarenal distribution of a water-soluble organic acid (para-aminohippuric acid (PAH-3H)) under constant-infusion, steady-state conditions in mouse and rat kidney in vivo. The technique described here has the following advantages: (a) Sectioning and freeze-drying are accomplished in a closed cryostat at temperatures below -40°C; (b) Handling of the section is facilitated by mounting of the section-to-be on adhesive-coated Saran Wrap prior to cutting; (c) Unembedded freeze-dried sections are attached to photographic film at ambient temperature in the dark room; (d) Fixation follows completion of radioautographic exposure and precedes photographic development; (e) Permanent close contact is maintained between tissue and film. Morphologic preservation compared favorably with that obtained by optimal fixation techniques, which, however, permit diffusion. Cellular accumulation of PAH-3H during secretion was demonstrated in the proximal tubule under steady-state conditions in vivo. The cellular concentration of PAH-3H was uniform throughout the length of the proximal tubule in mouse and rat kidney. PMID:4349130

Automatic segmentation and supervised learning-based selection of nuclei in cancer tissue images.

PubMed

Nandy, Kaustav; Gudla, Prabhakar R; Amundsen, Ryan; Meaburn, Karen J; Misteli, Tom; Lockett, Stephen J

2012-09-01

Analysis of preferential localization of certain genes within the cell nuclei is emerging as a new technique for the diagnosis of breast cancer. Quantitation requires accurate segmentation of 100-200 cell nuclei in each tissue section to draw a statistically significant result. Thus, for large-scale analysis, manual processing is too time consuming and subjective. Fortuitously, acquired images generally contain many more nuclei than are needed for analysis. Therefore, we developed an integrated workflow that selects, following automatic segmentation, a subpopulation of accurately delineated nuclei for positioning of fluorescence in situ hybridization-labeled genes of interest. Segmentation was performed by a multistage watershed-based algorithm and screening by an artificial neural network-based pattern recognition engine. The performance of the workflow was quantified in terms of the fraction of automatically selected nuclei that were visually confirmed as well segmented and by the boundary accuracy of the well-segmented nuclei relative to a 2D dynamic programming-based reference segmentation method. Application of the method was demonstrated for discriminating normal and cancerous breast tissue sections based on the differential positioning of the HES5 gene. Automatic results agreed with manual analysis in 11 out of 14 cancers, all four normal cases, and all five noncancerous breast disease cases, thus showing the accuracy and robustness of the proposed approach. Published 2012 Wiley Periodicals, Inc.

Needle endomicroscope with a plastic, achromatic objective to perform optical biopsies of breast tissue

NASA Astrophysics Data System (ADS)

Kyrish, Matthew; Dobbs, Jessica; Richards-Kortum, Rebecca; Tkaczyk, Tomasz

2013-03-01

In order to diagnose cancer in breast tissue, a sample must be removed, prepared, and examined under a microscope. To provide an alternative to conventional biopsies, an endomicroscope intended to perform optical biopsies is demonstrated. The system provides high resolution, high contrast images in real-time which could allow a diagnosis to be made during surgery without the need for tissue removal. Optical sectioning is achieved via structured illumination to reject out of focus light. An image is relayed between the sample plane and the imaging system by a coherent fiber bundle with an achromatized objective lens at the distal tip of the fiber bundle which is the diameter of a biopsy needle. The custom, plastic objective provides correction for both the excitation and emission wavelengths of proflavine (452 nm and 515 nm, respectively). It also magnifies the object onto the distal tip of the fiber bundle to increase lateral resolution. The lenses are composed of the optical plastics Zeonex E48R, PMMA, and polystyrene. The lenses are fabricated via single point diamond turning and assembled using a zero alignment technique. The lateral resolution and chromatic focal shift were measured and in vitro images of breast carcinoma cells stained with proflavine were captured. The optical biopsy system is able to achieve optical sectioning and to resolve smaller features than the current high resolution microendoscope.

Development of a Mechanical Scanning Device With High-Frequency Ultrasound Transducer for Ultrasonic Capsule Endoscopy.

PubMed

Wang, Xingying; Seetohul, Vipin; Chen, Ruimin; Zhang, Zhiqiang; Qian, Ming; Shi, Zhehao; Yang, Ge; Mu, Peitian; Wang, Congzhi; Huang, Zhihong; Zhou, Qifa; Zheng, Hairong; Cochran, Sandy; Qiu, Weibao

2017-09-01

Wireless capsule endoscopy has opened a new era by enabling remote diagnostic assessment of the gastrointestinal tract in a painless procedure. Video capsule endoscopy is currently commercially available worldwide. However, it is limited to visualization of superficial tissue. Ultrasound (US) imaging is a complementary solution as it is capable of acquiring transmural information from the tissue wall. This paper presents a mechanical scanning device incorporating a high-frequency transducer specifically as a proof of concept for US capsule endoscopy (USCE), providing information that may usefully assist future research. A rotary solenoid-coil-based motor was employed to rotate the US transducer with sectional electronic control. A set of gears was used to convert the sectional rotation to circular rotation. A single-element focused US transducer with 39-MHz center frequency was used for high-resolution US imaging, connected to an imaging platform for pulse generation and image processing. Key parameters of US imaging for USCE applications were evaluated. Wire phantom imaging and tissue phantom imaging have been conducted to evaluate the performance of the proposed method. A porcine small intestine specimen was also used for imaging evaluation in vitro. Test results demonstrate that the proposed device and rotation mechanism are able to offer good image resolution ( [Formula: see text]) of the lumen wall, and they, therefore, offer a viable basis for the fabrication of a USCE device.

Microwave fixation versus formalin fixation of surgical and autopsy tissue.

PubMed

Login, G R

1978-05-01

Microwave irradiation of surgical and autopsy tissue penetrates, fixes, and hardens the tissue almost immediately (the fluid media used in the microwave consisted of saline, ten percent phosphate buffered formalin, and distilled water). Tissue sections from a representative sample of organs were tested. Comparable sections were simultaneously fixed in a phosphate buffered ten percent formalin bath in a vaccum oven as a control. Hematoxylin and eosin were used to stain the sections. Results equal to and superior to the control method were obtained. Saline microwave fixation was superior to formalin microwave fixation. Tissues placed in Zenker's solution and fixed in standard microwave oven (for approximately one minute) yielded results at least equal to conventional Zenker fixation (approximately two hours). No tissue hardening resulted from Zenker microwave fixation. A unique time versus temperature graph (microwave heating curve) reduces individual variation with this technique.

Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non-Small-Cell Lung Cancer.

PubMed

McLaughlin, Joseph; Han, Gang; Schalper, Kurt A; Carvajal-Hausdorf, Daniel; Pelekanou, Vasiliki; Rehman, Jamaal; Velcheti, Vamsidhar; Herbst, Roy; LoRusso, Patricia; Rimm, David L

2016-01-01

Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression. To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies. PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non-small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined. PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study. Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02). Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.

Full scattering profile of tissues with elliptical cross sections

NASA Astrophysics Data System (ADS)

Duadi, H.; Feder, I.; Fixler, D.

2018-02-01

Light reflectance and transmission from soft tissue has been utilized in noninvasive clinical measurement devices such as the photoplethysmograph (PPG) and reflectance pulse oximeter. Most methods of near infrared (NIR) spectroscopy focus on the volume reflectance from a semi-infinite sample, while very few measure transmission. However, since PPG and pulse oximetry are usually measured on tissue such as earlobe, fingertip, lip and pinched tissue, we propose examining the full scattering profile (FSP), which is the angular distribution of exiting photons. The FSP provides more comprehensive information when measuring from a cylindrical tissue. In our work we discovered a unique point, that we named the iso-pathlength (IPL) point, which is not dependent on changes in the reduced scattering coefficient (µs'). This IPL point was observed both in Monte Carlo (MC) simulation and in experimental tissue mimicking phantoms. The angle corresponding to this IPL point depends only on the tissue geometry. In the case of cylindrical tissues this point linearly depends on the tissue diameter. Since the target tissues for clinically physiological measuring are not a perfect cylinder, in this work we will examine how the change in the tissue cross section geometry influences the FSP and the IPL point. We used a MC simulation to compare a circular to an elliptic tissue cross section. The IPL point can serve as a self-calibration point for optical tissue measurements such as NIR spectroscopy, PPG and pulse oximetery.

75 FR 54159 - Center for Scientific Review; Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-03

... Integrated Review Group; Musculoskeletal Tissue Engineering Study Section. Date: September 28-29, 2010. Time... Skin Sciences Integrated Review Group; Arthritis, Connective Tissue and Skin Study Section. Date...

Microcomputed tomography characterization of neovascularization in bone tissue engineering applications.

PubMed

Young, Simon; Kretlow, James D; Nguyen, Charles; Bashoura, Alex G; Baggett, L Scott; Jansen, John A; Wong, Mark; Mikos, Antonios G

2008-09-01

Vasculogenesis and angiogenesis have been studied for decades using numerous in vitro and in vivo systems, fulfilling the need to elucidate the mechanisms involved in these processes and to test potential therapeutic agents that inhibit or promote neovascularization. Bone tissue engineering in particular has benefited from the application of proangiogenic strategies, considering the need for an adequate vascular supply during healing and the challenges associated with the vascularization of scaffolds implanted in vivo. Conventional methods of assessing the in vivo angiogenic response to tissue-engineered constructs tend to rely on a two-dimensional assessment of microvessel density within representative histological sections without elaboration of the true vascular tree. The introduction of microcomputed tomography (micro-CT) has recently allowed investigators to obtain a diverse range of high-resolution, three-dimensional characterization of structures, including renal, coronary, and hepatic vascular networks, as well as bone formation within healing defects. To date, few studies have utilized micro-CT to study the vascular response to an implanted tissue engineering scaffold. In this paper, conventional in vitro and in vivo models for studying angiogenesis will be discussed, followed by recent developments in the use of micro-CT for vessel imaging in bone tissue engineering research. A new study demonstrating the potential of contrast-enhanced micro-CT for the evaluation of in vivo neovascularization in bony defects is described, which offers significant potential in the evaluation of bone tissue engineering constructs.

Methods for Improving the Curvature of Steerable Needles in Biological Tissue

PubMed Central

Adebar, Troy K.; Greer, Joseph D.; Laeseke, Paul F.; Hwang, Gloria L.; Okamura, Allison M.

2016-01-01

Robotic needle steering systems have the potential to improve percutaneous interventions such as radiofrequency ablation of liver tumors, but steering techniques described to date have not achieved sufficiently small radius of curvature in biological tissue to be relevant to this application. In this work, the impact of tip geometry on steerable needle curvature is examined. Finite-element simulations and experiments with bent-tip needles in ex vivo liver tissue demonstrate that selection of tip length and angle can greatly improve curvature, with radius of curvature below 5 cm in liver tissue possible through judicious selection of these parameters. Motivated by the results of this analysis, a new articulated-tip steerable needle is described, in which a distal section is actively switched by a robotic system between a straight tip (resulting in a straight path) and a bent tip (resulting in a curved path). This approach allows the tip length and angle to be increased, while the straight configuration allows the needle tip to still pass through an introducer sheath and rotate inside the body. Validation testing in liver tissue shows that the new articulated-tip steerable needle achieves smaller radius of curvature compared to bent-tip needles described in previous work. Steerable needles with optimized tip parameters, which can generate tight curves in liver tissue, increase the clinical relevance of needle steering to percutaneous interventions. PMID:26441438

Quantification of regional cerebral blood flow and volume with dynamic susceptibility contrast-enhanced MR imaging.

PubMed

Rempp, K A; Brix, G; Wenz, F; Becker, C R; Gückel, F; Lorenz, W J

1994-12-01

Quantification of regional cerebral blood flow (rCBF) and volume (rCBV) with dynamic magnetic resonance (MR) imaging. After bolus administration of a paramagnetic contrast medium, rapid T2*-weighted gradient-echo images of two sections were acquired for the simultaneous creation of concentration-time curves in the brain-feeding arteries and in brain tissue. Absolute rCBF and rCBV values were determined for gray and white brain matter in 12 subjects with use of principles of the indicator dilution theory. The mean rCBF value in gray matter was 69.7 mL/min +/- 29.7 per 100 g tissue and in white matter, 33.6 mL/min +/- 11.5 per 100 g tissue; the average rCBV was 8.0 mL +/- 3.1 per 100 g tissue and 4.2 mL +/- 1.0 per 100 g tissue, respectively. An age-related decrease in rCBF and rCBV for gray and white matter was observed. Preliminary data demonstrate that the proposed technique allows the quantification of rCBF and rCBV. Although the results are in good agreement with data from positron emission tomography studies, further evaluation is needed to establish the validity of method.

Tissue- and cell-specific expression of mouse xanthine oxidoreductase gene in vivo: regulation by bacterial lipopolysaccharide.

PubMed Central

Kurosaki, M; Li Calzi, M; Scanziani, E; Garattini, E; Terao, M

1995-01-01

The expression of the xanthine oxidoreductase gene was studied in various mouse organs and tissues, under basal conditions and on treatment with bacterial lipopolysaccharide. Levels of xanthine oxidoreductase protein and mRNA were compared in order to understand the molecular mechanisms regulating the expression of this enzyme system. The highest amounts of xanthine oxidoreductase and the respective mRNA are observed in the duodenum and jejunum, where the protein is present in an unusual form because of a specific proteolytic cleavage of the primary translation product present in all locations. Under basal conditions, multiple tissue-specific mechanisms of xanthine oxidoreductase regulation are evident. Lipopolysaccharide increases enzyme activity in some, but not all tissues, mainly via modulation of the respective transcript, although translational and post-translational mechanisms are also active. In situ hybridization studies on tissue sections obtained from mice under control conditions or with lipopolysaccharide treatment demonstrate that xanthine oxidoreductase is present in hepatocytes, predominantly in the proximal tubules of the kidney, epithelial layer of the gastrointestinal mucosa, the alveolar compartment of the lung, the pulpar region of the spleen and the vascular component of the heart. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:7864814

HFIP Extraction Followed by 2D CTAB/SDS-PAGE Separation: A New Methodology for Protein Identification from Tissue Sections after MALDI Mass Spectrometry Profiling for Personalized Medicine Research

PubMed Central

Longuespée, Rémi; Tastet, Christophe; Desmons, Annie; Kerdraon, Olivier; Day, Robert

2014-01-01

Abstract Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and profiling technology have become the easiest methods for quickly accessing the protein composition of a tissue area. Unfortunately, the demand for the identification of these proteins remains unmet. To overcome this bottleneck, we combined several strategies to identify the proteins detected via MALDI profiling including on-tissue protein extraction using hexafluoroIsopropanol (1,1,1,3,3,3-hexafluoro-2-propanol, HFIP) coupled with two-dimensional cetyl trimethylammonium bromide/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (2D CTAB/SDS-PAGE) for separation followed by trypsin digestion and MALDI-MS analyses for identification. This strategy was compared with an on-tissue bottom-up strategy that we previously developed. The data reflect the complementarity of the approaches. An increase in the number of specific proteins identified has been established. This approach demonstrates the potential of adapted extraction procedures and the combination of parallel identification approaches for personalized medicine applications. The anatomical context provides important insight into identifying biomarkers and may be considered a first step for tissue-based biomarker research, as well as the extemporaneous examination of biopsies during surgery. PMID:24841221

High resolution microphotonic needle for endoscopic imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Tadayon, Mohammad Amin; Mohanty, Aseema; Roberts, Samantha P.; Barbosa, Felippe; Lipson, Michal

2017-02-01

GRIN (Graded index) lens have revolutionized micro endoscopy enabling deep tissue imaging with high resolution. The challenges of traditional GRIN lenses are their large size (when compared with the field of view) and their limited resolution. This is because of the relatively weak NA in standard graded index lenses. Here we introduce a novel micro-needle platform for endoscopy with much higher resolution than traditional GRIN lenses and a FOV that corresponds to the whole cross section of the needle. The platform is based on polymeric (SU-8) waveguide integrated with a microlens micro fabricated on a silicon substrate using a unique molding process. Due to the high index of refraction of the material the NA of the needle is much higher than traditional GRIN lenses. We tested the probe in a fluorescent dye solution (19.6 µM Alexa Flour 647 solution) and measured a numerical aperture of 0.25, focal length of about 175 µm and minimal spot size of about 1.6 µm. We show that the platform can image a sample with the field of view corresponding to the cross sectional area of the waveguide (80x100 µm2). The waveguide size can in principle be modified to vary size of the imaging field of view. This demonstration, combined with our previous work demonstrating our ability to implant the high NA needle in a live animal, shows that the proposed system can be used for deep tissue imaging with very high resolution and high field of view.

Planar small-angle x-ray scattering imaging of phantoms and biological samples

NASA Astrophysics Data System (ADS)

Choi, M.; Badano, A.

2017-04-01

Coherent small-angle x-ray scattering (SAXS) provides molecular and nanometer-scale structural information. By capturing SAXS data at multiple locations across a sample, we obtained planar images and observed improved contrast given by the difference in the material scattering cross sections. We use phantoms made with 3D printing techniques, with tissue-mimicking plastic (PMMA), and with a highly scattering reference material (AgBe), which were chosen because of their well characterized scattering cross section to demonstrate and characterize the planar imaging of a laboratory SAXS system. We measure 1.07 and 2.14 nm-1 angular intensity maps for AgBe, 9.5 nm-1 for PMMA, and 12.3 nm-1 for Veroclear. The planar SAXS images show material discrimination based on their cross sectional features. The image signal-to-noise ratio (SNR) of each q image was dependent on exposure time and x-ray flux. We observed a lower SNR (91 ± 48) at q angles where no characteristic peaks for either material exist. To improve the visualization of the acquired data by utilizing all q-binned data, we describe a weighted-sum presentation method with a priori knowledge of relevant cross sections to improve the SNR (10 000 ± 6400) over the SNR from a single q-image at 1.07 nm-1 (1100 ± 620). In addition, we describe planar SAXS imaging of a mouse brain slice showing differentiation of tissue types as compared to a conventional absorption-based x-ray imaging technique.

Raman microspectroscopic study of oral buccal mucosa

NASA Astrophysics Data System (ADS)

Behl, Isha; Mamgain, Hitesh; Deshmukh, Atul; Kukreja, Lekha; Hole, Arti R.; Krishna, C. Murali

2014-03-01

Oral cancer is the most common cancer among Indian males, with 5-year- survival-rates of less than 50%. Efficacy of Raman spectroscopic methods in non-invasive and objective diagnosis of oral cancers and confounding factors has already been demonstrated. The present Raman microspectroscopic study was undertaken for in-depth and site-specific analysis of normal and tumor tissues. 10 normal and 10 tumors unstained sections from 20 tissues were accrued. Raman data of 160 x 60 μm and 140 x 140 μm in normal and tumor sections, respectively, were acquired using WITec alpha 300R equipped with 532 nm laser, 50X objective and 600 gr/mm grating. Spectral data were corrected for CCDresponse, background. First-derivitized and vector-normalized data were then subjected to K-mean cluster analysis to generate Raman maps and correlated with their respective histopathology. In normal sections, stratification among epithelial layers i.e. basal, intermediate, superficial was observed. Tumor, stromal and inflammatory regions were identified in case of tumor section. Extracted spectra of the pathologically annotated regions were subjected to Principal component analysis. Findings suggest that all three layers of normal epithelium can be differentiated against tumor cells. In epithelium, basal and superficial layers can be separated while intermediate layer show misclassifications. In tumors, discrimination of inflammatory regions from tumor cells and tumor-stroma regions were observed. Finding of the study indicate Raman mapping can lead to molecular level insights of normal and pathological states.

[Normal and pathological elastic tissue under the electron microscope on thin and ultrathin sections (author's transl)].

PubMed

Adnet, J J; Pinteaux, A; Pousse, G; Caulet, T

1976-04-01

Three simple methods (adapted from optical techniques) for normal and pathological elastic tissue caracterisation in electron microscopy on thin and ultrathin sections are proposed. Two of these methods (orcein and fuchsin resorcin) seem to have a specificity for arterial and breast cancer elastic tissue. Weigert's method gives the best contrast.

Substrate-Mediated Laser Ablation under Ambient Conditions for Spatially-Resolved Tissue Proteomics

PubMed Central

Fatou, Benoit; Wisztorski, Maxence; Focsa, Cristian; Salzet, Michel; Ziskind, Michael; Fournier, Isabelle

2015-01-01

Numerous applications of ambient Mass Spectrometry (MS) have been demonstrated over the past decade. They promoted the emergence of various micro-sampling techniques such as Laser Ablation/Droplet Capture (LADC). LADC consists in the ablation of analytes from a surface and their subsequent capture in a solvent droplet which can then be analyzed by MS. LADC is thus generally performed in the UV or IR range, using a wavelength at which analytes or the matrix absorb. In this work, we explore the potential of visible range LADC (532 nm) as a micro-sampling technology for large-scale proteomics analyses. We demonstrate that biomolecule analyses using 532 nm LADC are possible, despite the low absorbance of biomolecules at this wavelength. This is due to the preponderance of an indirect substrate-mediated ablation mechanism at low laser energy which contrasts with the conventional direct ablation driven by sample absorption. Using our custom LADC system and taking advantage of this substrate-mediated ablation mechanism, we were able to perform large-scale proteomic analyses of micro-sampled tissue sections and demonstrated the possible identification of proteins with relevant biological functions. Consequently, the 532 nm LADC technique offers a new tool for biological and clinical applications. PMID:26674367

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope.

PubMed

Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T C

2015-10-01

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2015-10-01

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Osteochondral Biopsy Analysis Demonstrates That BST-CarGel Treatment Improves Structural and Cellular Characteristics of Cartilage Repair Tissue Compared With Microfracture

PubMed Central

Méthot, Stéphane; Changoor, Adele; Tran-Khanh, Nicolas; Hoemann, Caroline D.; Stanish, William D.; Restrepo, Alberto; Shive, Matthew S.; Buschmann, Michael D.

2016-01-01

Objective The efficacy and safety of BST-CarGel, a chitosan-based medical device for cartilage repair, was compared with microfracture alone at 1 year during a multicenter randomized controlled trial (RCT) in the knee. The quality of repair tissue of osteochondral biopsies collected from a subset of patients was compared using blinded histological assessments. Methods The international RCT evaluated repair tissue quantity and quality by 3-dimensional quantitative magnetic resonance imaging as co-primary endpoints at 12 months. At an average of 13 months posttreatment, 21/41 BST-CarGel and 17/39 microfracture patients underwent elective second look arthroscopies as a tertiary endpoint, during which ICRS (International Cartilage Repair Society) macroscopic scoring was carried out, and osteochondral biopsies were collected. Stained histological sections were evaluated by blinded readers using ICRS I and II histological scoring systems. Collagen organization was evaluated using a polarized light microscopy score. Results BST-CarGel treatment resulted in significantly better ICRS macroscopic scores (P = 0.0002) compared with microfracture alone, indicating better filling, integration, and tissue appearance. Histologically, BST-CarGel resulted in a significant improvement of structural parameters—Surface Architecture (P = 0.007) and Surface/Superficial Assessment (P = 0.042)—as well as cellular parameters—Cell Viability (P = 0.006) and Cell Distribution (P = 0.032). No histological parameters were significantly better for the microfracture group. BST-CarGel treatment also resulted in a more organized repair tissue with collagen stratification more similar to native hyaline cartilage, as measured by polarized light microscopy scoring (P = 0.0003). Conclusion Multiple and independent analyses in this biopsy substudy demonstrated that BST-CarGel treatment results in improved structural and cellular characteristics of repair tissue at 1 year posttreatment compared with microfracture alone, supporting previously reported results by quantitative magnetic resonance imaging. PMID:26958314

Feasibility of using tissue microarray cores of paraffin-embedded breast cancer tissue for measurement of gene expression: a proof-of-concept study.

PubMed

Drury, Suzanne; Salter, Janine; Baehner, Frederick L; Shak, Steven; Dowsett, Mitch

2010-06-01

To determine whether 0.6 mm cores of formalin-fixed paraffin-embedded (FFPE) tissue, as commonly used to construct immunohistochemical tissue microarrays, may be a valid alternative to tissue sections as source material for quantitative real-time PCR-based transcriptional profiling of breast cancer. Four matched 0.6 mm cores of invasive breast tumour and two 10 microm whole sections were taken from eight FFPE blocks. RNA was extracted and reverse transcribed, and TaqMan assays were performed on the 21 genes of the Oncotype DX Breast Cancer assay. Expression of the 16 recurrence-related genes was normalised to the set of five reference genes, and the recurrence score (RS) was calculated. RNA yield was lower from 0.6 mm cores than from 10 microm whole sections, but was still more than sufficient to perform the assay. RS and single gene data from cores were highly comparable with those from whole sections (RS p=0.005). Greater variability was seen between cores than between sections. FFPE sections are preferable to 0.6 mm cores for RNA profiling in order to maximise RNA yield and to allow for standard histopathological assessment. However, 0.6 mm cores are sufficient and would be appropriate to use for large cohort studies.

One Size Fits All: Evaluation of the Transferability of a New "Learning" Histologic Image Analysis Application.

PubMed

Arlt, Janine; Homeyer, André; Sänger, Constanze; Dahmen, Uta; Dirsch, Olaf

2016-01-01

Quantitative analysis of histologic slides is of importance for pathology and also to address surgical questions. Recently, a novel application was developed for the automated quantification of whole-slide images. The aim of this study was to test and validate the underlying image analysis algorithm with respect to user friendliness, accuracy, and transferability to different histologic scenarios. The algorithm splits the images into tiles of a predetermined size and identifies the tissue class of each tile. In the training procedure, the user specifies example tiles of the different tissue classes. In the subsequent analysis procedure, the algorithm classifies each tile into the previously specified classes. User friendliness was evaluated by recording training time and testing reproducibility of the training procedure of users with different background. Accuracy was determined with respect to single and batch analysis. Transferability was demonstrated by analyzing tissue of different organs (rat liver, kidney, small bowel, and spleen) and with different stainings (glutamine synthetase and hematoxylin-eosin). Users of different educational background could apply the program efficiently after a short introduction. When analyzing images with similar properties, accuracy of >90% was reached in single images as well as in batch mode. We demonstrated that the novel application is user friendly and very accurate. With the "training" procedure the application can be adapted to novel image characteristics simply by giving examples of relevant tissue structures. Therefore, it is suitable for the fast and efficient analysis of high numbers of fully digitalized histologic sections, potentially allowing "high-throughput" quantitative "histomic" analysis.

3D confocal reconstruction of gene expression in mouse.

PubMed

Hecksher-Sørensen, J; Sharpe, J

2001-01-01

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

Joint Instability and Osteoarthritis

PubMed Central

Blalock, Darryl; Miller, Andrew; Tilley, Michael; Wang, Jinxi

2015-01-01

Joint instability creates a clinical and economic burden in the health care system. Injuries and disorders that directly damage the joint structure or lead to joint instability are highly associated with osteoarthritis (OA). Thus, understanding the physiology of joint stability and the mechanisms of joint instability-induced OA is of clinical significance. The first section of this review discusses the structure and function of major joint tissues, including periarticular muscles, which play a significant role in joint stability. Because the knee, ankle, and shoulder joints demonstrate a high incidence of ligament injury and joint instability, the second section summarizes the mechanisms of ligament injury-associated joint instability of these joints. The final section highlights the recent advances in the understanding of the mechanical and biological mechanisms of joint instability-induced OA. These advances may lead to new opportunities for clinical intervention in the prevention and early treatment of OA. PMID:25741184

Joint instability and osteoarthritis.

PubMed

Blalock, Darryl; Miller, Andrew; Tilley, Michael; Wang, Jinxi

2015-01-01

Joint instability creates a clinical and economic burden in the health care system. Injuries and disorders that directly damage the joint structure or lead to joint instability are highly associated with osteoarthritis (OA). Thus, understanding the physiology of joint stability and the mechanisms of joint instability-induced OA is of clinical significance. The first section of this review discusses the structure and function of major joint tissues, including periarticular muscles, which play a significant role in joint stability. Because the knee, ankle, and shoulder joints demonstrate a high incidence of ligament injury and joint instability, the second section summarizes the mechanisms of ligament injury-associated joint instability of these joints. The final section highlights the recent advances in the understanding of the mechanical and biological mechanisms of joint instability-induced OA. These advances may lead to new opportunities for clinical intervention in the prevention and early treatment of OA.

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue

PubMed Central

Koob, A.O.; Bruns, L.; Prassler, C.; Masliah, E.; Klopstock, T.; Bender, A.

2016-01-01

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. PMID:22402104

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

PubMed

Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

2012-06-15

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

Quantitative characterization of mechanically indented in vivo human skin in adults and infants using optical coherence tomography

NASA Astrophysics Data System (ADS)

Huang, Pin-Chieh; Pande, Paritosh; Shelton, Ryan L.; Joa, Frank; Moore, Dave; Gillman, Elisa; Kidd, Kimberly; Nolan, Ryan M.; Odio, Mauricio; Carr, Andrew; Boppart, Stephen A.

2017-03-01

Influenced by both the intrinsic viscoelasticity of the tissue constituents and the time-evolved redistribution of fluid within the tissue, the biomechanical response of skin can reflect not only localized pathology but also systemic physiology of an individual. While clinical diagnosis of skin pathologies typically relies on visual inspection and manual palpation, a more objective and quantitative approach for tissue characterization is highly desirable. Optical coherence tomography (OCT) is an interferometry-based imaging modality that enables in vivo assessment of cross-sectional tissue morphology with micron-scale resolution, which surpasses those of most standard clinical imaging tools, such as ultrasound imaging and magnetic resonance imaging. This pilot study investigates the feasibility of characterizing the biomechanical response of in vivo human skin using OCT. OCT-based quantitative metrics were developed and demonstrated on the human subject data, where a significant difference between deformed and nondeformed skin was revealed. Additionally, the quantified postindentation recovery results revealed differences between aged (adult) and young (infant) skin. These suggest that OCT has the potential to quantitatively assess the mechanically perturbed skin as well as distinguish different physiological conditions of the skin, such as changes with age or disease.

MR-guided conformal heating of canine prostate using interstitial applicators

NASA Astrophysics Data System (ADS)

Nau, William H.; Diederich, Chris J.; Ross, Anthony; Butts, R. K.; Rieke, Viola; Bouley, Donna; Gill, Harchi; Daniel, Bruce; Sommer, Graham

2003-06-01

MRI compatible, multi-element ultrasound applicators were fabricated using cylindrical piezoceramic transducers sectored to 180 degrees to provide angular directional heating. The applicators were designed to be inserted into standard 13 or 14 gage brachytherapy catheters integrated with water-cooling. Two applicators were inserted transperinealy into the posterior region of a canine prostate. Power output ranged from 5-15 W per element during the 15 minute heating period. Phase-sensitive gradient-recalled MR imaging was used to monitor the treatment in real-time on a 0.5 Tesla MRT system. Gadolinium-enhanced T1 weighted images and diffusion-weighted images were obtained to view the regions which had been ablated during the heating procedure. Upon euthanasia, the prostate was removed, axially sectioned, and stained with TTC to reveal any regions of remaining viable tissue. Results from this study indicated a large volume of ablated tissue within the prostate which was highly correlated to the regions in the T1-weighted and diffusion-weighted images which had decreased intensity, and to the 52C contour displayed in the images obtained during the treatment. This study demonstrates the ability to control thermal coagulation within a targeted tissue volume while protecting surrounding tissue from thermal damage.

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System.

PubMed

Poon, Candice C; Ebacher, Vincent; Liu, Katherine; Yong, Voon Wee; Kelly, John James Patrick

2018-05-03

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Imaging and Spectroscopy of Natural Fluorophores in Pine Needles

PubMed Central

Williams, Nari

2018-01-01

Many plant tissues fluoresce due to the natural fluorophores present in cell walls or within the cell protoplast or lumen. While lignin and chlorophyll are well-known fluorophores, other components are less well characterized. Confocal fluorescence microscopy of fresh or fixed vibratome-cut sections of radiata pine needles revealed the presence of suberin, lignin, ferulate, and flavonoids associated with cell walls as well as several different extractive components and chlorophyll within tissues. Comparison of needles in different physiological states demonstrated the loss of chlorophyll in both chlorotic and necrotic needles. Necrotic needles showed a dramatic change in the fluorescence of extractives within mesophyll cells from ultraviolet (UV) excited weak blue fluorescence to blue excited strong green fluorescence associated with tissue browning. Comparisons were made among fluorophores in terms of optimal excitation, relative brightness compared to lignin, and the effect of pH of mounting medium. Fluorophores in cell walls and extractives in lumens were associated with blue or green emission, compared to the red emission of chlorophyll. Autofluorescence is, therefore, a useful method for comparing the histology of healthy and diseased needles without the need for multiple staining techniques, potentially aiding visual screening of host resistance and disease progression in needle tissue. PMID:29393922

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens.

PubMed

Kim, Dong-Young; Sato, Ayuko; Fukuyama, Satoshi; Sagara, Hiroshi; Nagatake, Takahiro; Kong, Il Gyu; Goda, Kaoru; Nochi, Tomonori; Kunisawa, Jun; Sato, Shintaro; Yokota, Yoshifumi; Lee, Chul Hee; Kiyono, Hiroshi

2011-04-01

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

Detection, mapping, and quantification of single walled carbon nanotubes in histological specimens with photoacoustic microscopy.

PubMed

Avti, Pramod K; Hu, Song; Favazza, Christopher; Mikos, Antonios G; Jansen, John A; Shroyer, Kenneth R; Wang, Lihong V; Sitharaman, Balaji

2012-01-01

In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Optical-resolution (OR) and acoustic-resolution (AR)--Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs.

FTIR Imaging of Brain Tissue Reveals Crystalline Creatine Deposits Are an ex Vivo Marker of Localized Ischemia during Murine Cerebral Malaria: General Implications for Disease Neurochemistry

PubMed Central

2012-01-01

Phosphocreatine is a major cellular source of high energy phosphates, which is crucial to maintain cell viability under conditions of impaired metabolic states, such as decreased oxygen and energy availability (i.e., ischemia). Many methods exist for the bulk analysis of phosphocreatine and its dephosphorylated product creatine; however, no method exists to image the distribution of creatine or phosphocreatine at the cellular level. In this study, Fourier transform infrared (FTIR) spectroscopic imaging has revealed the ex vivo development of creatine microdeposits in situ in the brain region most affected by the disease, the cerebellum of cerebral malaria (CM) diseased mice; however, such deposits were also observed at significantly lower levels in the brains of control mice and mice with severe malaria. In addition, the number of deposits was observed to increase in a time-dependent manner during dehydration post tissue cutting. This challenges the hypotheses in recent reports of FTIR spectroscopic imaging where creatine microdeposits found in situ within thin sections from epileptic, Alzheimer’s (AD), and amlyoid lateral sclerosis (ALS) diseased brains were proposed to be disease specific markers and/or postulated to contribute to the brain pathogenesis. As such, a detailed investigation was undertaken, which has established that the creatine microdeposits exist as the highly soluble HCl salt or zwitterion and are an ex-vivo tissue processing artifact and, hence, have no effect on disease pathogenesis. They occur as a result of creatine crystallization during dehydration (i.e., air-drying) of thin sections of brain tissue. As ischemia and decreased aerobic (oxidative metabolism) are common to many brain disorders, regions of elevated creatine-to-phosphocreatine ratio are likely to promote crystal formation during tissue dehydration (due to the lower water solubility of creatine relative to phosphocreatine). The results of this study have demonstrated that although the deposits do not occur in vivo, and do not directly play any role in disease pathogenesis, increased levels of creatine deposits within air-dried tissue sections serve as a highly valuable marker for the identification of tissue regions with an altered metabolic status. In this study, the location of crystalline creatine deposits were used to identify whether an altered metabolic state exists within the molecular and granular layers of the cerebellum during CM, which complements the recent discovery of decreased oxygen availability in the brain during this disease. PMID:23259037

Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering

PubMed Central

Annabi, Nasim; Nichol, Jason W.; Zhong, Xia; Ji, Chengdong; Koshy, Sandeep; Khademhosseini, Ali

2010-01-01

Tissue engineering holds great promise for regeneration and repair of diseased tissues, making the development of tissue engineering scaffolds a topic of great interest in biomedical research. Because of their biocompatibility and similarities to native extracellular matrix, hydrogels have emerged as leading candidates for engineered tissue scaffolds. However, precise control of hydrogel properties, such as porosity, remains a challenge. Traditional techniques for creating bulk porosity in polymers have demonstrated success in hydrogels for tissue engineering; however, often the conditions are incompatible with direct cell encapsulation. Emerging technologies have demonstrated the ability to control porosity and the microarchitectural features in hydrogels, creating engineered tissues with structure and function similar to native tissues. In this review, we explore the various technologies for controlling the porosity and microarchitecture within hydrogels, and demonstrate successful applications of combining these techniques. PMID:20121414

M30 expression demonstrates apoptotic cells, correlates with in situ end-labeling, and is associated with Ki-67 expression in large intestinal neoplasms.

PubMed

Carr, N J

2000-12-01

The monoclonal antibody M30 recognizes a neoepitope of cytokeratin 18 produced during apoptosis. It is reactive in formalin-fixed, paraffin-embedded tissue and has great potential in the study of apoptosis in clinical and experimental material. To compare the results of M30 immunoexpression with a more established technique of demonstrating apoptosis in tissue sections, in situ end-labeling. A secondary objective was to compare the results with immunoexpression of the proliferation-associated antigen Ki-67. Retrospective analysis of adenomas and adenocarcinomas of the large intestine. Immunohistochemistry for M30 and Ki-67, and in situ end-labeling. Formalin-fixed, paraffin-embedded tissue was used. The number of cells positive for M30, Ki-67, and in situ end-labeling, expressed as a proportion of the total number of cells counted. A strong positive correlation was found between in situ end-labeling and expression of M30, although the counts were widely scattered around the regression line. Counts of Ki-67 were strongly correlated with both M30 expression and in situ end-labeling. Immunoexpression of M30 was generally easier to interpret than in situ end-labeling, and the procedures for M30 immunohistochemistry were technically less exacting. These findings support the application of M30 immunoreactivity in the study of apoptosis.

A stochastic step model of replicative senescence explains ROS production rate in ageing cell populations.

PubMed

Lawless, Conor; Jurk, Diana; Gillespie, Colin S; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F

2012-01-01

Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells.One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence.

A Stochastic Step Model of Replicative Senescence Explains ROS Production Rate in Ageing Cell Populations

PubMed Central

Lawless, Conor; Jurk, Diana; Gillespie, Colin S.; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F.

2012-01-01

Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells. One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence. PMID:22359661

Dendritic cells in Barrett's esophagus and esophageal adenocarcinoma.

PubMed

Bobryshev, Yuri V; Tran, Dinh; Killingsworth, Murray C; Buckland, Michael; Lord, Reginald V N

2009-01-01

Like other premalignant conditions that develop in the presence of chronic inflammation, the development and progression of Barrett's esophagus is associated with the development of an immune response, but how this immune response is regulated is poorly understood. A comprehensive literature search failed to find any report of the presence of dendritic cells in Barrett's intestinal metaplasia and esophageal adenocarcinoma and this prompted our study. We used immunohistochemical staining and electron microscopy to examine whether dendritic cells are present in Barrett's esophagus and esophageal adenocarcinoma. Immunohistochemical staining with CD83, a specific marker for dendritic cells, was performed on paraffin-embedded sections of Barrett's intestinal metaplasia (IM, n = 12), dysplasia (n = 11) and adenocarcinoma (n = 14). CD83+ cells were identified in the lamina propria surrounding intestinal type glands in Barrett's IM, dysplasia, and cancer tissues. Computerized quantitative analysis showed that the numbers of dendritic cells were significantly higher in cancer tissues. Double immunostaining with CD83, CD20, and CD3, and electron microscopy demonstrated that dendritic cells are present in Barrett's esophagus and form clusters with T cells and B cells directly within the lamina propria. These findings demonstrate that dendritic cells are present in Barrett's tissues, with a significant increase in density in adenocarcinoma compared to benign Barrett's esophagus. Dendritic cells may have a role in the pathogenesis and immunotherapy treatment of Barrett's esophagus and adenocarcinoma.

Cornea and ocular lens visualized with three-dimensional confocal microscopy

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1992-08-01

This paper demonstrates the advantages of three-dimensional reconstruction of the cornea and the ocular crystalline lens by confocal microscopy and volume rendering computer techniques. The advantages of noninvasive observation of ocular structures in living, unstained, unfixed tissue include the following: the tissue is in a natural living state without the artifacts of fixation, mechanical sectioning, and staining; the three-dimensional structure can be observed from any view point and quantitatively analyzed; the dynamics of morphological changes can be studied; and the use of confocal microscopic observation results in a reduction of the number of animals required for ocular morphometric studies. The main advantage is that the dynamic morphology of ocular structures can be investigated in living ocular tissue. A laser scanning confocal microscope was used in the reflected light mode to obtain the two- dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with 488 nm wavelength. The microscope objective was a Leitz 25X, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133, three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The under sampling resulted in a three-dimensional visualization rendering in which the corneal thickness (z-axis) is compressed. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their `beaded' cell borders, basal lamina, nerve plexus, nerve fibers, free nerve endings in the basal epithelial cells, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in-situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers.

Visualizing Epithelial Expression in Vertical and Horizontal Planes With Dual Axes Confocal Endomicroscope Using Compact Distal Scanner.

PubMed

Li, Gaoming; Li, Haijun; Duan, Xiyu; Zhou, Quan; Zhou, Juan; Oldham, Kenn R; Wang, Thomas D

2017-07-01

The epithelium is a thin layer of tissue that lines hollow organs, such as colon. Visualizing in vertical cross sections with sub-cellular resolution is essential to understanding early disease mechanisms that progress naturally in the plane perpendicular to the tissue surface. The dual axes confocal architecture collects optical sections in tissue by directing light at an angle incident to the surface using separate illumination and collection beams to reduce effects of scattering, enhance dynamic range, and increase imaging depth. This configuration allows for images to be collected in the vertical as well as horizontal planes. We designed a fast, compact monolithic scanner based on the principle of parametric resonance. The mirrors were fabricated using microelectromechanical systems (MEMS) technology and were coated with aluminum to maximize near-infrared reflectivity. We achieved large axial displacements [Formula: see text] and wide lateral deflections >20°. The MEMS chip has a 3.2×2.9 mm 2 form factor that allows for efficient packaging in the distal end of an endomicroscope. Imaging can be performed in either the vertical or horizontal planes with [Formula: see text] depth or 1 ×1 mm 2 area, respectively, at 5 frames/s. We systemically administered a Cy5.5-labeled peptide that is specific for EGFR, and collected near-infrared fluorescence images ex vivo from pre-malignant mouse colonic epithelium to reveal the spatial distribution of this molecular target. Here, we demonstrate a novel scanning mechanism in a dual axes confocal endomicroscope that collects optical sections of near-infrared fluorescence in either vertical or horizontal planes to visualize molecular expression in the epithelium.

Extraction efficiency and implications for absolute quantitation of propranolol in mouse brain, liver and kidney thin tissue sections using droplet-based liquid microjunction surface sampling-HPLC ESI-MS/MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kertesz, Vilmos; Weiskittel, Taylor M.; Vavek, Marissa

Currently, absolute quantitation aspects of droplet-based surface sampling for thin tissue analysis using a fully automated autosampler/HPLC-ESI-MS/MS system are not fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from thin tissue sections. Methods: Adjacent thin tissue sections of propranolol dosed mouse brain (10- μm-thick), kidney (10- μm-thick) and liver (8-, 10-, 16- and 24- μm-thick) were obtained. Absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and tandem mass spectrometric analysis. Thesemore » values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach. Results: Extraction efficiency of propranolol using 10- μm-thick brain, kidney and liver thin tissues using droplet-based surface sampling varied between ~45-63%. Extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 μm to 24 μm. Randomly selecting half of the samples as standards, precision and accuracy of propranolol concentrations obtained for the other half of samples as quality control metrics were determined. Resulting precision ( ±15%) and accuracy ( ±3%) values, respectively, were within acceptable limits. In conclusion, comparative quantitation of adjacent mouse thin tissue sections of different organs and of various thicknesses by droplet-based surface sampling and by bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided the required quantitation accuracy and precision for assay validations. Furthermore, this means that once the extraction efficiency was calibrated for a given tissue type and drug, the droplet-based approach provides a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.« less

Extraction efficiency and implications for absolute quantitation of propranolol in mouse brain, liver and kidney thin tissue sections using droplet-based liquid microjunction surface sampling-HPLC ESI-MS/MS

DOE PAGES

Kertesz, Vilmos; Weiskittel, Taylor M.; Vavek, Marissa; ...

2016-06-22

Currently, absolute quantitation aspects of droplet-based surface sampling for thin tissue analysis using a fully automated autosampler/HPLC-ESI-MS/MS system are not fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from thin tissue sections. Methods: Adjacent thin tissue sections of propranolol dosed mouse brain (10- μm-thick), kidney (10- μm-thick) and liver (8-, 10-, 16- and 24- μm-thick) were obtained. Absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and tandem mass spectrometric analysis. Thesemore » values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach. Results: Extraction efficiency of propranolol using 10- μm-thick brain, kidney and liver thin tissues using droplet-based surface sampling varied between ~45-63%. Extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 μm to 24 μm. Randomly selecting half of the samples as standards, precision and accuracy of propranolol concentrations obtained for the other half of samples as quality control metrics were determined. Resulting precision ( ±15%) and accuracy ( ±3%) values, respectively, were within acceptable limits. In conclusion, comparative quantitation of adjacent mouse thin tissue sections of different organs and of various thicknesses by droplet-based surface sampling and by bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided the required quantitation accuracy and precision for assay validations. Furthermore, this means that once the extraction efficiency was calibrated for a given tissue type and drug, the droplet-based approach provides a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.« less

Two distinct events, section compression and loss of particles ("lost caps"), contribute to z-axis distortion and bias in optical disector counting.

PubMed

Baryshnikova, Larisa M; Von Bohlen Und Halbach, Oliver; Kaplan, Suleyman; Von Bartheld, Christopher S

2006-09-01

Deformation of tissue sections in the z-axis can bias optical disector counting. When samples of particle densities are not representative for the entire tissue section, significant bias of estimated numbers can result. To assess the occurrence, prevalence, extent, sequence of events, and causes of z-axis distortion, the distribution of neuronal nucleoli in thick paraffin and vibratome sections was determined in chicken, rodent, and human brain tissues. When positions of neuronal nucleoli were measured in the z-axis, nucleoli were more frequent at the surfaces (bottom and top) of tissue sections than in the core. This nonlinear z-axis distribution was not lab-, equipment-, or investigator-specific, and was independent of age, fixation quality, coverslipping medium, or paraffin melting temperature, but in paraffin sections, was highly correlated with the tilt of the knife (cutting) angle. Manipulation of subsequent tissue processing steps revealed that two events contribute to z-axis distortion. Initially, a higher density of particles results at surfaces after sectioning, apparently due to section compression. Subsequently, particles can be lost to varying degrees from surfaces during floating or staining and dehydration, resulting in "lost caps." These results may explain different degrees of z-axis distortion between different types of sections and different labs, and reinforce the importance of checking z-axis distributions as a "quality control" prior to selection of guard zones in optical disector counting. Indirect approaches to assess section quality, such as resectioning in a perpendicular plane, yield additional artifacts, and should be replaced by a direct quantitative measurement of z-axis distribution of particles. (c) 2006 Wiley-Liss, Inc.

76 FR 13404 - Cancer Therapy Evaluation Program Intellectual Property Option to Collaborator

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-11

... with the Agent (including specimens obtained from NCI CTEP-funded tissue banks) (``Section B Inventions... tissue banks) (``Section B Inventions''): Institution agrees to grant to Collaborator(s): (i) a paid-up...

Optical feedback effects on terahertz quantum cascade lasers: modelling and applications

NASA Astrophysics Data System (ADS)

Rakić, Aleksandar D.; Lim, Yah Leng; Taimre, Thomas; Agnew, Gary; Qi, Xiaoqiong; Bertling, Karl; Han, She; Wilson, Stephen J.; Kundu, Iman; Grier, Andrew; Ikonić, Zoran; Valavanis, Alexander; Demić, Aleksandar; Keeley, James; Li, Lianhe H.; Linfield, Edmund H.; Davies, A. Giles; Harrison, Paul; Ferguson, Blake; Walker, Graeme; Prow, Tarl; Indjin, Dragan; Soyer, H. Peter

2016-11-01

Terahertz (THz) quantum cascade lasers (QCLs) are compact sources of radiation in the 1-5 THz range with significant potential for applications in sensing and imaging. Laser feedback interferometry (LFI) with THz QCLs is a technique utilizing the sensitivity of the QCL to the radiation reflected back into the laser cavity from an external target. We will discuss modelling techniques and explore the applications of LFI in biological tissue imaging and will show that the confocal nature of the QCL in LFI systems, with their innate capacity for depth sectioning, makes them suitable for skin diagnostics with the well-known advantages of more conventional confocal microscopes. A demonstration of discrimination of neoplasia from healthy tissue using a THz, LFI-based system in the context of melanoma is presented using a transgenic mouse model.

Boron absorption imaging in rat lung colon adenocarcinoma metastases

NASA Astrophysics Data System (ADS)

Altieri, S.; Bortolussi, S.; Bruschi, P.; Fossati, F.; Vittor, K.; Nano, R.; Facoetti, A.; Chiari, P.; Bakeine, J.; Clerici, A.; Ferrari, C.; Salvucci, O.

2006-05-01

Given the encouraging results from our previous work on the clinical application of BNCT on non-resectable, chemotherapy resistant liver metastases, we explore the possibility to extend our technique to lung metastases. A fundamental requirement for BNCT is achieving higher 10B concentrations in the metastases compared to those in healthy tissue. For this reason we developed a rat model with lung metastases in order to study the temporal distribution of 10B concentration in tissues and tumoral cells. Rats with induced lung metastases from colon adenocarcinoma were sacrificed two hours after intraperitoneal Boronphenylalanine infusion. The lungs were harvested, frozen in liquid nitrogen and subsequently histological sections underwent neutron autoradiography in the nuclear reactor Triga Mark II, University of Pavia. Our findings demonstrate higher Boron uptake in tumoral nodules compared to healthy lung parenchyma 2 hours after Boronphenylalanine infusion.

Elevated expression in situ of selectin and immunoglobulin superfamily type adhesion molecules in retroocular connective tissues from patients with Graves' ophthalmopathy.

PubMed Central

Heufelder, A E; Bahn, R S

1993-01-01

Activation of certain adhesion molecules within vascular endothelium and the surrounding extravascular space is a critical event in the recruitment and targeting of an inflammatory response or autoimmune attack to a particular tissue site. We have recently demonstrated that the adhesion of lymphocytes to cultured retroocular fibroblasts obtained from patients with Graves' ophthalmopathy (GO) is mediated predominantly by the interaction of lymphocyte function-associated antigen-1 (LFA-1), expressed on lymphocytes, with intercellular adhesion molecule-1 (ICAM-1), expressed by these cells following exposure to interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), IL-1 alpha or purified thyroid-stimulating immunoglobulins. We now report the expression and localization in situ of several adhesion molecules, ICAM-1, endothelial leucocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and LFA-3 in retroocular tissues derived from patients with severe GO (n = 4) and normal individuals (n = 3). Serial cryostat sections of tissue specimens were processed for immunoperoxidase staining using various MoAbs against ICAM-1, ELAM-1, VCAM-1 and LFA-3. In addition, consecutive sections were stained with MoAbs against LFA-1, CD45RO (UCHL-1)DR-human leucocyte antigen (HLA-DR), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150,95). In GO-retroocular tissues, strong immunoreactivity for ICAM-1 and LFA-3 was detected in blood vessels (> 90%), in perimysial fibroblasts surrounding extraocular muscle fibres, and in connective tissue distinct from extraocular muscle. No ICAM-1 or LFA-3 immunoreactivity was present in extraocular muscle cells themselves. ICAM-1 and LFA-3 immunoreactivity in normal tissues was minimal or absent both in connective and muscle tissues. Vascular endothelium was strongly positive for ELAM-1 and VCAM-1 in GO-retroocular tissues, while VCAM-1 immunoreactivity was minimal (< 5% of blood vessels) and ELAM-1 immunoreactivity was generally absent in normal retroocular tissue. LFA-1-expressing, activated mononuclear cells and memory T lymphocytes (CD3+/CD45RO+) were only detected in GO-retrocular tissues, and were mainly localized around blood vessels and in areas of ICAM-1-expressing connective and perimysial tissue. HLA-DR expression was restricted to GO-tissue specimens, with strong immunoreactivity detected in blood vessels, macrophages and connective tissue and perimysial fibroblasts. No HLA-DR was detectable in extraocular muscle cells. In conclusion, infiltration of the orbit in GO by mononuclear cells, and their targeting within the orbit, may depend upon the coordinate expression of certain adhesion and MHC molecules.(ABSTRACT TRUNCATED AT 400 WORDS) Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:7680294

Positron Spectroscopy Investigation of Normal Brain Section and Brain Section with Glioma Derived from a Rat Glioma Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, SH.; Ballmann, C.; Quarles, C. A.

2009-03-10

The application of positron annihilation lifetime spectroscopy (PALS) and Doppler broadening spectroscopy (DBS) to the study of animal or human tissue has only recently been reported [G. Liu, et al. phys. stat. sol. (C) 4, Nos. 10, 3912-3915 (2007)]. We have initiated a study of normal brain section and brain section with glioma derived from a rat glioma model. For the rat glioma model, 200,000 C6 cells were implanted in the basal ganglion of adult Sprague Dawley rats. The rats were sacrificed at 21 days after implantation. The brains were harvested, sliced into 2 mm thick coronal sections, and fixedmore » in 4% formalin. PALS lifetime runs were made with the samples soaked in formalin, and there was not significant evaporation of formalin during the runs. The lifetime spectra were analyzed into two lifetime components. While early results suggested a small decrease in ortho-Positronium (o-Ps) pickoff lifetime between the normal brain section and brain section with glioma, further runs with additional samples have showed no statistically significant difference between the normal and tumor tissue for this type of tumor. The o-Ps lifetime in formalin alone was lower than either the normal tissue or glioma sample. So annihilation in the formalin absorbed in the samples would lower the o-Ps lifetime and this may have masked any difference due to the glioma itself. DBS was also used to investigate the difference in positronium formation between tumor and normal tissue. Tissue samples are heterogeneous and this needs to be carefully considered if PALS and DBS are to become useful tools in distinguishing tissue samples.« less

Optical Histology: High-Resolution Visualization of Tissue Microvasculature

NASA Astrophysics Data System (ADS)

Moy, Austin Jing-Ming

Mammalian tissue requires the delivery of nutrients, growth factors, and the exchange of oxygen and carbon dioxide gases to maintain normal function. These elements are delivered by the blood, which travels through the connected network of blood vessels, known as the vascular system. The vascular system consists of large feeder blood vessels (arteries and veins) that are connected to the small blood vessels (arterioles and venules), which in turn are connected to the capillaries that are directly connected to the tissue and facilitate gas exchange and nutrient delivery. These small blood vessels and capillaries make up an intricate but organized network of blood vessels that exist in all mammalian tissues known as the microvasculature and are very important in maintaining the health and proper function of mammalian tissue. Due to the importance of the microvasculature in tissue survival, disruption of the microvasculature typically leads to tissue dysfunction and tissue death. The most prevalent method to study the microvasculature is visualization. Immunohistochemistry (IHC) is the gold-standard method to visualize tissue microvasculature. IHC is very well-suited for highly detailed interrogation of the tissue microvasculature at the cellular level but is unwieldy and impractical for wide-field visualization of the tissue microvasculature. The objective my dissertation research was to develop a method to enable wide-field visualization of the microvasculature, while still retaining the high-resolution afforded by optical microscopy. My efforts led to the development of a technique dubbed "optical histology" that combines chemical and optical methods to enable high-resolution visualization of the microvasculature. The development of the technique first involved preliminary studies to quantify optical property changes in optically cleared tissues, followed by development and demonstration of the methodology. Using optical histology, I successfully obtained high resolution, depth sectioned images of the microvasculature in mouse brain and the coronary microvasculature in mouse heart. Future directions of optical histology include the potential to facilitate visualization of the entire microvascular structure of an organ as well as visualization of other tissue molecular markers of interest.

Histomorphometrical analysis following augmentation of infected extraction sites exhibiting severe bone loss and primarily closed by intrasocket reactive soft tissue.

PubMed

Mardinger, Ofer; Vered, Marilena; Chaushu, Gavriel; Nissan, Joseph

2012-06-01

Intrasocket reactive soft tissue can be used for primary closure during augmentation of infected extraction sites exhibiting severe bone loss prior to implant placement. The present study evaluated the histological characteristics of the initially used intrasocket reactive soft tissue, the overlying soft tissue, and the histomorphometry of the newly formed bone during implant placement. Thirty-six consecutive patients (43 sites) were included in the study. Extraction sites demonstrating extensive bone loss on preoperative periapical and panoramic radiographs served as inclusion criteria. Forty-three implants were inserted after a healing period of 6 months. Porous bovine xenograft bone mineral was used as a single bone substitute. The intrasocket reactive soft tissue was sutured over the grafting material to seal the coronal portion of the socket. Biopsies of the intrasocket reactive soft tissue at augmentation, healed mucosa, and bone cores at implant placement were retrieved and evaluated. The intrasocket reactive soft tissue demonstrated features compatible with granulation tissue and long junctional epithelium. The mucosal samples at implant placement demonstrated histopathological characteristics of keratinized mucosa with no residual elements of granulation tissue. Histomorphometrically, the mean composition of the bone cores was - vital bone 40 ± 19% (13.7-74.8%); bone substitute 25.7 ± 13% (0.6-51%); connective tissue 34.3 ± 15% (13.8-71.9%). Intrasocket reactive soft tissue used for primary closure following ridge augmentation is composed of granulation tissue and long junctional epithelium. At implant placement, clinical and histological results demonstrate its replacement by keratinized gingiva. The histomorphometrical results reveal considerable bone formation. Fresh extraction sites of hopeless teeth demonstrating chronic infection and severe bone loss may be grafted simultaneously with their removal. © 2010 Wiley Periodicals, Inc.

Systematic approach to study of thinly and thickly sectioned melanoma tissues with scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Miyasaka, C.; Tittmann, B. R.; Tutwiler, R.; Tian, Y.; Maeva, E.; Shum, D.

2010-03-01

The present study is to investigate the feasibility of applying in-vivo acoustic microscopy to the analysis of cancerous tissue. The study was implemented with mechanical scanning reflection acoustic microscope (SAM) by the following procedures. First, we ultrasonically visualized thick sections of normal and tumor tissues to determine the lowest transducer frequency required for cellular imaging. We used skin for normal tissue and the tumor was a malignant melanoma. Thin sections of the tissue were also studied with the optical and high-frequency-ultrasonic imaging for pathological evaluation. Secondly, we ultrasonically visualized subsurface cellular details of thin tissue specimens with different modes (i.e., pulse and tone-burst wave modes) to obtain the highest quality ultrasonic images. The objective is to select the best mode for the future design of a future SAM for in-vivo examination. Thirdly, we developed a mathematical modeling technique based on an angular spectrum approach for improving image processing and comparing numerical to experimental results.

[Report on notifications according to section 8d of the German Transplantation Act (TPG) for the years 2009-2011].

PubMed

Schilling-Leiß, D; Witt, F; Tönjes, R R

2014-01-01

In Germany, the Tissue Act came into effect on 1 August 2007. Since then, every tissue establishment is legally obligated to keep a record of its activities according to section 8d subsection 3 of the Transplantation Act (TPG). An annual report must be submitted to the Paul Ehrlich Institute once a year up to 1 March of the subsequent year. The report should include the types and quantities of tissues procured, conditioned, processed, stored, distributed or otherwise disposed of, imported, and exported. The report should be made on a TPG-based notification form published on the Internet by the Paul Ehrlich Institute. The present report according to section 8d subsection 3 of the TPG is based on data of the reporting years 2009-2011. Six years after implementation of the TPG's reporting obligation for tissue establishments, the number of tissue establishments known by the Paul Ehrlich Institute has increased from 349 in 2007 to 949 in 2011. In the course of continuous optimization of the notification forms, including tissue-specific glossaries, the reported data of most of the tissues and tissue preparations have become more conclusive.

Lectin-Based Assay for Glycoform-Specific Detection of α2,6-sialylated Transferrin and Carcinoembryonic Antigen in Tissue and Body Fluid.

PubMed

Ito, Hiromi; Hoshi, Kyoka; Honda, Takashi; Hashimoto, Yasuhiro

2018-05-30

Antibodies are useful for detecting glycoprotein antigens, but a conventional antibody recognizes only a protein epitope rather than a glycan. Thus, glycan isoform detection generally requires time- and labor-consuming processes such as lectin affinity column chromatography followed by sandwich ELISA. We recently found antigen-antibody reactions that were inhibited by lectin binding to glycans on the glycoprotein antigen, leading to a convenient glycoform-specific assay. Indeed, Sambucus sieboldiana agglutinin (SSA) lectin, a binder to sialylα2,6galactose residue, inhibited antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform-specific. SSA inhibition was also applicable for visualizing localization of α2,6-sialylated-Tf in a liver section. This is the first immunohistochemical demonstration of glycoform localization in a tissue section. SSA inhibition was utilized for establishing ELISA to quantify α2,6-sialylated carcinoembryonic antigen (CEA), a marker for various cancers. In addition, α2,6-sialylated-CEA was visualized in a colonic adenocarcinoma section by SSA inhibition. The method would further be applicable to a simple and rapid estimation of other α2,6-sialylated glycoproteins and have a potential aid to histopathological diagnosis.

Laser Capture and Single Cell Genotyping from Frozen Tissue Sections.

PubMed

Kroneis, Thomas; Ye, Jody; Gillespie, Kathleen

2016-01-01

There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.

On-chip clearing of arrays of 3-D cell cultures and micro-tissues.

PubMed

Grist, S M; Nasseri, S S; Poon, T; Roskelley, C; Cheung, K C

2016-07-01

Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 μm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear(T2), and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear(T2); however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This on-chip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications.

Integration of Ion Mobility MSE after Fully Automated, Online, High-Resolution Liquid Extraction Surface Analysis Micro-Liquid Chromatography

PubMed Central

2017-01-01

Direct analysis by mass spectrometry (imaging) has become increasingly deployed in preclinical and clinical research due to its rapid and accurate readouts. However, when it comes to biomarker discovery or histopathological diagnostics, more sensitive and in-depth profiling from localized areas is required. We developed a comprehensive, fully automated online platform for high-resolution liquid extraction surface analysis (HR-LESA) followed by micro–liquid chromatography (LC) separation and a data-independent acquisition strategy for untargeted and low abundant analyte identification directly from tissue sections. Applied to tissue sections of rat pituitary, the platform demonstrated improved spatial resolution, allowing sample areas as small as 400 μm to be studied, a major advantage over conventional LESA. The platform integrates an online buffer exchange and washing step for removal of salts and other endogenous contamination that originates from local tissue extraction. Our carry over–free platform showed high reproducibility, with an interextraction variability below 30%. Another strength of the platform is the additional selectivity provided by a postsampling gas-phase ion mobility separation. This allowed distinguishing coeluted isobaric compounds without requiring additional separation time. Furthermore, we identified untargeted and low-abundance analytes, including neuropeptides deriving from the pro-opiomelanocortin precursor protein and localized a specific area of the pituitary gland (i.e., adenohypophysis) known to secrete neuropeptides and other small metabolites related to development, growth, and metabolism. This platform can thus be applied for the in-depth study of small samples of complex tissues with histologic features of ∼400 μm or more, including potential neuropeptide markers involved in many diseases such as neurodegenerative diseases, obesity, bulimia, and anorexia nervosa. PMID:28945354

Internalization of Escherichia coli O157:H7 gfp+ in rocket and Swiss chard baby leaves as affected by abiotic and biotic damage.

PubMed

Hartmann, R; Fricke, A; Stützel, H; Mansourian, S; Dekker, T; Wohanka, W; Alsanius, B

2017-07-01

Internalization of human pathogens in edible parts of vegetables eaten raw is a major concern, since once internalized they are protected from sanitizing treatments. In this study, we examined the invasion of gfp-labelled Escherichia coli O157:H7 into intact and biotically (infection with Xanthomonas campestris/Pseudomonas syringae) and abiotically (grating with silicon carbide) damaged leaves of wild rocket (Diplotaxis tenuifolia) and Swiss chard (Beta vulgaris subsp. cicla) using laser scanning confocal microscopy. Bacterial cells were found in internal locations of the tissue, irrespective of tissue health status. Contaminated leaf sections of biotically and abiotically damaged wild rocket leaves showed higher susceptibility to microbial invasion, while the pathogen was internalized in greater numbers into intact Swiss chard leaf sections when abiotically, but not biotically, damaged. The greatest differences were observed between the plant species; after surface sanitization, E. coli O157:H7 was still detected in wild rocket leaves, but not in Swiss chard leaves. Contamination of leafy vegetables with Escherichia coli O157:H7 is a growing problem, as reported outbreaks are increasing. However, establishment of this human pathogen in the phyllosphere is not completely understood. Using laser scanning confocal microscopy, we demonstrated that E. coli O157:H7gfp+ can invade plant tissue of Swiss chard and wild rocket leaves and that the bacterium is more sensitive to surface sanitization of Swiss chard leaves. Damage to leaf tissue promoted leaf invasion, but the nature of the damage (abiotic or biotic) and plant species had an impact. © 2017 The Society for Applied Microbiology.

Extraneous tissue in surgical pathology: a College of American Pathologists Q-Probes study of 275 laboratories.

PubMed

Gephardt, G N; Zarbo, R J

1996-11-01

To develop a multi-institutional reference database of extraneous tissue (contaminants) in surgical pathology. In 1994, participants in the College of American Pathologists Q-Probes quality improvement program performed prospective and retrospective evaluations of extraneous tissue found in surgical pathology microscopic sections for a period of 4 weeks or until 1000 slides were reviewed in each participating laboratory. Two hundred seventy-five surgical pathology laboratories institutions, predominantly from North America. Extraneous tissue contamination rate for slides in prospective and retrospective reviews; staffing and practice procedures; location of extraneous tissue on slides; type of extraneous tissue (normal, abnormal, nonneoplastic, neoplasm, microorganisms, etc); class of extraneous tissue (slide or block contaminants); source of extraneous tissue (different or same case); origin of extraneous tissue (pathology laboratory, physician's office or operating room); and degree of diagnostic difficulty caused by extraneous tissue. Three hundred twenty-one thousand seven hundred fifty-seven slides were reviewed in the prospective study and 57083 slides in the retrospective study. There was an overall extraneous tissue rate of 0.6% of slides (2074/321757) in the prospective study and 2.9% of slides (1653/57083) in the retrospective study. Of those slides with extraneous tissue, the extraneous tissue was located near diagnostic tissue sections in 59.5% of the slides reviewed prospectively and in 25.3% of slides reviewed retrospectively; deeper sections were performed to evaluate extraneous tissue in 12.2% of prospective cases and in 3.1% of retrospective cases. Of the laboratories, 98% had written guidelines for changing solution in tissue processors, and 64.9% had guidelines for maintaining water baths free of extraneous tissue. A total of 98.9% used lens paper, filter bags, or sponges for processing fragmented and small specimens. Written protocols for documentation of extraneous tissue in surgical pathology reports were established in 6.1% of laboratories, for removal of extraneous tissue from blocks in 5.7%, and for removal of extraneous tissue from microscopic slides in 4.7%. In 24% of laboratories no comment or record was kept to document extraneous tissue. Extraneous tissue consisted of neoplasm in 12.7% of the prospectively reviewed slides and in 6.0% of the retrospectively reviewed slides. For the prospective study, 59.4% of extraneous tissue was classified as slide contaminants, and 28.4% was found to be contaminants within the paraffin block; for the retrospective study, 72.9% was classified as slide contaminants and 15.9% as block contaminants. For the prospective study, 63.2% of extraneous tissue was presumed to be from a different case, and in the retrospective study, 48.5% was presumed to be from a different case. Over 90% of extraneous tissue was thought to originate from the pathology laboratory. The degree of diagnostic difficulty caused by extraneous tissue was judged to be severe in 0.4% of slides in the prospective study and 0.1% of slides in the retrospective study. In the prospective study, it could not be determined whether the tissue in the diagnostic sections was extraneous in 0.6% of slides, and in the retrospective study, it could not be determined whether tissue in the diagnostic sections was extraneous in 0.1%. This study has documented the frequency, type, origin, source, and diagnostic difficulty of extraneous tissue and presents benchmarks of extraneous tissue experienced in the general practice of surgical pathology.

Staining paraffin embedded sections of scald of barley before paraffin removal.

PubMed

Xi, K; Burnett, P A

1997-07-01

Staining of paraffin embedded sections with periodic acid-Schiff reagent and fast green before paraffin removal resulted in differentiation of barley seed and leaf tissue from fungal structures of Rhynchosporium secalis. Crystal violet, toluidine blue O and antiline blue also successfully stained fungal structures of R. secalis in barley leaf tissues. Staining of embedded sections before paraffin removal allows simple processing of a series of sections, saves time and reduces solvent consumption.

Viruses and interstitial cystitis: adenovirus genomes cannot be demonstrated in urinary bladder biopsies.

PubMed

Hukkanen, V; Haarala, M; Nurmi, M; Klemi, P; Kiilholma, P

1996-01-01

Microbes may be involved in the pathogenesis of interstitial cystitis (IC). Adenoviruses and BK virus (BKV) can infect epithelial cells in urinary bladder and they are causative agents for hemorrhagic cystitis. We therefore studied the presence of adenovirus and BKV genomes in urinary bladder tissue specimens of patients with IC using polymerase chain reaction (PCR) and in situ hybridization (ISH). Controls were specimens from cases with transitional cell carcinoma of the bladder. Nucleic acids were extracted from paraffin sections of the bladder tissue for PCR. Primers detecting all adenovirus types were used. In situ hybridization was carried out for the paraffin sections using digoxigenin-labeled DNA probes for adenovirus and BKV. The adenovirus DNA PCR was able to detect one to two infected cells/specimen. All the seven IC cases studied and six controls were negative for adenovirus DNA by PCR and ISH. The ISH test for BKV genomes was also considered negative in IC cases and controls. The specimens which were negative in PCR tests yielded a signal with beta-globin primers, thus being amplifiable. We conclude that adenovirus and BKV do not play a major pathogenetic role in interstitial cystitis.

Affinity cytochemistry of vascular endothelia in brain tumors by biotinylated Ulex europaeus type I lectin (UEA I).

PubMed

Weber, T; Seitz, R J; Liebert, U G; Gallasch, E; Wechsler, W

1985-01-01

The vascularization of 50 tumors of the central nervous system (CNS) including 17 meningiomas, 25 neuroectodermal tumors, i.e., astrocytomas, oligodendrogliomas, mixed gliomas, glioblastomas, medulloblastomas, seven metastatic carcinomas, and one malignant hemangioendothelioma were investigated using biotinylated Ulex europaeus type I lectin (UEA I) in an indirect avidinbiotin-peroxidase procedure. The cytochemical staining pattern of UEA I on paraffin sections was compared with that of biotinylated Dolichos biflorus lectin (DBA), and with the immunocytochemical staining of factor VIII related antigen (F VIII/RAG) by polyclonal antisera using the PAP technique. UEA I visualized the endothelia of blood vessels with equal intensity, sensitivity, and reliability in normal brain and in tumor tissue with neovascularization. While large, medium, and small vessels were equally well demonstrated by UEA I and antibodies against FVIII/RAG, capillaries and endothelial sprouts were stained more consistently and intensely by UEA I. No reliable cytochemical staining could be obtained by DBA regardless of tissue or cell type investigated. It is concluded that UEA I is a highly useful cytochemical marker for the identification of vascular endothelia in paraffin sections of human brain tumors.

In Vivo Fiber-Optic Raman Mapping Of Metastases In Mouse Brains

NASA Astrophysics Data System (ADS)

Stelling, A.; Kirsch, M.; Steiner, G.; Krafft, C.; Schackert, G.; Salzer, R.

2010-08-01

Vibrational spectroscopy, in particular Raman spectroscopy, has potential applications in the field of in vivo diagnostics. Raman and FT-IR spectroscopy analyze the complete biochemical information at any given pixel within the visual field. Here we demonstrate the feasibility of performing Raman spectroscopic measurements on living mice brains using a fiber-optic probe with a nominal spatial resolution of 60 μm. The objectives of this study were to 1) evaluate preclinical models, namely murine brain slices containing experimental tumors, 2) optimize the preparation of pristine brain tissue to obtain reference information, to 3) optimize the conditions for introducing a fiber-optic probe to acquire Raman maps in vivo, and 4) to transfer results obtained from human brain tumors to an animal model. Disseminated brain metastases of malignant melanomas were induced by injecting tumor cells into the carotid artery of mice. The procedure mimicked hematogenous tumor spread in one brain hemisphere while the other hemisphere remained tumor free. Three series of sections were prepared consecutively from whole mouse brains: pristine, 2-mm thick sections for Raman mapping and dried, thin sections for FT-IR imaging, hematoxylin and eosin-stained thin sections for histopathological assessment. Raman maps were collected serially using a spectrometer coupled to a fiber-optic probe. FT-IR images were recorded using a spectrometer with a multi-channel detector. The FT-IR images and the Raman maps were evaluated by multivariate data analysis. The results obtained from the thin section studies were employed to guide measurements of murine brains in vivo. Raman maps with an acquisition time of over an hour could be performed on the living animals. No damage to the tissue was observed.

Temporal focusing microscopy combined with three-dimensional structured illumination

NASA Astrophysics Data System (ADS)

Isobe, Keisuke; Toda, Keisuke; Song, Qiyuan; Kannari, Fumihiko; Kawano, Hiroyuki; Miyawaki, Atsushi; Midorikawa, Katsumi

2017-05-01

Temporal focusing microscopy provides the optical sectioning capability in wide-field two-photon fluorescence imaging. Here, we demonstrate temporal focusing microscopy combined with three-dimensional structured illumination, which enables us to enhance the three-dimensional spatial resolution and reject the background fluorescence. Experimentally, the periodic pattern of the illumination was produced not only in the lateral direction but also in the axial direction by the interference between three temporal focusing pulses, which were easily generated using a digital micromirror device. The lateral resolution and optical sectioning capability were successfully enhanced by factors of 1.6 and 3.6, respectively, compared with those of temporal focusing microscopy. In the two-photon fluorescence imaging of a tissue-like phantom, the out-of-focus background fluorescence and the scattered background fluorescence could also be rejected.

Confocal microscopy imaging of solid tissue

EPA Science Inventory

Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ...

Molecular structure of human aortic valve by μSR- FTIR microscopy

NASA Astrophysics Data System (ADS)

Borkowska, Anna M.; Nowakowski, Michał; Lis, Grzegorz J.; Wehbe, Katia; Cinque, Gianfelice; Kwiatek, Wojciech M.

2017-11-01

Aortic valve is a part of the heart most frequently affected by pathological processes in humans what constitute a very serious health problem. Therefore, studies of morphology and molecular microstructure of the AV are needed. μSR- FTIR spectroscopy and microscopy represent unique tools to study chemical composition of the tissue and to identify spectroscopic markers characteristic for structural and functional features. Normal AV reveals a multi-layered structure and the compositional and structural changes within particular layers may trigger degenerative processes within the valve. Thus, deep insight into the structure of the valve to understand pathological processes occurring in AV is needed. In order to identify differences between three layers of human AV, tissue sections of macroscopically normal AV were studied using μSR- FTIR spectroscopy in combination with histological and histochemical stainings. Tissue sections deposited onto CaF2 substrates were mapped and representative set of IR spectra collected from fibrosa, spongiosa and ventricularis were analysed by Principal Component Analysis (PCA) in the spectral range between 1850-1000 cm-1 and 3050-2750 cm-1. PCA revealed a layered molecular structure of the valve and it was possible to identify IR bands associated to different tissue parts. Spongiosa layer was well differentiated from other two layers mainly based on IR bands characteristic for the distribution of glycosaminoglycans (GAGs) in the tissue - like 1170 cm-1 (υas(C-O-S)) and 1380 cm-1 (acetyl amino group). Additionally, it was distinguished from fibrosa and ventricularis based on 1085 cm-1 and 1240 cm-1 bands characteristic for GAGs and for carbohydrates- ν(C-O) and ν(C-O-C) respectively and nucleic acids -νsym(PO2-) and νasym(PO2-) respectively, which were less specific for this layer. The use of μSR- FTIR spectroscopy demonstrated co-localization of GAGs and lipids in spongiosa layer what may indicate their contribution in the very early phase of aortic valve calcific degeneration.

Observations on the preparation of sections of dental hard and soft tissues without conventional embedding procedures.

PubMed

Mok, Y C; Fearnhead, R W

1985-09-01

Inexpensive thin copper discs loaded with diamonds embedded in small slits around the periphery, may be used to cut sections from unembedded tooth samples without disrupting the cellular and extracellular components intimately associated with hard tissue interfaces. The tissue may be unfixed, fixed or cut using fixation or dye solutions as the lubricant. The use of these discs therefore opens up new avenues of histochemical investigation of hard tissue unrestricted by those artefacts associated with conventional or traditional methods of preparation.

A teaching phantom for sonographers.

PubMed

Zagzebski, J A; Madsen, E L; Frank, G R

1991-01-01

An anthropomorphic torso section phantom is described that is intended for use during initial stages of ultrasonographer training. The phantom represents a section of the upper abdomen, with simulated ribs, liver, kidney with fat pad, gallbladder, aorta, and bowel gas. Positioned in the liver are ten simulated soft tissue masses, which produce a variety of typical echographic patterns. All simulated soft tissue components are formed of tissue-mimicking materials that match their corresponding tissue counterparts in terms of speed of sound, ultrasonic attenuation, and density. Construction details are presented and examples of images are shown.

[Histochemical stains for minerals by hematoxylin-lake method].

PubMed

Miyagawa, Makoto

2013-04-01

The present study was undertaken to establish the experimental animal model by histological staining methods for minerals. After intraperitoneal injections of minerals, precipitates deposited on the surface of the liver. Liver tissues were fixed in paraformaldehyde, embedded in paraffin and cut into thin sections which were used as minerals containing standard section. Several reagents for histological stains and spectrophotometry for minerals were applied in both test-tube experiments and stainings of tissue sections to test for minerals. Hematoxylin-lake was found of capable of staining minerals in tissue. A simple technique used was described for light microscopic detection of minerals.

Boric acid-enhanced embedding medium for cryomicrotomy.

PubMed

Lim, Jin Ik; Park, Hun-Kuk

2012-05-01

A polyvinyl alcohol (PVA)/polyethylene glycol (PEG)-based resin is commonly used as a cryoembedding medium for the histological analysis of frozen tissue sections. However, it is not easy to obtain sufficient numbers of satisfactory reproducible sections owing to the differences between the mechanical properties of the medium and embedded tissue and the low cohesive force of the medium. We describe a modified PVA-based cryoembedding medium, composed of PVA (10wt% and 15wt%) with the addition of boric acid (from 0 to 5wt%), that can improve the sectioning properties and efficiency of frozen tissue for histological analysis. The amount of load under the same compressive displacement as well as cohesive force increased with increasing boric acid and PVA contents. 15wt% PVA and 3wt% boric acid was determined as an optimal composition for cryoembedding material based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices and the amount of load under the same compressive displacement test. On the basis of the results of routine hematoxylin and eosin staining of cryosections of tissue embedded in a medium with 3wt% boric acid and PVA, it was concluded that the modified PVA cryoembedding medium can improve the efficiency of cryosectioning for subsequent histological or histochemical analysis of various tissues. Copyright © 2011 Elsevier GmbH. All rights reserved.

An enhanced droplet-based liquid microjunction surface sampling system coupled with HPLC-ESI-MS/MS for spatially resolved analysis

DOE PAGES

Van Berkel, Gary J.; Weiskittel, Taylor M.; Kertesz, Vilmos

2014-11-07

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparatemore » height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. Lastly, the merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue.« less

An enhanced droplet-based liquid microjunction surface sampling system coupled with HPLC-ESI-MS/MS for spatially resolved analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Berkel, Gary J.; Weiskittel, Taylor M.; Kertesz, Vilmos

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparatemore » height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. Lastly, the merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue.« less

Microcomputed Tomography Characterization of Neovascularization in Bone Tissue Engineering Applications

PubMed Central

Young, Simon; Kretlow, James D.; Nguyen, Charles; Bashoura, Alex G.; Baggett, L. Scott; Jansen, John A.; Wong, Mark

2008-01-01

Abstract Vasculogenesis and angiogenesis have been studied for decades using numerous in vitro and in vivo systems, fulfilling the need to elucidate the mechanisms involved in these processes and to test potential therapeutic agents that inhibit or promote neovascularization. Bone tissue engineering in particular has benefited from the application of proangiogenic strategies, considering the need for an adequate vascular supply during healing and the challenges associated with the vascularization of scaffolds implanted in vivo. Conventional methods of assessing the in vivo angiogenic response to tissue-engineered constructs tend to rely on a two-dimensional assessment of microvessel density within representative histological sections without elaboration of the true vascular tree. The introduction of microcomputed tomography (micro-CT) has recently allowed investigators to obtain a diverse range of high-resolution, three-dimensional characterization of structures, including renal, coronary, and hepatic vascular networks, as well as bone formation within healing defects. To date, few studies have utilized micro-CT to study the vascular response to an implanted tissue engineering scaffold. In this paper, conventional in vitro and in vivo models for studying angiogenesis will be discussed, followed by recent developments in the use of micro-CT for vessel imaging in bone tissue engineering research. A new study demonstrating the potential of contrast-enhanced micro-CT for the evaluation of in vivo neovascularization in bony defects is described, which offers significant potential in the evaluation of bone tissue engineering constructs. PMID:18657028

Image-guided feedback for ophthalmic microsurgery using multimodal intraoperative swept-source spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography

NASA Astrophysics Data System (ADS)

Li, Jianwei D.; Malone, Joseph D.; El-Haddad, Mohamed T.; Arquitola, Amber M.; Joos, Karen M.; Patel, Shriji N.; Tao, Yuankai K.

2017-02-01

Surgical interventions for ocular diseases involve manipulations of semi-transparent structures in the eye, but limited visualization of these tissue layers remains a critical barrier to developing novel surgical techniques and improving clinical outcomes. We addressed limitations in image-guided ophthalmic microsurgery by using microscope-integrated multimodal intraoperative swept-source spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography (iSS-SESLO-OCT). We previously demonstrated in vivo human ophthalmic imaging using SS-SESLO-OCT, which enabled simultaneous acquisition of en face SESLO images with every OCT cross-section. Here, we integrated our new 400 kHz iSS-SESLO-OCT, which used a buffered Axsun 1060 nm swept-source, with a surgical microscope and TrueVision stereoscopic viewing system to provide image-based feedback. In vivo human imaging performance was demonstrated on a healthy volunteer, and simulated surgical maneuvers were performed in ex vivo porcine eyes. Denselysampled static volumes and volumes subsampled at 10 volumes-per-second were used to visualize tissue deformations and surgical dynamics during corneal sweeps, compressions, and dissections, and retinal sweeps, compressions, and elevations. En face SESLO images enabled orientation and co-registration with the widefield surgical microscope view while OCT imaging enabled depth-resolved visualization of surgical instrument positions relative to anatomic structures-of-interest. TrueVision heads-up display allowed for side-by-side viewing of the surgical field with SESLO and OCT previews for real-time feedback, and we demonstrated novel integrated segmentation overlays for augmented-reality surgical guidance. Integration of these complementary imaging modalities may benefit surgical outcomes by enabling real-time intraoperative visualization of surgical plans, instrument positions, tissue deformations, and image-based surrogate biomarkers correlated with completion of surgical goals.

New approach in evaluation of ceramic-polymer composite bioactivity and biocompatibility.

PubMed

Borkowski, Leszek; Sroka-Bartnicka, Anna; Polkowska, Izabela; Pawlowska, Marta; Palka, Krzysztof; Zieba, Emil; Slosarczyk, Anna; Jozwiak, Krzysztof; Ginalska, Grazyna

2017-09-01

Regeneration of bone defects was promoted by a novel β-glucan/carbonate hydroxyapatite composite and characterized by Raman spectroscopy, microCT and electron microscopy. The elastic biomaterial with an apatite-forming ability was developed for bone tissue engineering and implanted into the critical-size defects of rabbits' tibiae. The bone repair process was analyzed on non-decalcified bone/implant sections during a 6-month regeneration period. Using spectroscopic methods, we were able to determine the presence of amides, lipids and assign the areas of newly formed bone tissue. Raman spectroscopy was also used to assess the chemical changes in the composite before and after the implantation process. SEM analyses showed the mineralization degree in the defect area and that the gap size decreased significantly. Microscopic images revealed that the implant debris were interconnected to the poorly mineralized inner side of a new bone tissue. Our study demonstrated that the composite may serve as a biocompatible background for collagen ingrowth and exhibits the advantages of applying Raman spectroscopy, SEM and microCT in studying these samples.

Construction of an efficient two-photon fluorescent probe for imaging nitroreductase in live cells and tissues

NASA Astrophysics Data System (ADS)

Zhou, Liyi; Gong, Liang; Hu, Shunqin

2018-06-01

Compared with traditional confocal microscopy, two-photon fluorescence microscopy (TPFM), which excites a two-photon (TP) fluorophore by near-infrared light, provides improved three-dimensional image resolution with increased tissue-image depth (>500 μm) and an extended observation time. Therefore, the development of novel functional TP fluorophores has attracted great attention in recent years. Herein, a novel TP fluorophore CM-NH2, which have the donor-π-acceptor (D-π-A)-structure, was designed and synthesized. We further used this dye developed a new type of TP fluorescent probe CM-NO2 for detecting nitroreductase (NTR). Upon incubated with NTR for 15 min, CM-NO2 displayed a 90-fold fluorescence enhancement at 505 nm and the maximal TP action cross-section value after reaction was detected and calculated to be 200 GM at 760 nm. The probe exhibited excellent properties such as high sensitivity, high selectivity, low cytotoxicity, and high photostability. Moreover, the probe was utilized to image the tumor hypoxia in live HeLa cells. Finally, using the CM-NO2 to image NTR in tissues was demonstrated.

Embedding and Chemical Reactivation of Green Fluorescent Protein in the Whole Mouse Brain for Optical Micro-Imaging

PubMed Central

Gang, Yadong; Zhou, Hongfu; Jia, Yao; Liu, Ling; Liu, Xiuli; Rao, Gong; Li, Longhui; Wang, Xiaojun; Lv, Xiaohua; Xiong, Hanqing; Yang, Zhongqin; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2017-01-01

Resin embedding has been widely applied to fixing biological tissues for sectioning and imaging, but has long been regarded as incompatible with green fluorescent protein (GFP) labeled sample because it reduces fluorescence. Recently, it has been reported that resin-embedded GFP-labeled brain tissue can be imaged with high resolution. In this protocol, we describe an optimized protocol for resin embedding and chemical reactivation of fluorescent protein labeled mouse brain, we have used mice as experiment model, but the protocol should be applied to other species. This method involves whole brain embedding and chemical reactivation of the fluorescent signal in resin-embedded tissue. The whole brain embedding process takes a total of 7 days. The duration of chemical reactivation is ~2 min for penetrating 4 μm below the surface in the resin-embedded brain. This protocol provides an efficient way to prepare fluorescent protein labeled sample for high-resolution optical imaging. This kind of sample was demonstrated to be imaged by various optical micro-imaging methods. Fine structures labeled with GFP across a whole brain can be detected. PMID:28352214

Histopathological observations of human periimplantitis lesions.

PubMed

Berglundh, Tord; Gislason, Orn; Lekholm, Ulf; Sennerby, Lars; Lindhe, Jan

2004-05-01

The aim of the present study was to analyze some characteristics of advanced and progressive periimplantitis lesions in man. Soft tissue biopsies were obtained from 12 implants in six patients. The implants had been in function between 4 and 21 years and were, with one exception, located in the maxilla. The radiographic examination performed prior to biopsy revealed that all sites exhibited advanced bone loss. Further, clinical signs of severe inflammation, such as suppuration, swelling and/or fistula formation were detected in the majority of sites and seven of the 12 implants were found to be mobile at biopsy. Each biopsy was following fixation embedded in epoxy resin and sections were prepared for histometric and morphometric analysis. It was demonstrated (i). that all soft tissue units harbored large inflammatory cell infiltrates (ICT) that extended to a position apical of a pocket epithelium and (ii). that about 60% of the lesions were occupied by inflammatory cells, among which plasma cells dominated. Numerous amounts of PMN cells occurred not only in the pocket epithelium and adjacent connective tissue areas, but were also present in peri-vascular compartments in more central areas of the ICT.

Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.

PubMed

Yuan, Yinyin; Failmezger, Henrik; Rueda, Oscar M; Ali, H Raza; Gräf, Stefan; Chin, Suet-Feung; Schwarz, Roland F; Curtis, Christina; Dunning, Mark J; Bardwell, Helen; Johnson, Nicola; Doyle, Sarah; Turashvili, Gulisa; Provenzano, Elena; Aparicio, Sam; Caldas, Carlos; Markowetz, Florian

2012-10-24

Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin-stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. To deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy number profiles between samples. We next devised a predictor for survival in estrogen receptor-negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

Concept and setup for intraoperative imaging of tumorous tissue via Attenuated Total Reflection spectrosocopy with Quantum Cascade Lasers

NASA Astrophysics Data System (ADS)

Geiger, Florian B.; Koerdel, Martin; Schick, Anton; Heimann, Axel; Matiasek, Kaspar; Herkommer, Alois M.

2015-03-01

A major challenge in tumor surgery is the differentiation between normal and malignant tissue. Since an incompletely resected tumor easily leads to recidivism, the gold standard is to remove malignant tissue with a sufficient safety margin and send it to pathology for examination with patho-histological techniques (rapid section diagnosis). This approach, however, exhibits several disadvantages: The removal of additional tissue (safety margin) means additional stress to the patient; the correct interpretation of proper tumor excision relies on the pathologist's experience and the waiting time between resection and pathological result can be more than 45 minutes. This last aspect implies unnecessary occupation of cost-intensive operating room staff as well as longer anesthesia for the patient. Various research groups state that hyperspectral imaging in the mid-infrared, especially in the so called "fingerprint region", allows spatially resolved discrimination between normal and malignant tissue. All these experiments, though, took place in a laboratory environment and were conducted on dried, ex vivo tissue and on a microscopic scale. It is therefore our aim to develop a system incorporating the following properties: Intraoperatively and in vivo applicable, measurement time shorter than one minute, based on mid infrared spectroscopy, providing both spectral and spatial information and no use of external fluorescence markers. Theoretical assessment of different concepts and experimental studies show that a setup based on a tunable Quantum Cascade Laser and Attenuated Total Reflection seems feasible for in vivo tissue discrimination via imaging. This is confirmed by experiments with a first demonstrator.

Differential distribution of annexins-I, -II, -IV, and -VI in synovium.

PubMed Central

Goulding, N J; Dixey, J; Morand, E F; Dodds, R A; Wilkinson, L S; Pitsillides, A A; Edwards, J C

1995-01-01

OBJECTIVES--To examine the distribution of four annexins in non-inflamed rheumatoid arthritic and osteoarthritic synovial tissue. METHODS--Frozen sections were stained with monoclonal antibodies (MAb) specific for annexins-I, -II, -IV, and -VI, and for cell lineage related markers including CD68 and CD14 (macrophages), prolyl hydroxylase (fibroblasts), and CD3 (T cells). RESULTS--Each of the annexins was present in synovial tissues in significant amounts in the three groups studied. Annexin-I was predominantly found within the synovial lining layer and double labelling showed it to be present predominantly in cells of the macrophage lineage. In rheumatoid specimens there was increased staining within the lining layer, perivascularly and on macrophages within the tissue stroma. Annexin-II was present in a distribution similar to that of annexin-I, but with more prominent perivascular staining. Annexins-IV and -VI were seen chiefly in association with areas of lymphocyte infiltration in rheumatoid tissue, whereas annexins-I and -II were absent from these areas. Endothelial cells stained weakly positive for annexins-I and -II, and more strongly for -IV and -VI. CONCLUSIONS--This study demonstrates that annexins (particularly annexin-I, a putative mediator of the anti-inflammatory activities of glucocorticoids) are abundant in rheumatoid and non-rheumatoid synovial tissue, annexins-IV and -VI having a distribution distinct from that of -I and -II. Images PMID:7492225

Detection of Metastatic Breast and Thyroid Cancer in Lymph Nodes by Desorption Electrospray Ionization Mass Spectrometry Imaging

NASA Astrophysics Data System (ADS)

Zhang, Jialing; Feider, Clara L.; Nagi, Chandandeep; Yu, Wendong; Carter, Stacey A.; Suliburk, James; Cao, Hop S. Tran; Eberlin, Livia S.

2017-06-01

Ambient ionization mass spectrometry has been widely applied to image lipids and metabolites in primary cancer tissues with the purpose of detecting and understanding metabolic changes associated with cancer development and progression. Here, we report the use of desorption electrospray ionization mass spectrometry (DESI-MS) to image metastatic breast and thyroid cancer in human lymph node tissues. Our results show clear alterations in lipid and metabolite distributions detected in the mass spectra profiles from 42 samples of metastatic thyroid tumors, metastatic breast tumors, and normal lymph node tissues. 2D DESI-MS ion images of selected molecular species allowed discrimination and visualization of specific histologic features within tissue sections, including regions of metastatic cancer, adjacent normal lymph node, and fibrosis or adipose tissues, which strongly correlated with pathologic findings. In thyroid cancer metastasis, increased relative abundances of ceramides and glycerophosphoinisitols were observed. In breast cancer metastasis, increased relative abundances of various fatty acids and specific glycerophospholipids were seen. Trends in the alterations in fatty acyl chain composition of lipid species were also observed through detailed mass spectra evaluation and chemical identification of molecular species. The results obtained demonstrate DESI-MSI as a potential clinical tool for the detection of breast and thyroid cancer metastasis in lymph nodes, although further validation is needed. [Figure not available: see fulltext.

Quantitative proteome analysis reveals the correlation between endocytosis-associated proteins and hepatocellular carcinoma dedifferentiation.

PubMed

Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Reis, Henning; Ahrens, Maike; Turewicz, Michael; Eisenacher, Martin; Tautges, Stephanie; Canbay, Ali E; Meyer, Helmut E; Weber, Frank; Baba, Hideo A; Sitek, Barbara

2016-11-01

The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression. Copyright © 2016 Elsevier B.V. All rights reserved.

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp, Cyprinus carpio L. using in situ hybridization.

PubMed

Monaghan, S J; Thompson, K D; Adams, A; Kempter, J; Bergmann, S M

2015-05-01

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody-based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin-embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post-infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp. © 2014 John Wiley & Sons Ltd.

Physical models, cross sections, and numerical approximations used in MCNP and GEANT4 Monte Carlo codes for photon and electron absorbed fraction calculation.

PubMed

Yoriyaz, Hélio; Moralles, Maurício; Siqueira, Paulo de Tarso Dalledone; Guimarães, Carla da Costa; Cintra, Felipe Belonsi; dos Santos, Adimir

2009-11-01

Radiopharmaceutical applications in nuclear medicine require a detailed dosimetry estimate of the radiation energy delivered to the human tissues. Over the past years, several publications addressed the problem of internal dose estimate in volumes of several sizes considering photon and electron sources. Most of them used Monte Carlo radiation transport codes. Despite the widespread use of these codes due to the variety of resources and potentials they offered to carry out dose calculations, several aspects like physical models, cross sections, and numerical approximations used in the simulations still remain an object of study. Accurate dose estimate depends on the correct selection of a set of simulation options that should be carefully chosen. This article presents an analysis of several simulation options provided by two of the most used codes worldwide: MCNP and GEANT4. For this purpose, comparisons of absorbed fraction estimates obtained with different physical models, cross sections, and numerical approximations are presented for spheres of several sizes and composed as five different biological tissues. Considerable discrepancies have been found in some cases not only between the different codes but also between different cross sections and algorithms in the same code. Maximum differences found between the two codes are 5.0% and 10%, respectively, for photons and electrons. Even for simple problems as spheres and uniform radiation sources, the set of parameters chosen by any Monte Carlo code significantly affects the final results of a simulation, demonstrating the importance of the correct choice of parameters in the simulation.

Tissue Permeability Effects Associated with the Use of Mucoadhesive Cationic Nanoformulations of Docetaxel in the Bladder.

PubMed

Pandey, Rakhi; Jackson, John K; Mugabe, Clement; Liggins, Richard; Burt, Helen M

2016-08-01

Recently, efficacy studies in mice have shown that amine-terminated cationic (CNP) nanoparticulate carriers of DTX offer an improved formulation of the drug for intravesical delivery. It is hypothesized that this improved efficacy may arise from a carrier mediated bladder exfoliation process that removes the urothelial barrier allowing for increased drug uptake into bladder tissue. The objective of this study was to investigate exfoliation processes in fresh pig's bladders (ex vivo) exposed to three cationic polyglycerols with increasing degrees of amination (denoted 350, 580 and 780). The study also compared the tissue depth profile of DTX uptake into these tissues using these different carriers. Aminated polyglycerols were synthesized and characterized in the laboratory with low (CNP-360), medium (CNP-580) and high (CNP-780) levels of amine content. CNP-based DTX solutions and commercial DTX solutions in polysorbate 80 (Taxotere®) were doped with (3)H-radiolabeled DTX and prepared by solvent evaporation from acetonitrile, followed by drying and reconstitution in pH 6.4 buffer. Sections of fresh pig's bladder tissue were clamped into Franz diffusion cells and the urothelial side was exposed to the DTX solutions for 2 h. Tissue sections were then frozen for sectioning by cryotome sectioning and subsequently processed for drug analysis by liquid scintillation counting. Alternatively tissue sections were fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer for the purposes of scanning electron microscopy (SEM). Exposure of the urothelial surface to the amine-terminated polyglycerol solutions resulted in the exfoliation of bladder tissues in a time- and concentration-dependent manner. Exfoliation was significantly more pronounced when using CNPs with a medium or high levels of amination whereas only minor levels of exfoliation were seen with low levels. Following incubation of tissues in Tween-based commercial formulations (Taxotere) of DTX (0.5 mg/mL) the drug was detectable at low levels (10-40 μg/g tissue) in all depths of tissue. Similar drug uptake was observed using the CNP-360 formulation. However drug uptake levels were increased to 60-100 μg/g tissue when samples were incubated with either the CNP-580 or CNP-780 formulations. The use of cationic polyglycerols with higher levels of amine termination allows for an enhanced uptake of DTX into bladder tissues as compared to commercial (Taxotere) formulations. These increased drug levels probably arise from exfoliation processes resulting in a temporary elimination of the urothelial permeability barrier and increased drug penetration into the tissue.

Real-time sound speed correction using golden section search to enhance ultrasound imaging quality

NASA Astrophysics Data System (ADS)

Yoon, Chong Ook; Yoon, Changhan; Yoo, Yangmo; Song, Tai-Kyong; Chang, Jin Ho

2013-03-01

In medical ultrasound imaging, high-performance beamforming is important to enhance spatial and contrast resolutions. A modern receive dynamic beamfomer uses a constant sound speed that is typically assumed to 1540 m/s in generating receive focusing delays [1], [2]. However, this assumption leads to degradation of spatial and contrast resolutions particularly when imaging obese patients or breast since the sound speed is significantly lower than the assumed sound speed [3]; the true sound speed in the fatty tissue is around 1450 m/s. In our previous study, it was demonstrated that the modified nonlinear anisotropic diffusion is capable of determining an optimal sound speed and the proposed method is a useful tool to improve ultrasound image quality [4], [5]. In the previous study, however, we utilized at least 21 iterations to find an optimal sound speed, which may not be viable for real-time applications. In this paper, we demonstrates that the number of iterations can be dramatically reduced using the GSS(golden section search) method with a minimal error. To evaluate performances of the proposed method, in vitro experiments were conducted with a tissue mimicking phantom. To emulate a heterogeneous medium, the phantom was immersed in the water. From the experiments, the number of iterations was reduced from 21 to 7 with GSS method and the maximum error of the lateral resolution between direct and GSS was less than 1%. These results indicate that the proposed method can be implemented in real time to improve the image quality in the medical ultrasound imaging.

Preventive effect of resistant starch on activated carbon-induced constipation in mice

PubMed Central

QIAN, YU; ZHAO, XIN; KAN, JIANQUAN

2013-01-01

The aim of this study was to investigate the effects of resistant starch (RS) on activated carbon-induced constipation in ICR mice. ICR mice were fed on diet containing 15% RS of type RS2, RS3 or RS4 for 9 days. Gastrointestinal transit, defecation time and intestinal tissue histopathological sections, as well as motilin (MTL), gastrin (Gas), endothelin (ET), somatostatin (SS), acetylcholinesterase (AChE), substance P (SP) and vasoactive intestinal peptide (VIP) levels in serum were used to evaluate the preventive effects of RS on constipation. Bisacodyl, a laxative drug, was used as a positive control. The time to the first black stool defecation for normal, control, bisacodyl-treated (100 mg/kg, oral administration) and RS2-, RS3- and RS4-treated mice was 78, 208, 109, 181, 144 and 173 min, respectively. Following the consumption of RS2, RS3 and RS4 or the oral administration of bisacodyl (100 mg/kg), the gastrointestinal transit was reduced to 37.7, 52.1, 39.3 and 87.3%, respectively, of the transit in normal mice, respectively. Histopathological sections of intestinal tissue also underscored the protective effect of RS3. The serum levels of MTL, Gas, ET, AChE, SP and VIP were significantly increased and the serum levels of SS were reduced in the mice treated with RS compared with those in the untreated control mice (P<0.05). These results demonstrate that RS has preventive effects on mouse constipation and RS3 demonstrated the best functional activity. PMID:23935751

Use of a tissue sectioner to expose internal structures of biological samples for scanning electron microscopy.

PubMed

Brown, M F; Brotzman, H G; Kinden, D A

1976-09-01

A procedure yielding sections of unembedded biological samples for observation by scanning electron microscopy is described. Sections of samples, fixed and hardened in OsO4, were obtained in quantity with a tissue sectioner. Subsequent treatments to osmium-coat cut surfaces were employed prior to critical point drying. The procedure yields cleanly cut surfaces through cells and cytoplasmic organelles which are retained in their normal position. Sections of apple leaf and mouse kidney are illustrated. Sections can be readily cut in a desired plane with less structural damage than is typically encountered by other sectioning or dissection techniques.

Effect of Antigen Retrieval Methods on Nonspecific Binding of Antibody-Metal Nanoparticle Conjugates on Formalin-Fixed Paraffin-Embedded Tissue.

PubMed

Zhang, Yuying; Wang, Xin-Ping; Perner, Sven; Bankfalvi, Agnes; Schlücker, Sebastian

2018-01-02

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissues provides important diagnostic and prognostic information in pathology. Metal nanoparticles (NPs) and, in particular, surface-enhanced Raman scattering (SERS) nanotags as a new class of labeling reagents are promising to be used for multiplexed protein profiling on tissue sections. However, nonspecific binding of NPs onto the tissue specimens greatly hampers their clinical applications. In this study, we found that the antigen retrieval method strongly influences the extent of nonspecific binding of the antibody-SERS NP conjugates to the tissue. Our SERS labels comprised ca. 70 nm Au nanostars coated with ethylene glycol-modified Raman reporter molecules for hydrophilic stabilization and subsequent covalent bioconjugation to antibodies. We systematically investigated the influence of heat- and protease-induced epitope retrieval (HIER and PIER, respectively) on the immunostaining quality of prostate-specific antigen (PSA) on human prostate tissue sections. The best staining results were obtained with PIER. Pretreatment of the tissue sections by HIER led to selective but nonspecific adsorption of the antibody-Au nanostar conjugates onto epithelial cells, while enzymatic treatment within PIER did not. In addition to gold nanostars, also other types of metal NPs with different shapes and sizes (including ca. 20 nm quasi-spherical Au NPs and ca. 60 nm quasi-spherical Au/Ag nanoshells) as well as tissue sections from different organs (including prostate and breast) were tested; in each case the same tendency was observed, i.e., PIER yielded better results than HIER. Therefore, we recommend PIER for future NP-based tissue immunostaining such as immuno-SERS microscopy. Alternatively, for antigens that can only be unmasked by heating, PEGylation of the NPs is recommended to avoid nonspecific binding.

Blind Biobanking of the Prostatectomy Specimen: Critical Evaluation of the Existing Techniques and Development of the New 4-Level Tissue Extraction Model With High Sampling Efficacy.

PubMed

Tolkach, Yuri; Eminaga, Okyaz; Wötzel, Fabian; Huss, Sebastian; Bettendorf, Olaf; Eltze, Elke; Abbas, Mahmoud; Imkamp, Florian; Semjonow, Axel

2017-03-01

Fresh tissue is mandatory to perform high-quality translation studies. Several models for tissue extraction from prostatectomy specimens without guidance by frozen sections are already introduced. However, little is known about the sampling efficacy of these models, which should provide representative tissue in adequate volumes, account for multifocality and heterogeneity of tumor, not violate the routine final pathological examination, and perform quickly without frozen section-based histological control. The aim of the study was to evaluate the sampling efficacy of the existing tissue extraction models without guidance by frozen sections ("blind") and to develop an optimized model for tissue extraction. Five hundred thirty-three electronic maps of the tumor distribution in prostates from a single-center cohort of the patients subjected to radical prostatectomy were used for analysis. Six available models were evaluated in silico for their sampling efficacy. Additionally, a novel model achieving the best sampling efficacy was developed. The available models showed high efficacies for sampling "any part" from the tumor (up to 100%), but were uniformly low in efficacy to sample all tumor foci from the specimens (with the best technique sampling only 51.6% of the all tumor foci). The novel 4-level extraction model achieved a sampling efficacy of 93.1% for all tumor foci. The existing "blind" tissue extraction models from prostatectomy specimens without frozen sections control are suitable to target tumor tissues but these tissues do not represent the whole tumor. The novel 4-level model provides the highest sampling efficacy and a promising potential for integration into routine. Prostate 77: 396-405, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Increased protein expression of LHCG receptor and 17α-hydroxylase/17-20-lyase in human polycystic ovaries.

PubMed

Comim, F V; Teerds, K; Hardy, K; Franks, S

2013-11-01

Does the expression of LHCG receptor (LHCGR) protein and key enzymes in the androgen biosynthetic pathway differ in normal human versus polycystic ovarian tissue? LHCGR and 17α-hydroxylase/17-20-lyase (CYP17A1) protein levels are increased in polycystic ovaries (PCOs). The predominant source of excess androgen secretion in women with polycystic ovary syndrome (PCOS) is ovarian theca cells but few studies have directly assessed the presence and abundance of protein for key molecules involved in androgen production by theca, including LHCGR and the rate-limiting enzyme in androgen production, CYP17A1. This is a laboratory-based, cross-sectional study comparing protein expression of key molecules in the androgen biosynthetic pathway in archived ovarian tissue from women with normal ovaries (n = 10) with those with PCOs (n = 16). A quantitative morphometric study was performed using sections of archived human ovaries (n = 26) previously characterized as normal or polycystic. The distribution and abundance of LHCGR, CYP17A1, 3β-hydroxysteroid dehydrogenase type 2 (3βHSDII) and 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5) proteins were evaluated by immunohistochemistry and quantified. A higher proportion of theca cells from anovulatory PCO expressed LHCGR protein when compared with control ovaries (P = 0.01). A significant increase in the intensity of immunostaining for CYP17A1 was identified in antral follicles in sections of PCO compared with ovaries from normal women (P = 0.04). As the study used formalin-fixed ovarian tissue sections, it was not possible to carry out studies 'in vitro' using the same ovarian tissues in order to also demonstrate increased functional activity of LHCGR and CYP17A1. The data are in keeping with the results of previous studies in isolated theca cells and support the notion of an intrinsic abnormality of theca cell androgen production in women with PCOS. The research was supported by a Programme Grant, G0802782, from the Medical Research Council (MRC) UK and by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. F.V.C was supported by Capes Foundation (Brazilian Ministry of Education). The authors have no conflicts of interest to disclose.

Assessment of eye bank-prepared posterior lamellar corneal tissue for endothelial keratoplasty.

PubMed

Rose, Linda; Briceño, César A; Stark, Walter J; Gloria, Dante G; Jun, Albert S

2008-02-01

To evaluate eye bank-prepared tissue for Descemet's stripping automated endothelial keratoplasty (DSAEK). Experimental study and retrospective case series. Seventeen human donor corneas and 4 recipient patients undergoing DSAEK surgery. Corneal-scleral discs were obtained. Specular microscopy and pachymetry were performed. A designated Tissue Banks International technician used a microkeratome to prepare a flap. Posterior bed thickness was measured. The sectioned tissue was stored, and at 24 and 48 hours, pachymetry was repeated. At 48 hours, specular microscopy was repeated, and endothelial cell viability was assessed with trypan blue. Descemet's stripping automated endothelial keratoplasty was performed in 4 patients using eye bank-prepared posterior lamellar tissue. Corneal tissue was assessed with the following parameters: corneal thickness measured with ultrasonic pachymetry, cell density counts measured with a keratoanalyzer, and cell viability as observed with trypan blue exclusion. Patient outcomes were measured by changes in visual acuity (VA) and the presence of a clear graft. Donor corneal pachymetry before sectioning averaged 599+/-52 microm. Immediately after sectioning with a microkeratome set at a depth of 300 microm, mean posterior bed thickness was 328+/-95 microm. Thus, the mean cutting depth achieved by the microkeratome when set at 300 micrometers averaged 271+/-83 microm. After storage for 24 hours, the posterior beds measured 352 microm, an average swelling of 24 (7%) microm (P = 0.14). After 48 hours, the posterior beds measured 382 microm, an average swelling of 54 (16%) microm (P = 0.02). Cell counts 48 hours after sectioning decreased by an average of 11% (P = 0.10). Endothelial cell staining confirmed improvement in postsectioning morphology and survival with increased technician experience. All 4 patients receiving eye bank-prepared DSAEK tissue showed uncomplicated postoperative results, with improvement in VA. The microkeratome cutting depth was moderately accurate. Pachymetry, cell density, and cell viability of sectioned tissue after 48 hours in storage were encouraging overall. Initial clinical results of eye bank-prepared DSAEK tissue showed uncomplicated postoperative courses and improved VA. Additional studies are needed to follow the long-term outcomes in the recipients of these tissues.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Bova, G Steven; Eltoum, Isam A; Kiernan, John A; Siegal, Gene P; Frost, Andra R; Best, Carolyn J M; Gillespie, John W; Su, Gloria H; Emmert-Buck, Michael R

2005-02-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Submicron-resolution photoacoustic microscopy of endogenous light-absorbing biomolecules

NASA Astrophysics Data System (ADS)

Zhang, Chi

Photoacoustic imaging in biomedicine has the unique advantage of probing endogenous light absorbers at various length scales with a 100% relative sensitivity. Among the several modalities of photoacoustic imaging, optical-resolution photoacoustic microscopy (OR-PAM) can achieve high spatial resolution, on the order of optical wavelength, at <1 mm depth in biological tissue (the optical ballistic regime). OR-PAM has been applied successfully to structural and functional imaging of blood vasculature and red blood cells in vivo. Any molecules which absorb sufficient light at certain wavelengths can potentially be imaged by PAM. Compared with pure optical imaging, which typically targets fluorescent markers, label-free PAM avoids the major concerns that the fluorescent labeling probes may disturb the function of biomolecules and may have an insufficient density. This dissertation aims to advance label-free OR-PAM to the subcellular scale. The first part of this dissertation describes the technological advancement of PAM yielding high spatial resolution in 3D. The lateral resolution was improved by using optical objectives with high numerical apertures for optical focusing. The axial resolution was improved by using broadband ultrasonic transducers for ultrasound detection. We achieved 220 nm lateral resolution in transmission mode, 0.43 microm lateral resolution in reflection mode, 7.6 microm axial resolution in normal tissue, and 5.8 microm axial resolution with silicone oil immersion/injection. The achieved lateral resolution and axial resolution were the finest reported at the time. With high-resolution in 3D, PAM was demonstrated to resolve cellular and subcellular structures in vivo, such as red blood cells and melanosomes in melanoma cells. Compared with previous PAM systems, our high-resolution PAM could resolve capillaries in mouse ears more clearly. As an example application, we demonstrated intracellular temperature imaging, assisted by fluorescence signal detection, with sub-degree temperature resolution and sub-micron lateral resolution. The second part of this dissertation describes the exploration of endogenous light-absorbing biomolecules for PAM. We demonstrated cytochromes and myoglobin as new absorption contrasts for PAM and identified the corresponding optimal wavelengths for imaging. Fixed fibroblasts on slides and mouse ear sections were imaged by PAM at 422 nm and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard hematoxylin and eosin (H&E) histology. By imaging a blood-perfused mouse heart at 532 nm down to 150 microm in depth, we derived the myocardial sheet thickness and the cleavage height from an undehydrated heart for the first time. The findings promote PAM at new wavelengths and open up new possibilities for characterizing biological tissue. Of particular interest, dual-wavelength PAM around 250 nm and 420 nm wavelengths is analogous to H&E histology. The last part of this dissertation describes the development of sectioning photoacoustic microscopy (SPAM), based on the advancement in spatial resolution and new contrasts for PAM, with applications in brain histology. Label-free SPAM, assisted by a microtome, acquires serial distortion-free images of a specimen on the surface. By exciting cell nuclei at 266 nm wavelength with high resolution, SPAM could pinpoint cell nuclei sensitively and specifically in the mouse brain section, as confirmed by H&E histology. SPAM was demonstrated to generate high-resolution 3D images, highlighting cell nuclei, of formalin-fixed paraffin-embedded mouse brains without tissue staining or clearing. SPAM can potentially serve as a high-throughput and minimal-artifact substitute for histology, probe many other biomolecules and cells, and become a universal tool for animal or human whole-organ microscopy, with diverse applications in life sciences.

Targeted sections in either XY or XZ plane with dual-axes confocal endomicroscope (Conference Presentation)

NASA Astrophysics Data System (ADS)

Li, Gaoming; Li, Haijun; Duan, Xiyu; Zhou, Quan; Zhou, Juan; Oldham, Kenn R.; Wang, Thomas D.

2017-02-01

We demonstrate a dual axes confocal architecture, which can be used to collect horizontal(XY-plane) or vertical cross-sectional(XZ-plane) images for tissue. This scanner head is 5.5mm in outer diameter(OD), and integrates a 3D MEMS scanner with a compact chip size of 3.2×2.9mm2. To realize the miniaturization, there are some obstacles of the small size of 3D MEMS scanner, MEMS wire bundle, the air pressure effect for MEMS motion, the processing of parabolic mirror, and optical alignment to come over. In our probe, separation mechanical structure for optical alignment was adopted and a step shape MEMS holder was designed to deal with the difficult of MEMS wire bundle. Peptides have been demonstrated tremendous potential for in vivo use to detect colonic dysplasia. This class of in vivo molecular probe can be labeled with near-infrared (NIR) dyes for visualizing the full depth of the epithelium in small animals. To confirm our probe performance, we take use of USAF 1951 resolution target to test its lateral and axial resolution. It has lateral and axial resolution of 2.49um and 4.98um, respectively. When we collect the fluorescence imaging of colon, it shows that the field of view are 1000um×1000um (horizontal) and 1000um×430um (vertical). The horizontal and vertical cross-sectional images of fresh mouse colonic mucosa demonstrate imaging performance with this miniature instrument.

Evaluating mononuclear cells as nanoparticle delivery vehicles for the treatment of breast tumors

NASA Astrophysics Data System (ADS)

Murton, Jaclyn K.; Hu, Chelin; Ahmed, Mona M.; Hathaway, Helen J.; Nysus, Monique; Anderson Daniels, Tamara; Norenberg, Jeffrey P.; Adolphi, Natalie L.

2015-08-01

In breast cancer, certain types of circulating immune cells respond to long-range chemical signals from tumors by leaving the blood stream to actively infiltrate tumor tissue. The aim of this study was to evaluate whether immune cells could be used to deliver therapeutic nanoparticles into breast tumors in mice. Mononuclear splenocytes (MS) were harvested from donor mice, labeled with Indium-111, injected intravenously into immune-competent recipient mice (3 tumor-bearing and 3 control), and imaged longitudinally by SPECT/CT. For comparison, the biodistribution of bonemarrow derived macrophages (BMDM) in one pair of mice was also imaged. Quantitative analysis of the SPECT images demonstrates that, after 24 hours, the concentration of MS detected in mammary tumors is more than 3-fold higher than the concentration detected in normal mammary glands. The ratio of MS concentration in mammary tissue to MS concentration in non-target tissues (muscle, lung, heart, liver, spleen, and kidney) was enhanced in tumor-bearing mice (compared to controls), with statistical significance achieved for mammary/muscle (p<0.01), mammary/lung (p<0.05), and mammary/kidney (p<0.05). By contrast, BMDM did not show a different affinity for tumors relative to normal mammary tissue. MS were incubated with 100 nm red fluorescent nanoparticles, and flow cytometry demonstrated that ~35% of the MS population exhibited strong phagocytic uptake of the nanoparticles. After intravenous injection into tumor-bearing mice, fluorescence microscopy images of tumor sections show qualitatively that nanoparticle-loaded MS retain the ability to infiltrate mammary tumors. Taken together, these results suggest that MS carriers are capable of actively targeting therapeutic nanoparticles to breast tumors.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2001-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Final samples from Mir and Earth appeared histologically cartilaginous throughout their entire cross sections (5-8 mm thick), with the exception of fibrous outer capsules. Constructs grown on Earth (A) appeared to have a more organized extracellular matrix with more uniform collagen orientation as compared with constructs grown on Mir (B), but the average collagen fiber diameter was similar in the two groups (22 +- 2 nm) and comparable to that previously reported for developing articular cartilage. Randomly oriented collagen in Mir samples would be consistent with previous reports that microgravity disrupts fibrillogenesis. These are transmission electron micrographs of constructs from Mir (A) and Earth (B) groups at magnifications of x3,500 and x120,000 (Inset). The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Credit: Proceedings of the National Academy of Sciences.

The Connective Tissue Components of Optic Nerve Head Cupping in Monkey Experimental Glaucoma Part 1: Global Change

PubMed Central

Yang, Hongli; Ren, Ruojin; Lockwood, Howard; Williams, Galen; Libertiaux, Vincent; Downs, Crawford; Gardiner, Stuart K.; Burgoyne, Claude F.

2015-01-01

Purpose To characterize optic nerve head (ONH) connective tissue change within 21 monkey experimental glaucoma (EG) eyes, so as to identify its principal components. Methods Animals were imaged three to five times at baseline then every 2 weeks following chronic unilateral IOP elevation, and euthanized early through end-stage confocal scanning laser tomographic change. Optic nerve heads were serial-sectioned, three-dimensionally (3D) reconstructed, delineated, and quantified. Overall EG versus control eye differences were assessed by general estimating equations (GEE). Significant, animal-specific, EG eye change was required to exceed the maximum physiologic intereye differences in six healthy animals. Results Overall EG eye change was significant (P < 0.0026) and animal-specific EG eye change most frequent, for five phenomena (number of EG eyes and range of animal-specific change): posterior laminar deformation (21, −29 to −437 μm), laminar thickening (11, 20–73 μm) and thinning (3, −23 to −31 μm), scleral canal expansion (17, 20–139 μm), outward anterior (16, −16 to −124 μm) and posterior (17, −22 to −279 μm) laminar insertion migration, and peripapillary scleral bowing (11, 21–77 μm). Experimental glaucoma versus control eye laminar thickness differences were bimodal in behavior, being thickened in most EG eyes demonstrating the least deformation and less thickened or thinned in most EG eyes demonstrating the greatest deformation. Conclusions Our postmortem studies retrospectively identify five connective tissue components of ONH “cupping” in monkey EG which serve as targets for longitudinally staging and phenotyping ONH connective tissue alteration within all forms of monkey and human optic neuropathy. PMID:26641545

Structural normalization of the lymphoid tissue in asymptomatic HIV-infected patients after 48 weeks of potent antiretroviral therapy.

PubMed

Macías, J; Japón, M A; Leal, M; Sáez, C; Pineda, J A; Segura, D I; Ortega, J; Lissen, E

2001-12-07

The hallmark of HIV infection is the involution and destruction of lymphoid tissue. However, very little information exists on the effect of highly active antiretroviral therapy (HAART) on lymphoid tissue structure. To evaluate the effect of a HAART regimen after 48 weeks on the architecture and cell regeneration of tonsil lymphoid tissue in HIV-infected patients with CD4 T cell counts > or = 500/microl. From June 1997 to February 1998 all asymptomatic HIV-infected patients with CD4 T cell counts > or = 500/microl seen at our unit were offered quadruple antiretroviral therapy. Tonsil biopsies were obtained at baseline and at 48 weeks. Tonsil tissue sections were examined to evaluate structural and immunohistochemical changes by two blinded and independent pathologists. Cell numbers were counted for selected markers in T-dependent zones. Eleven patients were evaluable, six were excluded because of insufficient or inadequate sampling in at least one of the biopsies. Cellular depletion, plasma cell accumulation and prominent vessels were observed in all cases; three excluded patients with evaluable baseline biopsies showed similar tissue lesions. Follow-up biopsies demonstrated some degree of improvement in all patients. Germinal centres appeared in seven cases that were not seen at baseline. CD4 cell counts increased and CD8 cell counts decreased significantly in lymphoid tissue. An increase in CD45RA+ cells was observed; however, the proportion of CD45+Ki67+ cells did not differ between baseline and 48 weeks. This study shows an unexpected range of moderate to severe lymphoid tissue lesions in mildly immunosuppressed HIV-infected patients, which was partly restored after 48 weeks of HAART.

Morphological and biochemical studies of the elastic fibres in the craniomandibular joint articular disc of the tight-skin mouse.

PubMed

O'dell, N L; Burlison, S K; Starcher, B C; Pennington, C B

1996-05-01

The tight-skin (TSK) mouse is characterized by the hyperplasia of loose connective tissues, and of excessive growth of cartilage and of bones including the mandible. Since the fibroelastic connective tissues of the craniomandibular joint (CMJ) are essential to the functions of this joint, the present histological study compared the presence and general distribution of elastic fibres in CMJ discal tissues of TSK and normal mice. The excised CMJs were processed for light microscopy. The tissues were fixed, demineralized, embedded in paraffin, sectioned and then stained with resorcin-fuchsin to demonstrate elastic fibres. There were no obvious histological differences in either the amount or the distribution of elastic fibres in the discs from the two groups. In both groups, elastic fibres were found in the disc and in many of the attachments of the disc to the mandible and squamosal bone. In addition to the morphological preparations, articular discs and samples of lung tissue were excised from other mice and subjected to a radioimmunoassay for desmosine in order to estimate the amounts of elastin in these tissues; the amount of elastin was significantly reduced in the TSK lung, but the amounts of elastin in the TSK and normal CMJ discal tissues were not significantly different statistically. These morphological and histochemical results suggest that the distribution and quantity of elastic fibres in the TSK mouse disc are not significantly different from those in the normal mouse articular disc. Moreover, these data may be interpreted to either suggest a differential effect on the elastic fibres in different TSK tissues, or to support the suggestion that abnormal degradation of elastic fibres may not be characteristic of the TSK mouse.

Characterizing the heterogeneity of tumor tissues from spatially resolved molecular measures

PubMed Central

Zavodszky, Maria I.

2017-01-01

Background Tumor heterogeneity can manifest itself by sub-populations of cells having distinct phenotypic profiles expressed as diverse molecular, morphological and spatial distributions. This inherent heterogeneity poses challenges in terms of diagnosis, prognosis and efficient treatment. Consequently, tools and techniques are being developed to properly characterize and quantify tumor heterogeneity. Multiplexed immunofluorescence (MxIF) is one such technology that offers molecular insight into both inter-individual and intratumor heterogeneity. It enables the quantification of both the concentration and spatial distribution of 60+ proteins across a tissue section. Upon bioimage processing, protein expression data can be generated for each cell from a tissue field of view. Results The Multi-Omics Heterogeneity Analysis (MOHA) tool was developed to compute tissue heterogeneity metrics from MxIF spatially resolved tissue imaging data. This technique computes the molecular state of each cell in a sample based on a pathway or gene set. Spatial states are then computed based on the spatial arrangements of the cells as distinguished by their respective molecular states. MOHA computes tissue heterogeneity metrics from the distributions of these molecular and spatially defined states. A colorectal cancer cohort of approximately 700 subjects with MxIF data is presented to demonstrate the MOHA methodology. Within this dataset, statistically significant correlations were found between the intratumor AKT pathway state diversity and cancer stage and histological tumor grade. Furthermore, intratumor spatial diversity metrics were found to correlate with cancer recurrence. Conclusions MOHA provides a simple and robust approach to characterize molecular and spatial heterogeneity of tissues. Research projects that generate spatially resolved tissue imaging data can take full advantage of this useful technique. The MOHA algorithm is implemented as a freely available R script (see supplementary information). PMID:29190747

Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

PubMed

Lebelo, Ramokone L; Thys, Sofie; Benoy, Ina; Depuydt, Christophe E; Bogers, John-Paul; Bida, Meshack N; Mphahlele, M Jeffrey

2015-10-01

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections. © 2015 Wiley Periodicals, Inc.

Association between HIV replication and serum leptin levels: an observational study of a cohort of HIV-1-infected South African women

PubMed Central

2010-01-01

Background Advanced HIV infection can result in lipoatrophy and wasting, even in the absence of ongoing opportunistic infections, suggesting that HIV may directly affect adipose tissue amount and distribution. Methods We assessed the relationship of fat (measured using anthropometry, DEXA, MRI scans) or markers related to glucose and lipid metabolism with viral load in a cross-sectional sample of 83 antiretroviral-naïve HIV-1-infected South African women. A multivariable linear model was fitted to log10VL to assess the combined effect of these variables. Results In addition to higher T cell activation, women with viral load greater than the population median had lower waist circumference, body mass index and subcutaneous abdominal fat, as well as lower serum leptin. We demonstrate that leptin serum levels are inversely associated with viral replication, independent of the amount of adipose tissue. This association is maintained after adjusting for multiple variables associated with disease progression (i.e., cellular activation and innate immunity effector levels). Conclusions Our results demonstrate that serum leptin levels are inversely associated with viral replication, independent of disease progression: we postulate that leptin may affect viral replication. PMID:20822522

Introducing nuclei scatterer patterns into histology based intravascular ultrasound simulation framework

NASA Astrophysics Data System (ADS)

Kraft, Silvan; Karamalis, Athanasios; Sheet, Debdoot; Drecoll, Enken; Rummeny, Ernst J.; Navab, Nassir; Noël, Peter B.; Katouzian, Amin

2013-03-01

Medical ultrasonic grayscale images are formed from acoustic waves following their interactions with distributed scatterers within tissues media. For accurate simulation of acoustic wave propagation, a reliable model describing unknown parameters associated with tissues scatterers such as distribution, size and acoustic properties is essential. In this work, we introduce a novel approach defining ultrasonic scatterers by incorporating a distribution of cellular nuclei patterns in biological tissues to simulate ultrasonic response of atherosclerotic tissues in intravascular ultrasound (IVUS). For this reason, a virtual phantom is generated through manual labeling of different tissue types (fibrotic, lipidic and calcified) on histology sections. Acoustic properties of each tissue type are defined by assuming that the ultrasound signal is primarily backscattered by the nuclei of the organic cells within the intima and media of the vessel wall. This resulting virtual phantom is subsequently used to simulate ultrasonic wave propagation through the tissue medium computed using finite difference estimation. Subsequently B-mode images for a specific histological section are processed from the simulated radiofrequency (RF) data and compared with the original IVUS of the same tissue section. Real IVUS RF signals for these histological sections were obtained using a single-element mechanically rotating 40MHz transducer. Evaluation is performed by trained reviewers subjectively assessing both simulated and real B-mode IVUS images. Our simulation platform provides a high image quality with a very promising correlation to the original IVUS images. This will facilitate to better understand progression of such a chronic disease from micro-level and its integration into cardiovascular disease-specific models.

The Impact of Repeated Freeze-Thaw Cycles on the Quality of Biomolecules in Four Different Tissues.

PubMed

Ji, Xiaoli; Wang, Min; Li, Lingling; Chen, Fang; Zhang, Yanyang; Li, Qian; Zhou, Junmei

2017-10-01

High-quality biosamples are valuable resources for biomedical research. However, some tissues are stored without being sectioned into small aliquots and have to undergo repeated freeze-thaw cycles throughout prolonged experimentation. Little is known regarding the effects of repeated freeze-thaw cycles on the quality of biomolecules in tissues. The aim of this study was to evaluate the impact of repeated freeze-thaw (at room temperature or on ice) cycles on biomolecules and gene expression in four different types of tissues. Each fresh tissue was sectioned into seven aliquots and snap-frozen before undergoing repeated freeze-thaw cycles at room temperature or on ice. Biomolecules were extracted and analyzed. Both relative and absolute quantification were used to detect the changes in gene expression. The results indicated that the impact of repeated freeze-thaw cycles on RNA integrity varied by tissue type. Gene expression, including the housekeeping gene, was affected in RNA-degraded samples according to absolute quantification rather than relative quantification. Furthermore, our results suggest that thawing on ice could protect RNA integrity compared with thawing at room temperature. No obvious degradation of protein or DNA was observed with repeated freeze-thaw cycles either at room temperature or on ice. This research provides ample evidence for the necessity of sectioning fresh tissues into small aliquots before snap-freezing, thus avoiding degradation of RNA and alteration of gene expression resulting from repeated freeze-thaw cycles. For frozen tissue samples that were already in storage and had to be used repeatedly during their lifecycle, thawing on ice or sectioned at ultralow temperature is recommended.

Low proton conductance of plant cuticles and its relevance to the acid-growth theory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dreyer, S.A.; Seymour, V.; Cleland, R.E.

1981-09-01

Evidence obtained on the relation between the pH of the medium and the growth of intact stem sections is compatible with the acid-growth theory only if the proton conductance of the cuticle is an effective barrier to the entry or exit of protons from the tissue. By measuring the rate at which protons cross frozen-thawed epidermal strips of sunflower (Helianthus annus L.) and soybean hypocotyls (Glycine max Morr.) and enzymically isolated cuticles of Berberis aquifolium Persh. and tomato (Lycopersicum esculentum Mill.) fruit, we have now demonstrated the low proton conductance of the cuticular layer. Unless the conductance is enhanced bymore » abrasion of the cuticle or by removal of the cuticular waxes, proton movement into and out of a tissue across the cuticle will be significant only over long time periods.« less

Imaging of zymogen granules in fully wet cells: evidence for restricted mechanism of granule growth.

PubMed

Hammel, Ilan; Anaby, Debbie

2007-09-01

The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules.

Induction of Female-to-Male Sex Change in Adult Zebrafish by Aromatase Inhibitor Treatment

NASA Astrophysics Data System (ADS)

Takatsu, Kanae; Miyaoku, Kaori; Roy, Shimi Rani; Murono, Yuki; Sago, Tomohiro; Itagaki, Hideyuki; Nakamura, Masaru; Tokumoto, Toshinobu

2013-12-01

This study investigated whether undifferentiated germ and/or somatic stem cells remain in the differentiated ovary of a species that does not undergo sex changes under natural conditions and retain their sexual plasticity. The effect of aromatase inhibitor (AI)-treatment on sexually mature female zebrafish was examined. A 5-month AI treatment caused retraction of the ovaries after which testes-like organs appeared, and cyst structures filled with spermatozoa-like cells were observed in sections of these tissues. Electron microscopic observations revealed that these cells appeared as large sperm heads without tails. Sperm formation was re-examined after changing the diet to an AI-free food. A large number of normal sperm were obtained after eight weeks, and no formation of ovarian tissue was observed. Artificial fertilization using sperm from the sex-changed females was successful. These results demonstrated that sex plasticity remains in the mature ovaries of this species.

High-speed polarization sensitive optical frequency domain imaging with frequency multiplexing

PubMed Central

Yun, S.H.; Vakoc, B.J.; Shishkov, M.; Desjardins, A.E.; Park, B.H.; de Boer, J.F.; Tearney, G.J.; Bouma, B.E.

2009-01-01

Polarization sensitive optical coherence tomography (PS-OCT) provides a cross-sectional image of birefringence in biological samples that is complementary in many applications to the standard reflectance-based image. Recent ex vivo studies have demonstrated that birefringence mapping enables the characterization of collagen and smooth muscle concentration and distribution in vascular tissues. Instruments capable of applying these measurements percutaneously in vivo may provide new insights into coronary atherosclerosis and acute myocardial infarction. We have developed a polarization sensitive optical frequency domain imaging (PS-OFDI) system that enables high-speed intravascular birefringence imaging through a fiber-optic catheter. The novel design of this system utilizes frequency multiplexing to simultaneously measure reflectance of two incident polarization states, overcoming concerns regarding temporal variations of the catheter fiber birefringence and spatial variations in the birefringence of the sample. We demonstrate circular cross-sectional birefringence imaging of a human coronary artery ex vivo through a flexible fiber-optic catheter with an A-line rate of 62 kHz and a ranging depth of 6.2 mm. PMID:18542183

2-Phenylbenzothiazole conjugated with cyclopentadienyl tricarbonyl [CpM(CO)3] (M = Re, (99m)Tc) complexes as potential imaging probes for β-amyloid plaques.

PubMed

Jia, Jianhua; Cui, Mengchao; Dai, Jiapei; Liu, Boli

2015-04-14

Technetium-99m-labeled cyclopentadienyl tricarbonyl complexes conjugated with the 2-phenylbenzothiazole binding motif were synthesized. The rhenium surrogates , , and were demonstrated to have moderate to high affinities for Aβ1-42 aggregates with Ki values of 142, 76, 64 and 24 nM, respectively. During the fluorescence staining of brain sections of transgenic mice and patients with Alzheimer's disease, these rhenium complexes demonstrated perfect and intense labeling of Aβ plaques. Moreover, in in vitro autoradiography, (99m)Tc-labeled complexes clearly detected β-amyloid plaques on sections of brain tissue from transgenic mice, which confirmed the sufficient affinity of these tracers for Aβ plaques. However, these compounds did not show desirable properties in vivo, especially showing poor brain uptake (below 0.5% ID g(-1)), which will hinder the further development of these tracers as brain imaging agents. Nonetheless, it is encouraging that these (99m)Tc-labeled complexes designed by a conjugate approach displayed sufficient affinities for Aβ plaques.

21 CFR 878.4010 - Tissue adhesive.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., is a device used for adhesion of internal tissues and vessels. (2) Classification. Class III... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue adhesive. 878.4010 Section 878.4010 Food... DEVICES GENERAL AND PLASTIC SURGERY DEVICES Surgical Devices § 878.4010 Tissue adhesive. (a) Tissue...

21 CFR 878.4010 - Tissue adhesive.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., is a device used for adhesion of internal tissues and vessels. (2) Classification. Class III... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Tissue adhesive. 878.4010 Section 878.4010 Food... DEVICES GENERAL AND PLASTIC SURGERY DEVICES Surgical Devices § 878.4010 Tissue adhesive. (a) Tissue...

Comparison of Combined Aerobic and High-Force Eccentric Resistance Exercise With Aerobic Exercise Only for People With Type 2 Diabetes Mellitus

PubMed Central

Marcus, Robin L; Smith, Sheldon; Morrell, Glen; Addison, Odessa; Dibble, Leland E; Wahoff-Stice, Donna; LaStayo, Paul C

2008-01-01

Background and Purpose: The purpose of this study was to compare the outcomes between a diabetes exercise training program using combined aerobic and high-force eccentric resistance exercise and a program of aerobic exercise only. Subjects and Methods: Fifteen participants with type 2 diabetes mellitus (T2DM) participated in a 16-week supervised exercise training program: 7 (mean age=50.7 years, SD=6.9) in a combined aerobic and eccentric resistance exercise program (AE/RE group) and 8 (mean age=58.5 years, SD=6.2) in a program of aerobic exercise only (AE group). Outcome measures included thigh lean tissue and intramuscular fat (IMF), glycosylated hemoglobin, body mass index (BMI), and 6-minute walk distance. Results: Both groups experienced decreases in mean glycosylated hemoglobin after training (AE/RE group: −0.59% [95% confidence interval (CI)=−1.5 to 0.28]; AE group: −0.31% [95% CI=−0.60 to −0.03]), with no significant between-group differences. There was an interaction between group and time with respect to change in thigh lean tissue cross-sectional area, with the AE/RE group gaining more lean tissue (AE/RE group: 15.1 cm2 [95% CI=7.6 to 22.5]; AE group: −5.6 cm2 [95% CI=−10.4 to 0.76]). Both groups experienced decreases in mean thigh IMF cross-sectional area (AE/RE group: −1.2 cm2 [95% CI=−2.6 to 0.26]; AE group: −2.2 cm2 [95% CI=−3.5 to −0.84]) and increases in 6-minute walk distance (AE/RE group: 45.5 m [95% CI=7.5 to 83.6]; AE group: 29.9 m [95% CI=−7.7 to 67.5]) after training, with no between-group differences. There was an interaction between group and time with respect to change in BMI, with the AE/RE group experiencing a greater decrease in BMI. Discussion and Conclusion: Significant improvements in long-term glycemic control, thigh composition, and physical performance were demonstrated in both groups after participating in a 16-week exercise program. Subjects in the AE/RE group demonstrated additional improvements in thigh lean tissue and BMI. Improvements in thigh lean tissue may be important in this population as a means to increase resting metabolic rate, protein reserve, exercise tolerance, and functional mobility. PMID:18801851

Mast cells and angiogenesis in wound healing.

PubMed

Gaber, Mohamed A; Seliet, Iman A; Ehsan, Nermin A; Megahed, Mohamed A

2014-02-01

To investigate the role of mast cells and vascular endothelial growth factor (VEGF) as a mediator of angiogenesis to promote wound healing in surgical and pathological scars. The study was carried out on 40 patients who presented with active scar lesions. They were subdivided into 4 groups. They included granulation tissue (10 cases), surgical scar (10 cases), hypertrophic scar (10 cases), and keloid scar (10 cases). Also 10 healthy volunteers of the same age and sex were selected as a control group. Skin biopsies were taken from the patients and the control group. Skin biopsies from clinically assessed studied groups were processed for routine histology and embedded in paraffin. Four sections were prepared from each paraffin block. The first section was stained with hematoxylin and eosin for histological evaluation. The second and third sections were processed for immunostaining of mast cells that contain chymase (MCCs) and mast cells that contain tryptase (MCTs). The fourth section was processed for immunostaining of VEGF. MCCs exhibited mild expression in normal tissue, granulation tissue, and surgical, hypertrophic and keloid scars. MCTs exhibited mild expression in normal tissue, granulation tissue and keloid, whereas moderate expression was exhibited in hypertrophic and surgical scars. VEGF expression was absent in normal tissue, mild in keloid, surgical and hypertrophic scars, and moderate in keloids and granulation tissue. Mast cell expression variation among different scar types signals the pathological evolution of the lesion, and hence may guide the need for therapeutic intervention.

O-GlcNAcylation in oral squamous cell carcinoma.

PubMed

Kongkaew, Tassaporn; Aung, Win Pa Pa; Supanchart, Chayarop; Makeudom, Anupong; Langsa-Ard, Sarawat; Sastraruji, Thanapat; Chaiyarit, Ponlatham; Krisanaprakornkit, Suttichai

2018-03-01

Two post-translational mechanisms commonly demonstrated in various cancers are protein phosphorylation and glycosylation by O-linked β-N-acetylglucosamine (O-GlcNAc). However, only phosphorylation of the epidermal growth factor receptor (EGFR)/Akt pathway has been reported in oral squamous cell carcinoma (OSCC). Therefore, we aimed to determine both post-translational modifications in OSCC tissues and in oral cancer cells compared to normal tissues and oral keratinocytes and to find correlations of these modifications with histological grading. Thirty-two OSCC and ten normal formalin-fixed and paraffin-embedded sections were probed with the anti-O-GlcNAc, anti-O-GlcNAc transferase (OGT), anti-phosphorylated-EGFR tyr1173 , and anti-phosphorylated-Akt ser473 antibodies following standard immunohistochemistry. The immunohistochemical (IHC) score was determined using the Fromowitz standard. Whole cell lysates of oral cancer cells and normal oral keratinocytes were immunoblotted with the anti-O-GlcNAc antibody. The median IHC scores of O-GlcNAc or OGT between OSCC and normal tissues were not different, whereas those of phosphorylated-EGFR tyr1173 and phosphorylated-Akt ser473 were significantly higher in OSCC than normal tissues (P < .001 and P < .01, respectively). Similarly, expression of O-GlcNAcylated proteins in oral cancer cells and normal oral keratinocytes did not differ. In the OSCC group, the median IHC scores of O-GlcNAc and OGT were significantly lower than those of phosphorylated-EGFR tyr1173 and phosphorylated-Akt ser473 (P < .01 and P < .001, respectively). The IHC scores of O-GlcNAc or OGT were not determined to correlate with histological grading. Unlike other types of cancers, our findings demonstrate that the levels of O-GlcNAcylation are not significantly increased in OSCC tissues or in oral cancer cells and are not associated with the histological grading of OSCC. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Code of Federal Regulations, 2010 CFR

2010-04-01

... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

Bone marrow lesions in hip osteoarthritis are characterized by increased bone turnover and enhanced angiogenesis.

PubMed

Shabestari, M; Vik, J; Reseland, J E; Eriksen, E F

2016-10-01

Bone marrow lesions (BML), previously denoted bone marrow edema, are detected as water signals by magnetic resonance imaging (MRI). Previous histologic studies were unable to demonstrate any edematous changes at the tissue level. Therefore, our aim was to investigate the underlying biological mechanisms of the water signal in MRI scans of bone affected by BML. Tetracycline labeling in addition to water sensitive MRI scans of 30 patients planned for total hip replacement surgery was undertaken. Twenty-one femoral heads revealed BML on MRI, while nine were negative and used as controls (CON). Guided by the MRI images cylindrical biopsies were extracted from areas with BML in the femoral heads. Tissue sections from the biopsies were subjected to histomorphometric image analyses of the cancellous bone envelope. Patients with BML exhibited an average 40- and 18-fold increase of bone formation rate and mineralizing surface, respectively. Additionally, samples with BML demonstrated 2-fold reduction of marrow fat and 28-fold increase of woven bone. Immunohistochemical analysis showed a 4-fold increase of angiogenesis markers CD31 and von Willebrand Factor (vWF) in the BML-group compared to CON. This study indicates that BML are characterized by increased bone turnover, vascularity and angiogenesis in keeping with it being a reparatory process. Thus, the water signal, which is the hallmark of BML on MRI, is most probably reflecting increased tissue vascularity accompanying increased remodeling activity. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Comparison of the relaxation effect in vitro of nitroglycerin vs. fenoterol on human myometrial strips.

PubMed

David, M; Hamann, C; Chen, F C; Bruch, L; Lichtenegger, W

2000-01-01

Substance dose-related comparison of relaxation effect of nitroglycerin (GTN) and the beta 2-mimetic substance fenoterol in human myometrial tissue. Test criterion is the isometric force development of isolated human myometrial strips. These muscle strips were removed from the lower uterine segment at cesarean section. Fenoterol in concentrations of 3 x 10(-8)-10(-5) mol/l or GTN in concentrations of 1.7 x 10(-8)-5.8 x 10(-4) mol/l were applied to the 2 x 2 x 10-mm strips, which were fixed and maintained in tissue baths. The curves were plotted on line. The integral or the "area under the curve" (AUC) served as the parameter for muscle strip activity. A total of 100 strips from 20 patients were used. GTN demonstrated a significant relaxation effect in the in vitro model on human myometrial strips from pregnant women already treated with oxytocin. The effect was able to be enhanced to a point where oxytocin-induced contractions were completely absent. A relatively clear connection was demonstrated between dose and effect whereby increased muscle relaxation resulted at increased concentrations. Compared to GTN application, muscle strip relaxation was less pronounced under fenoterol; a complete inhibition of myometrial activity was not achieved under fenoterol. With respect to relaxation of the myometrial tissue samples the NO donor GTN is at least as potent as the standard tocolytic agent fenoterol in the in vitro model.

Early Prediction of Cancer Progression by Depth-Resolved Nanoscale Mapping of Nuclear Architecture from Unstained Tissue Specimens.

PubMed

Uttam, Shikhar; Pham, Hoa V; LaFace, Justin; Leibowitz, Brian; Yu, Jian; Brand, Randall E; Hartman, Douglas J; Liu, Yang

2015-11-15

Early cancer detection currently relies on screening the entire at-risk population, as with colonoscopy and mammography. Therefore, frequent, invasive surveillance of patients at risk for developing cancer carries financial, physical, and emotional burdens because clinicians lack tools to accurately predict which patients will actually progress into malignancy. Here, we present a new method to predict cancer progression risk via nanoscale nuclear architecture mapping (nanoNAM) of unstained tissue sections based on the intrinsic density alteration of nuclear structure rather than the amount of stain uptake. We demonstrate that nanoNAM detects a gradual increase in the density alteration of nuclear architecture during malignant transformation in animal models of colon carcinogenesis and in human patients with ulcerative colitis, even in tissue that appears histologically normal according to pathologists. We evaluated the ability of nanoNAM to predict "future" cancer progression in patients with ulcerative colitis who did and did not develop colon cancer up to 13 years after their initial colonoscopy. NanoNAM of the initial biopsies correctly classified 12 of 15 patients who eventually developed colon cancer and 15 of 18 who did not, with an overall accuracy of 85%. Taken together, our findings demonstrate great potential for nanoNAM in predicting cancer progression risk and suggest that further validation in a multicenter study with larger cohorts may eventually advance this method to become a routine clinical test. ©2015 American Association for Cancer Research.

The feasibility of non-contact ultrasound for medical imaging.

PubMed

Clement, G T; Nomura, H; Adachi, H; Kamakura, T

2013-09-21

High intensity focused ultrasound in air may provide a means for medical and biological imaging without direct coupling of an ultrasound probe. In this study, an approach based on highly focused ultrasound in air is described and the feasibility of the technique is assessed. The overall method is based on the observations that (1) ultrasound in air has superior focusing ability and stronger nonlinear harmonic generation as compared to tissue propagation and (2) a tightly focused field directed into tissue causes point-like spreading that may be regarded as a source for generalized diffraction tomography. Simulations of a spherically-curved transducer are performed, where the transducer's radiation pattern is directed from air into tissue. It is predicted that a focal pressure of 162 dB (2.5 kPa) is sufficient to direct ultrasound through the body, and provide a small but measurable signal (∼1 mPa) upon exit. Based on the simulations, a 20 cm diameter array consisting of 298 transducers is constructed. For this feasibility study, a 40 kHz resonance frequency is selected based on the commercial availability of such transducers. The array is used to focus through water and acrylic phantoms, and the time history of the exiting signal is evaluated. Sufficient data are acquired to demonstrate a low-resolution tomographic reconstruction. Finally, to demonstrate the feasibility to record a signal in vivo, a 75 mm × 55 mm section of a human hand is imaged in a C-mode configuration.

Immunohistochemical detection of chlamydiae in formalin-fixed tissue sections: comparison of a monoclonal antibody with yolk derived antibodies (IgY).

PubMed

Kunz, U S; Pospischil, A; Paccaud, M F

1991-06-01

Immunohistological detection of chlamydiae in formalin-fixed and paraffin-embedded sections of various organs from several species is described. In a retrospective study, two antisera, a commercially available monoclonal murine antibody (IgMur) and vitelline immunoglobulins (IgY), extracted from the egg yolk of immunized hens, were compared and tested for their applicability under routine condition. Both antisera were applied to tissues from which chlamydiae had been isolated or in which the presence of chlamydiae had been suspected in specially stained sections. Antigen labelling was optimal with the monoclonal antibody. Vitelline immunoglobulins produced some unspecific reactions, especially in lung tissue sections. Because of the antigenic relationship between the vitelline antibodies and tissues of birds, IgY are not suitable for the detection of psittacosis on avian substrates, when using an indirect immunological method. Staining in other tissues e.g. intestine or placenta was of equal quality as that attained with monoclonal antibodies. Depending on the advantages and disadvantages in every individual case, one of the two antibodies may be chosen for further studies. Vitelline antibodies should be preferred with respect to animal welfare.

Automated detection of breast cancer in resected specimens with fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Phipps, Jennifer E.; Gorpas, Dimitris; Unger, Jakob; Darrow, Morgan; Bold, Richard J.; Marcu, Laura

2018-01-01

Re-excision rates for breast cancer lumpectomy procedures are currently nearly 25% due to surgeons relying on inaccurate or incomplete methods of evaluating specimen margins. The objective of this study was to determine if cancer could be automatically detected in breast specimens from mastectomy and lumpectomy procedures by a classification algorithm that incorporated parameters derived from fluorescence lifetime imaging (FLIm). This study generated a database of co-registered histologic sections and FLIm data from breast cancer specimens (N  =  20) and a support vector machine (SVM) classification algorithm able to automatically detect cancerous, fibrous, and adipose breast tissue. Classification accuracies were greater than 97% for automated detection of cancerous, fibrous, and adipose tissue from breast cancer specimens. The classification worked equally well for specimens scanned by hand or with a mechanical stage, demonstrating that the system could be used during surgery or on excised specimens. The ability of this technique to simply discriminate between cancerous and normal breast tissue, in particular to distinguish fibrous breast tissue from tumor, which is notoriously challenging for optical techniques, leads to the conclusion that FLIm has great potential to assess breast cancer margins. Identification of positive margins before waiting for complete histologic analysis could significantly reduce breast cancer re-excision rates.

Corals Use Similar Immune Cells and Wound-Healing Processes as Those of Higher Organisms

PubMed Central

Palmer, Caroline V.; Traylor-Knowles, Nikki G.; Willis, Bette L.; Bythell, John C.

2011-01-01

Sessile animals, like corals, frequently suffer physical injury from a variety of sources, thus wound-healing mechanisms that restore tissue integrity and prevent infection are vitally important for defence. Despite the ecological importance of reef-building corals, little is known about the cells and processes involved in wound healing in this group or in phylogenetically basal metazoans in general. A histological investigation into wound healing of the scleractinian coral Porites cylindrica at 0 h, 6 h, 24 h and 48 h after injury revealed differences in cellular components between injured and healthy tissues. Cell counts of the obligate endosymbiont, Symbiodinium, and melanin volume fraction analysis revealed rapid declines in both Symbiodinium abundance and tissue cross-sectional area occupied by melanin-containing granular cells after injury. Four phases of wound healing were identified, which are similar to phases described for both vertebrates and invertebrates. The four phases included (i) plug formation via the degranulation of melanin-containing granular cells; (ii) immune cell infiltration (inflammation); (iii) granular tissue formation (proliferation); and (iv) maturation. This study provides detailed documentation of the processes involved in scleractinian wound healing for the first time and further elucidates the roles of previously-described immune cells, such as fibroblasts. These results demonstrate the conservation of wound healing processes from anthozoans to humans. PMID:21887359

Polarization properties of amyloid-beta plaques in Alzheimer's disease (Conference Presentation)

NASA Astrophysics Data System (ADS)

Baumann, Bernhard; Wöhrer, Adelheid; Ricken, Gerda; Pircher, Michael; Kovacs, Gabor G.; Hitzenberger, Christoph K.

2016-03-01

In histopathological practice, birefringence is used for the identification of amyloidosis in numerous tissues. Amyloid birefringence is caused by the parallel arrangement of fibrous protein aggregates. Since neurodegenerative processes in Alzheimer's disease (AD) are also linked to the formation of amyloid-beta (Aβ) plaques, optical methods sensitive to birefringence may act as non-invasive tools for Aβ identification. At last year's Photonics West, we demonstrated polarization-sensitive optical coherence tomography (PS-OCT) imaging of ex vivo cerebral tissue of advanced stage AD patients. PS-OCT provides volumetric, structural imaging based on both backscatter contrast and tissue polarization properties. In this presentation, we report on polarization-sensitive neuroimaging along with numerical simulations of three-dimensional Aβ plaques. High speed PS-OCT imaging was performed using a spectral domain approach based on polarization maintaining fiber optics. The sample beam was interfaced to a confocal scanning microscope arrangement. Formalin-fixed tissue samples as well as thin histological sections were imaged. For comparison to the PS-OCT results, ray propagation through plaques was modeled using Jones analysis and various illumination geometries and plaque sizes. Characteristic polarization patterns were found. The results of this study may not only help to understand PS-OCT imaging of neuritic Aβ plaques but may also have implications for polarization-sensitive imaging of other fibrillary structures.

Oxidized LDL accumulation in experimental renal ischemia reperfusion injury model.

PubMed

Kulah, Eyup; Tascilar, Oge; Acikgoz, Serefden; Tekin, Ishak Ozel; Karadeniz, Guldeniz; Can, Murat; Gun, Banu; Barut, Figen; Comert, Mustafa

2007-01-01

The aim of this study was to identify oxidative damage of kidney during ischemia reperfusion injury (IRI) by evaluating changes in lipid peroxidation markers in tissue and blood by an experimental model. Oxidized LDL (ox-LDL) was used as an oxidative stress biomarker, whereas paraoxonase (PON-1) activity was used as an antioxidative biomarker. Sixty-three male Wistar rats were randomly assigned into three groups: renal IRI, sham, and control. In the renal IRI group, the right kidney was removed and the artery and vein of the left kidney were clamped for 90 minutes. The presence of ox-LDL in the kidney tissue sections was determined by using an immunofluorescent staining method. The plasma ox-LDL levels did not increase significantly at the 24th hour following IRI, made a peak at the 48th hour, and declined at the 72nd hour. Accumulation of ox-LDL was detected in the kidney tissue on the 24th, 48th, and 72nd hours of the renal IRI. Serum PON-1 levels have peaked on the 24th hour and then declined. This study demonstrates the accumulation of ox-LDL molecules in the renal tissues of the IRI model. Future strategies aimed to reduce the lipid peroxidation during the initial hours of renal IRI may be useful to prevent complications of ischemia.

Endophytic colonization of olive roots by the biocontrol strain Pseudomonas fluorescens PICF7.

PubMed

Prieto, Pilar; Mercado-Blanco, Jesús

2008-05-01

Confocal microscopy combined with three-dimensional olive root tissue sectioning was used to provide evidence of the endophytic behaviour of Pseudomonas fluorescens PICF7, an effective biocontrol strain against Verticillium wilt of olive. Two derivatives of the green fluorescent protein (GFP), the enhanced green and the red fluorescent proteins, have been used to visualize simultaneously two differently fluorescently tagged populations of P. fluorescens PICF7 within olive root tissues at the single cell level. The time-course of colonization events of olive roots cv. Arbequina by strain PICF7 and the localization of tagged bacteria within olive root tissues are described. First, bacteria rapidly colonized root surfaces and were predominantly found in the differentiation zone. Thereafter, microscopy observations showed that PICF7-tagged populations eventually disappeared from the root surface, and increasingly colonized inner root tissues. Localized and limited endophytic colonization by the introduced bacteria was observed over time. Fluorescent-tagged bacteria were always visualized in the intercellular spaces of the cortex region, and no colonization of the root xylem vessels was detected at any time. To the best of our knowledge, this is the first time this approach has been used to demonstrate endophytism of a biocontrol Pseudomonas spp. strain in a woody host such as olive using a nongnotobiotic system.

Detection, Mapping, and Quantification of Single Walled Carbon Nanotubes in Histological Specimens with Photoacoustic Microscopy

PubMed Central

Mikos, Antonios G.; Jansen, John A.; Shroyer, Kenneth R.; Wang, Lihong V.; Sitharaman, Balaji

2012-01-01

Aims In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Materials and Methods Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Results Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. Conclusions The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs. PMID:22496892

Morph-X-Select: Morphology-based tissue aptamer selection for ovarian cancer biomarker discovery

PubMed Central

Wang, Hongyu; Li, Xin; Volk, David E.; Lokesh, Ganesh L.-R.; Elizondo-Riojas, Miguel-Angel; Li, Li; Nick, Alpa M.; Sood, Anil K.; Rosenblatt, Kevin P.; Gorenstein, David G.

2016-01-01

High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5′dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of human ovarian tissue and human microvascular endothelial cells. This new Morph-X-Select method allows us to select high-affinity aptamers and their associated target proteins in a specific and accurate way, and could be used for personalized biomarker discovery to improve medical decision-making and to facilitate the development of targeted therapies to achieve more favorable outcomes. PMID:27839510

Quantification of carotid atherosclerotic plaque components using feature space analysis and magnetic resonance imaging.

PubMed

Karmonik, Christof; Basto, Pamela; Morrisett, Joel D

2006-01-01

Atherosclerosis is one of the main causes of cardiovascular disease, accounting for more than one third of all deaths in the United States, there is a growing need to develop non-invasive techniques to assess the severity of atherosclerotic plaque burden. Recent research has suggested that not the size of the atherosclerotic plaque but rather its composition is indicative for plaque rupture as the underlying event of stroke and acute coronary syndrome. With its excellent soft-tissue contrast, magnetic resonance imaging (MRI) is a favored modality for examining plaque composition. In an ex-vivo study, aimed to show the feasibility of quantifying the components of carotid atherosclerotic plaques in-vivo, we acquired multi-contrast MRI images of 13 freshly excised endarterectomy tissues with commercially available MRI sequences and a human surface coil. Feature space analysis (FSA) was utilized in four representative tissues to determine the total relative abundance of calcific, lipidic, fibrotic, thrombotic and normal components as well as in consecutive 2 mm sections across the carotid bifurcation in each tissue. Excellent qualitative agreement between the FSA results and the results obtained from histological methods was observed. This study demonstrates the feasibility of combining MRI with FSA to quantify carotid atherosclerotic plaques in-vivo.

Matrix Sublimation/Recrystallization for Imaging Proteins by Mass Spectrometry at High Spatial Resolution

PubMed Central

Yang, Junhai; Caprioli, Richard M.

2011-01-01

We have employed matrix deposition by sublimation for protein image analysis on tissue sections using a hydration/recrystallization process that produces high quality MALDI mass spectra and high spatial resolution ion images. We systematically investigated different washing protocols, the effect of tissue section thickness, the amount of sublimated matrix per unit area and different recrystallization conditions. The results show that an organic solvent rinse followed by ethanol/water rinses substantially increased sensitivity for the detection of proteins. Both the thickness of tissue section and amount of sinapinic acid sublimated per unit area have optimal ranges for maximal protein signal intensity. Ion images of mouse and rat brain sections at 50, 20 and 10 µm spatial resolution are presented and are correlated with H&E stained optical images. For targeted analysis, histology directed imaging can be performed using this protocol where MS analysis and H&E staining are performed on the same section. PMID:21639088

Application of tissue mesodissection to molecular cancer diagnostics.

PubMed

Krizman, David; Adey, Nils; Parry, Robert

2015-02-01

To demonstrate clinical application of a mesodissection platform that was developed to combine advantages of laser-based instrumentation with the speed/ease of manual dissection for automated dissection of tissue off standard glass slides. Genomic analysis for KRAS gene mutation was performed on formalin fixed paraffin embedded (FFPE) cancer patient tissue that was dissected using the mesodissection platform. Selected reaction monitoring proteomic analysis for quantitative Her2 protein expression was performed on FFPE patient tumour tissue dissected by a laser-based instrument and the MilliSect instrument. Genomic analysis demonstrates highly confident detection of KRAS mutation specifically in lung cancer cells and not the surrounding benign, non-tumour tissue. Proteomic analysis demonstrates Her2 quantitative protein expression in breast cancer cells dissected manually, by laser-based instrumentation and by MilliSect instrumentation (mesodissection). Slide-mounted tissue dissection is commonly performed using laser-based instruments or manually scraping tissue by scalpel. Here we demonstrate that the mesodissection platform as performed by the MilliSect instrument for tissue dissection is cost-effective; it functions comparably to laser-based dissection and which can be adopted into a clinical diagnostic workflow. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Quantum Cascade Laser-Based Infrared Microscopy for Label-Free and Automated Cancer Classification in Tissue Sections.

PubMed

Kuepper, Claus; Kallenbach-Thieltges, Angela; Juette, Hendrik; Tannapfel, Andrea; Großerueschkamp, Frederik; Gerwert, Klaus

2018-05-16

A feasibility study using a quantum cascade laser-based infrared microscope for the rapid and label-free classification of colorectal cancer tissues is presented. Infrared imaging is a reliable, robust, automated, and operator-independent tissue classification method that has been used for differential classification of tissue thin sections identifying tumorous regions. However, long acquisition time by the so far used FT-IR-based microscopes hampered the clinical translation of this technique. Here, the used quantum cascade laser-based microscope provides now infrared images for precise tissue classification within few minutes. We analyzed 110 patients with UICC-Stage II and III colorectal cancer, showing 96% sensitivity and 100% specificity of this label-free method as compared to histopathology, the gold standard in routine clinical diagnostics. The main hurdle for the clinical translation of IR-Imaging is overcome now by the short acquisition time for high quality diagnostic images, which is in the same time range as frozen sections by pathologists.

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for papillomavirus DNA and antigen.

PubMed

Kidney, B A; Haines, D M; Ellis, J A; Burnham, M L; Teifke, J P; Czerwinski, G; Jackson, M L

2001-06-01

To determine whether vaccine site-associated sarcomas (VSS) from cats contain papillomavirus antigen or DNA. 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. Sections from each tissue block were evaluated for papillomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-bovine papillomavirus type-1 antibody. The DNA was extracted from sections of each tissue block, and polymerase chain reaction assays were performed, using primers designed to amplify regions of the E5 gene of bovine papillomavirus and consensus primers designed to amplify a region of the L1 gene of animal papillomaviruses. Sections from 20 of the tissue blocks were evaluated by use of nonradioactive in situ hybridization for bovine papillomavirus DNA. Papillomavirus antigen and DNA were not detected in any of the VSS. Results suggest that papillomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

Digital quantification of fibrosis in liver biopsy sections: description of a new method by Photoshop software.

PubMed

Dahab, Gamal M; Kheriza, Mohamed M; El-Beltagi, Hussien M; Fouda, Abdel-Motaal M; El-Din, Osama A Sharaf

2004-01-01

The precise quantification of fibrous tissue in liver biopsy sections is extremely important in the classification, diagnosis and grading of chronic liver disease, as well as in evaluating the response to antifibrotic therapy. Because the recently described methods of digital image analysis of fibrosis in liver biopsy sections have major flaws, including the use of out-dated techniques in image processing, inadequate precision and inability to detect and quantify perisinusoidal fibrosis, we developed a new technique in computerized image analysis of liver biopsy sections based on Adobe Photoshop software. We prepared an experimental model of liver fibrosis involving treatment of rats with oral CCl4 for 6 weeks. After staining liver sections with Masson's trichrome, a series of computer operations were performed including (i) reconstitution of seamless widefield images from a number of acquired fields of liver sections; (ii) image size and solution adjustment; (iii) color correction; (iv) digital selection of a specified color range representing all fibrous tissue in the image and; (v) extraction and calculation. This technique is fully computerized with no manual interference at any step, and thus could be very reliable for objectively quantifying any pattern of fibrosis in liver biopsy sections and in assessing the response to antifibrotic therapy. It could also be a valuable tool in the precise assessment of antifibrotic therapy to other tissue regardless of the pattern of tissue or fibrosis.

An inexpensive alternative to routine section adhesives for histology: the mucilaginous substance of the Assyrian plum.

PubMed

Al-Shammary, Hayfaa A

2014-05-01

We found that the mucilaginous substance of the Assyrian plum, Cordia myxa, can be used as an adhesive for attaching sections of animal tissues to slides. Unlike Mayer's albumen, this material left no stainable residue and had no noticeable effect on the histological structure of the tissue sections. The mucilaginous substance of C. myxa is a useful and inexpensive alternative to standard adhesives.

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

PubMed

Joshi, Molishree; Keith Pittman, H; Haisch, Carl; Verbanac, Kathryn

2008-09-01

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain.

PubMed

Terr, L I

1986-09-01

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.

Soft tissue wound healing at teeth, dental implants and the edentulous ridge when using barrier membranes, growth and differentiation factors and soft tissue substitutes.

PubMed

Vignoletti, Fabio; Nunez, Javier; Sanz, Mariano

2014-04-01

To review the biological processes of wound healing following periodontal and periimplant plastic surgery when different technologies are used in a) the coverage of root and implant dehiscences, b) the augmentation of keratinized tissue (KT) and c) the augmentation of soft tissue volume. An electronic search from The National Library of Medicine (MEDLINE-PubMed) was performed: English articles with research focus in oral soft tissue regeneration, providing histological outcomes, either from animal experimental studies or human biopsy material were included. Barrier membranes, enamel matrix derivatives, growth factors, allogeneic and xenogeneic soft tissue substitutes have been used in soft tissue regeneration demonstrating different degrees of regeneration. In root coverage, these technologies were able to improve new attachment, although none has shown complete regeneration. In KT augmentation, tissue-engineered allogenic products and xenogeneic collagen matrixes demonstrated integration within the host connective tissue and promotion of keratinization. In soft tissue augmentation and peri-implant plastic surgery there are no histological data currently available. Soft tissue substitutes, growth differentiation factors demonstrated promising histological results in terms of soft tissue regeneration and keratinization, whereas there is a need for further studies to prove their added value in soft tissue augmentation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rotational distortion correction in endoscopic optical coherence tomography based on speckle decorrelation

PubMed Central

Uribe-Patarroyo, Néstor; Bouma, Brett E.

2015-01-01

We present a new technique for the correction of nonuniform rotation distortion in catheter-based optical coherence tomography (OCT), based on the statistics of speckle between A-lines using intensity-based dynamic light scattering. This technique does not rely on tissue features and can be performed on single frames of data, thereby enabling real-time image correction. We demonstrate its suitability in a gastrointestinal balloon-catheter OCT system, determining the actual rotational speed with high temporal resolution, and present corrected cross-sectional and en face views showing significant enhancement of image quality. PMID:26625040

Prostaglandin release from in vitro guinea-pig gallbladder.

PubMed

Booker, M L; LaMorte, W W

1983-02-01

In order to study prostaglandin release from guinea pig gallbladder, full thickness tissue sections were incubated for one hour in Krebs solution. Extraction and two dimensional chromatography of incubation media obtained in the presence of radio-labelled arachidonic acid demonstrated the presence of PGE2, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane B2. These results were supported by radioimmunoassay of incubations conducted in the absence of exogenous arachidonate and in the presence of varying concentrations of unlabelled exogenous arachidonate. The previously reported predominance of PGE2 was only seen at high concentrations of exogenous arachidonate.

Optical Coherence Tomography

PubMed Central

Huang, David; Swanson, Eric A.; Lin, Charles P.; Schuman, Joel S.; Stinson, William G.; Chang, Warren; Hee, Michael R.; Flotte, Thomas; Gregory, Kenton; Puliafito, Carmen A.; Fujimoto, James G.

2015-01-01

A technique called optical coherence tomography (OCT) has been developed for noninvasive cross-sectional imaging in biological systems. OCT uses low-coherence interferometry to produce a two-dimensional image of optical scattering from internal tissue microstructures in a way that is analogous to ultrasonic pulse-echo imaging. OCT has longitudinal and lateral spatial resolutions of a few micrometers and can detect reflected signals as small as ~10−10 of the incident optical power. Tomographic imaging is demonstrated in vitro in the peripapillary area of the retina and in the coronary artery, two clinically relevant examples that are representative of transparent and turbid media, respectively. PMID:1957169

Automatic segementation of histological structures in normal and neoplastic mammary gland tissue sections

NASA Astrophysics Data System (ADS)

Fernandez-Gonzalez, Rodrigo; Deschamps, Thomas; Idica, Adam; Malladi, Ravikanth; Ortiz de Solorzano, Carlos

2003-07-01

In this paper we present a scheme for real time segmentation of histological structures in microscopic images of normal and neoplastic mammary gland sections. Paraffin embedded or frozen tissue blocks are sliced, and sections are stained with hematoxylin and eosin (H&E). The sections are then imaged using conventional bright field microscopy. The background of the images is corrected by arithmetic manipulation using a "phantom." Then we use the fast marching method with a speed function that depends on the brightness gradient of the image to obtain a preliminary approximation to the boundaries of the structures of interest within a region of interest (ROI) of the entire section manually selected by the user. We use the result of the fast marching method as the initial condition for the level set motion equation. We run this last method for a few steps and obtain the final result of the segmentation. These results can be connected from section to section to build a three-dimensional reconstruction of the entire tissue block that we are studying.

Visualisation of Collagen in fixed skeletal muscle tissue using fluorescently tagged Collagen binding protein CNA35.

PubMed

Mohammadkhah, Melika; Simms, Ciaran K; Murphy, Paula

2017-02-01

Detection and visualisation of Collagen structure are important to understand the relationship between mechanical behaviour and microstructure in skeletal muscle since Collagen is the main structural protein in animal connective tissues, and is primarily responsible for their passive load-bearing properties. In the current study, the direct detection and visualization of Collagen using fluorescently tagged CNA35 binding protein (fused to EGFP or tdTomato) is reported for the first time on fixed skeletal muscle tissue. This Technical Note also establishes a working protocol by examining tissue preparation, dilution factor, exposure time etc. for sensitivity and specificity. Penetration of the binding protein into intact mature skeletal muscle was found to be very limited, but detection works well on tissue sections with higher sensitivity on wax embedded sections compared to frozen sections. CNA35 fused to tdTomato has a higher sensitivity than CNA35 fused to EGFP but both show specific detection. Best results were obtained with 15μm wax embedded sections, with blocking of non-specific binding in 1% BSA and antigen retrieval in Sodium Citrate. There was a play-off between dilution of the binding protein and time of incubation but both CNA35-tdTomato and CNA35-EGFP worked well with approximately 100μg/ml of purified protein with overnight incubation, while CNA35-tdTomato could be utilized at 5 fold less concentration. This approach can be applied to study the relationship between skeletal muscle micro-structure and to observe mechanical response to applied deformation. It can be used more broadly to detect Collagen in a variety of fixed tissues, useful for structure-functions studies, constitutive modelling, tissue engineering and assessment of muscle tissue pathologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Placenta-specific drug delivery by trophoblast-targeted nanoparticles in mice

PubMed Central

Zhang, Baozhen; Tan, Lunbo; Yu, Yan; Wang, Baobei; Chen, Zhilong; Han, Jinyu; Li, Mengxia; Chen, Jie; Xiao, Tianxia; Ambati, Balamurali K; Cai, Lintao; Yang, Qing; Nayak, Nihar R; Zhang, Jian; Fan, Xiujun

2018-01-01

Rationale: The availability of therapeutics to treat pregnancy complications is severely lacking, mainly due to the risk of harm to the fetus. In placental malaria, Plasmodium falciparum-infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) on the surfaces of trophoblasts. Based on this principle, we have developed a method for targeted delivery of payloads to the placenta using a synthetic placental CSA-binding peptide (plCSA-BP) derived from VAR2CSA, a CSA-binding protein expressed on IEs. Methods: A biotinylated plCSA-BP was used to examine the specificity of plCSA-BP binding to mouse and human placental tissue in tissue sections in vitro. Different nanoparticles, including plCSA-BP-conjugated nanoparticles loaded with indocyanine green (plCSA-INPs) or methotrexate (plCSA-MNPs), were administered intravenously to pregnant mice to test their efficiency at drug delivery to the placenta in vivo. The tissue distribution and localization of the plCSA-INPs were monitored in live animals using an IVIS imaging system. The effect of plCSA-MNPs on fetal and placental development and pregnancy outcome were examined using a small-animal high-frequency ultrasound (HFUS) imaging system, and the concentrations of methotrexate in fetal and placental tissues were measured using high-performance liquid chromatography (HPLC). Results: plCSA-BP binds specifically to trophoblasts and not to other cell types in the placenta or to CSA-expressing cells in other tissues. Moreover, we found that intravenously administered plCSA-INPs accumulate in the mouse placenta, and ex vivo analysis of the fetuses and placentas confirmed placenta-specific delivery of these nanoparticles. We also demonstrate successful delivery of methotrexate specifically to placental cells by plCSA-BP-conjugated nanoparticles, resulting in dramatic impairment of placental and fetal development. Importantly, plCSA-MNPs treatment had no apparent adverse effects on maternal tissues. Conclusion: These results demonstrate that plCSA-BP-guided nanoparticles could be used for the targeted delivery of payloads to the placenta and serve as a novel placenta-specific drug delivery option. PMID:29774074

[Experimental xenogenic immune pancreatitis. --Immunohistological, enzyme histochemical and ultrastructural studies (author's transl)].

PubMed

Nizze, H

1975-01-01

Repeated intraperitoneal injections of anti-mouse pancreas rabbit serum or of anti-mouse pancreas guinea pig serum produce a chronical sclerotizing pancreatitis. This study has the aim to contribute to the further elucidation of the changes which occur in the acinar cells, as well as to the etiology and pathogenesis of immune pancreatitis, by means of immunohistological, enzyme histochemical and electron microscopic studies. Anti-mouse pancreas rabbit serum was obtained by sensitization of rabbits with an admixture of AB-mouse pancreas extract (100,000 g - supernatant) and complete Freund's adjuvant [details see NIZZE, Exp. Path. (1975a)]. The presence of precipitating mouse pancreas antibodies in the rabbit serum was ascertained by the agargel diffusion test according to Duchterlony (1958). The experiments were performed with 54 adult male white mice (AB colony strain) of 22 to 30 g.b.s. (averagely 26 g). The animals were divided into 4 groups which were treated as follows: 1. 24 mice with anti-mouse pancreas rabbit serum, 2. 12 mice with rabbit normal serum, 3. 12 mice with physiological saline, 4. 6 mice remained untreated (controls) Always 4 animals of the group 1 as well as each 2 of the groups 2 and 3 were administered in total 1, 3, 5, 9, 17 or 33 intraperitoneal injections of 0.3 ml of the correspondent serum or with physiological saline within 3 hours, 1, 2, 4, 8 or 16 days. The last injection was regularly applied 3 hours before sacrification by decapitation. The time of sacrification was always at 11.00 o'clock a.m. For immunohistological and enzyme histochemical investigations 10 mum thick cryostat sections were prepared consisting of pancreatic specimens piled up to a bloc. In each case the tissue samples were taken from the experimental animals and from one control animal sacrificed at the same day. The sections were incubated in FITC-labelled anti-rabbit globulin goat serum at room temperature for 30 min in a moist chamber. For control of specificity were employed: a) initial incubation of equal sections with unlabelled anti-rabbit globulin goat serum for 30 min ("blocking test''), b) pancreatic tissue specimens of each one untreated control animal present in the cryostat sections and thus incubated like the pancreatic tissue of the experimental animals, c) native nonincubated cryostat sections from the same bloc to exclude nonspecific autofluorescence. Evaluation of the sections was done in a Zeiss-Lg-microscope with HBO-50 high pressure mercury lamp. Exciter filters were UG 1/3.5 and 1/1.5, the eyepiece was screened with a GG 9/1 filter photographs were taken on ORWO X-ray film RS 2 (VEB Filmfabrik Wolfen). The enzyme histochemical studies were performed on cryostat sections of the same tissue bloc using the following methods: lead nitrate- or calcium-Co-method after GOMORI (1952) for demonstration of acid and alkaline phosphatase, naphthylacetate method (NACHLAS and SELIGMAN 1945) for nonspecific esterase, MTT-co-method (PEARSE et al...

Optically sectioned wide-field fluorescence lifetime imaging endoscopy enabled by structured illumination (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hinsdale, Taylor; Malik, Bilal H.; Rico-Jimenez, Jose J.; Jo, Javier A.; Maitland, Kristen C.

2016-03-01

We present a wide-field fluorescence lifetime imaging (FLIM) system with optical sectioning by structured illumination microscopy (SIM). FLIM measurements were made using a time gated ICCD camera in conjunction with a pulsed nitrogen dye laser operating at 450 nm. Intensity images were acquired at multiple time delays from a trigger initiated by a laser pulse to create a wide-field FLIM image, which was then combined with three phase SIM to provide optical sectioning. Such a mechanism has the potential to increase the reliability and accuracy of the FLIM measurements by rejecting background intensity. SIM also provides the opportunity to create volumetric FLIM images with the incorporation of scanning mechanisms for the sample plane. We present multiple embodiments of such a system: one as a free space endoscope and the other as a fiber microendoscope enabled by the introduction of a fiber bundle. Finally, we demonstrate the efficacy of such an imaging system by imaging dyes embedded in a tissue phantom.

Periodontal plastic surgery: thermal effect analysis using Erbium:YAG Kesler's handpiece. Histochemical evaluation by Picrosirius red stain and polarization microscopy for collagen determination: in

NASA Astrophysics Data System (ADS)

Kesler, Gavriel; Koren, Rumelia; Kesler, Anat; Kristt, Don; Gal, Rivka

2000-03-01

Recent technological advances lead to an increase in the options for the treatment of the periodontal diseases. Lasers utilized for gingival soft tissue resurfacing mainly for esthetics purposes, require careful histopathological evaluation of the effects on tissue. Up to date no comparative clinical or histological studies have been performed, aiming at demonstration of the effects of laser irradiation on connective tissue, especially its most important component -- the collagen fibers. The alteration in the structures of this tissue plays the most important role in the healing process. The aim of the present study is to evaluate the influence of Erbium: YAG - Kesler's hand piece on gingival tissue. This handpiece is designed for gingival resurfacing, in cases of 'Gummy smile' and gingival pigmentation. The following irradiation parameters were used: energy per pulse -- 500 mJ, repetition rate 10 pps, spot size 3 mm. Gingival biopsies specimens of 10 patients, 6 with 'Gummy smile' and 4 with gingival pigmentation were examined before laser treatment, and at 7 and 14 days after laser treatment. The tissues were fixed in LNRS, embedded in paraffin, and sectioned into 5 micrometer thickness, dewaxed in xylol and stained with H&E and Picrosirius Red (PSR). The sections were examined by polarization microscopy. PSR is a collagen stain that differentiates collagen fiber density by the range of colors from green through yellow to red, and/or fiber size. This was utilized in the present study to evaluate the hypothesis that Erbium -- YAG (Er: YAG) laser energy is capable of remodeling the collagen fibers in the gingival connective tissue through a photothermal process. We found a significant difference between the structures of collagen fibers at the first week and at 14 days post treatment. In the normal gingiva the predominant polarization colors were in the red-orange range, signifying tightly packed, mature collagen. During the first postoperative week, collagen fibers exhibit mainly polarization colors in the green to green-yellow range implying less packed intermediate collagen fibers. After two weeks, collagen fibers required their preoperative polarizing texture. These findings strongly suggest that the 'quality' rather than the quantity of gingival collagen fibers play an important role in order for collagen shrinkage and reproduction to occur.

Healing of human intrabony defects following regenerative periodontal therapy with a bovine-derived xenograft and guided tissue regeneration.

PubMed

Sculean, A; Stavropoulos, A; Windisch, P; Keglevich, T; Karring, T; Gera, I

2004-06-01

The purpose of the present study was to histologically evaluate the healing of human intrabony defects following treatment with either a bovine-derived xenograft (BDX) and guided tissue regeneration (GTR) [BDX + GTR] or a bovine-derived xenograft mixed with collagen (BDX Coll) and GTR [BDX Coll + GTR]. Eight patients with chronic periodontitis and each with one very deep intrabony defect around a tooth scheduled for extraction were treated with either a combination of BDX + GTR (five patients) or with BDX Coll + GTR (three patients). The postoperative healing was uneventful in all eight cases. After a healing period of 6 months, the teeth or roots were extracted together with some of their surrounding soft and hard tissues and subsequently fixed in 10% buffered formalin. Following decalcification in EDTA, the specimens were embedded in paraffin and 8-microm histological sections were cut in the mesio-distal direction, parallel to the long axes of the teeth. The sections were alternatively stained with hematoxylin and eosin, van Giesson's connective tissue stain or with the Ladevig's connective tissue staining method and examined under the light microscope. Generally, formation of new cementum with inserting collagen fibers was found in seven out of the eight treated cases, whereas in the remaining case (treated with BDX + GTR) the healing was characterized by formation of a long junctional epithelium along the debrided root surface and no formation of cementum or bone. In the specimens demonstrating periodontal regeneration the new cementum was always of a cellular type. In most cases, the graft particles were surrounded by bone. In some areas, the bone tissue around the graft particles was connected by perpendicularly inserting collagen fibers to the newly formed cementum on the root surface. The epithelium downgrowth stopped always at the most coronal part of the newly formed cementum. No remnants of the membrane material were observed in any of the biopsies. Connective tissue encapsulation of the graft particles was rarely observed and was limited to the most coronal part of the defects. The findings of the present study provide evidence that treatment of intrabony defects with both BDX + GTR and BDX Coll + GTR may enhance periodontal regeneration in humans.

Investigation of factors affecting hypothermic pelvic tissue cooling using bio-heat simulation based on MRI-segmented anatomic models.

PubMed

Lin, Yuting; Lin, Wei-Ching; Fwu, Peter T; Shih, Tzu-Ching; Yeh, Lee-Ren; Su, Min-Ying; Chen, Jeon-Hor

2015-10-01

This study applied a simulation method to map the temperature distribution based on magnetic resonance imaging (MRI) of individual patients, and investigated the influence of different pelvic tissue types as well as the choice of thermal property parameters on the efficiency of endorectal cooling balloon (ECB). MR images of four subjects with different prostate sizes and pelvic tissue compositions, including fatty tissue and venous plexus, were analyzed. The MR images acquired using endorectal coil provided a realistic geometry of deformed prostate that resembled the anatomy in the presence of ECB. A single slice with the largest two-dimensional (2D) cross-sectional area of the prostate gland was selected for analysis. The rectal wall, prostate gland, peri-rectal fatty tissue, peri-prostatic fatty tissue, peri-prostatic venous plexus, and urinary bladder were manually segmented. Pennes' bioheat thermal model was used to simulate the temperature distribution dynamics, by using an in-house finite element mesh based solver written in MATLAB. The results showed that prostate size and periprostatic venous plexus were two major factors affecting ECB cooling efficiency. For cases with negligible amount of venous plexus and small prostate, the average temperature in the prostate and neurovascular bundles could be cooled down to 25 °C within 30 min. For cases with abundant venous plexus and large prostate, the temperature could not reach 25 °C at the end of 3 h cooling. Large prostate made the cooling difficult to propagate through. The impact of fatty tissue on cooling effect was small. The filling of bladder with warm urine during the ECB cooling procedure did not affect the temperature in the prostate or NVB. In addition to the 2D simulation, in one case a 3D pelvic model was constructed for volumetric simulation. It was found that the 2D slice with the largest cross-sectional area of prostate had the most abundant venous plexus, and was the most difficult slice to cool, thus it may provide a conservative prediction of the cooling effect. This feasibility study demonstrated that the simulation tool could potentially be used for adjusting the setting of ECB for individual patients during hypothermic radical prostatectomy. Further studies using MR thermometry are required to validate the in silico results obtained using simulation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Investigation of factors affecting hypothermic pelvic tissue cooling using bio-heat simulation based on MRI-segmented anatomic models

PubMed Central

Lin, Yuting; Lin, Wei-Ching; Fwu, Peter T.; Shih, Tzu-Ching; Yeh, Lee-Ren; Su, Min-Ying; Chen, Jeon-Hor

2015-01-01

This study applied a simulation method to map the temperature distribution based on magnetic resonance imaging (MRI) of individual patients, and investigated the influence of different pelvic tissue types as well as the choice of thermal property parameters on the efficiency of endorectal cooling balloon (ECB). MR images of four subjects with different prostate sizes and pelvic tissue compositions, including fatty tissue and venous plexus, were analyzed. The MR images acquired using endorectal coil provided a realistic geometry of deformed prostate that resembled the anatomy in the presence of ECB. A single slice with the largest two-dimensional (2D) cross-sectional area of the prostate gland was selected for analysis. The rectal wall, prostate gland, peri-rectal fatty tissue, peri-prostatic fatty tissue, peri-prostatic venous plexus, and urinary bladder were manually segmented. Pennes’ bioheat thermal model was used to simulate the temperature distribution dynamics, by using an in-house finite element mesh based solver written in Matlab. The results showed that prostate size and periprostatic venous plexus were two major factors affecting ECB cooling efficiency. For cases with negligible amount of venous plexus and small prostate, the averaged temperature in the prostate and neurovascular bundles could be cooled down to 25°C within 30 minutes. For cases with abundant venous plexus and large prostate, the temperature could not reach 25°C at the end of 3 hours cooling. Large prostate made the cooling difficult to propagate through. The impact of fatty tissue on cooling effect was small. The filling of bladder with warm urine during the ECB cooling procedure did not affect the temperature in the prostate or NVB. In addition to the 2D simulation, in one case a 3D pelvic model was constructed for volumetric simulation. It was found that the 2D slice with the largest cross-sectional area of prostate had the most abundant venous plexus, and was the most difficult slice to cool, thus it may provide a conservative prediction of the cooling effect. This feasibility study demonstrated that the simulation tool could potentially be used for adjusting the setting of ECB for individual patients during hypothermic radical prostatectomy. Further studies using MR thermometry are required to validate the in silico results obtained using simulation. PMID:26198131

Cross-Reactivity of Antibodies against Leptospiral Recurrent Uveitis-Associated Proteins A and B (LruA and LruB) with Eye Proteins

PubMed Central

Verma, Ashutosh; Kumar, Pawan; Babb, Kelly; Timoney, John F.; Stevenson, Brian

2010-01-01

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be α-crystallin B and vimentin. Similarly, mass spectrometric analyses identified β-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human α-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant β-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized α-crystallin B, β-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis. PMID:20689825

Cross-reactivity of antibodies against leptospiral recurrent uveitis-associated proteins A and B (LruA and LruB) with eye proteins.

PubMed

Verma, Ashutosh; Kumar, Pawan; Babb, Kelly; Timoney, John F; Stevenson, Brian

2010-08-03

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be alpha-crystallin B and vimentin. Similarly, mass spectrometric analyses identified beta-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human alpha-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant beta-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized alpha-crystallin B, beta-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis.

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

PubMed

Ginsberg, Stephen D; Che, Shaoli

2004-08-01

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

Automatic registration of multi-modal microscopy images for integrative analysis of prostate tissue sections.

PubMed

Lippolis, Giuseppe; Edsjö, Anders; Helczynski, Leszek; Bjartell, Anders; Overgaard, Niels Chr

2013-09-05

Prostate cancer is one of the leading causes of cancer related deaths. For diagnosis, predicting the outcome of the disease, and for assessing potential new biomarkers, pathologists and researchers routinely analyze histological samples. Morphological and molecular information may be integrated by aligning microscopic histological images in a multiplex fashion. This process is usually time-consuming and results in intra- and inter-user variability. The aim of this study is to investigate the feasibility of using modern image analysis methods for automated alignment of microscopic images from differently stained adjacent paraffin sections from prostatic tissue specimens. Tissue samples, obtained from biopsy or radical prostatectomy, were sectioned and stained with either hematoxylin & eosin (H&E), immunohistochemistry for p63 and AMACR or Time Resolved Fluorescence (TRF) for androgen receptor (AR). Image pairs were aligned allowing for translation, rotation and scaling. The registration was performed automatically by first detecting landmarks in both images, using the scale invariant image transform (SIFT), followed by the well-known RANSAC protocol for finding point correspondences and finally aligned by Procrustes fit. The Registration results were evaluated using both visual and quantitative criteria as defined in the text. Three experiments were carried out. First, images of consecutive tissue sections stained with H&E and p63/AMACR were successfully aligned in 85 of 88 cases (96.6%). The failures occurred in 3 out of 13 cores with highly aggressive cancer (Gleason score ≥ 8). Second, TRF and H&E image pairs were aligned correctly in 103 out of 106 cases (97%).The third experiment considered the alignment of image pairs with the same staining (H&E) coming from a stack of 4 sections. The success rate for alignment dropped from 93.8% in adjacent sections to 22% for sections furthest away. The proposed method is both reliable and fast and therefore well suited for automatic segmentation and analysis of specific areas of interest, combining morphological information with protein expression data from three consecutive tissue sections. Finally, the performance of the algorithm seems to be largely unaffected by the Gleason grade of the prostate tissue samples examined, at least up to Gleason score 7.

Tissue Integration of a Volume-Stable Collagen Matrix in an Experimental Soft Tissue Augmentation Model.

PubMed

Ferrantino, Luca; Bosshardt, Dieter; Nevins, Myron; Santoro, Giacomo; Simion, Massimo; Kim, David

Reducing the need for a connective tissue graft by using an efficacious biomaterial is an important task for dental professionals and patients. This experimental study aimed to test the soft tissue response to a volume-stable new collagen matrix. The device demonstrated good stability during six different time points ranging from 0 to 90 days of healing with no alteration of the wound-healing processes. The 90-day histologic specimen demonstrates eventual replacement of most of the matrix with new connective tissue fibers.

Strongly Acidic Auxin Indole-3-Methanesulfonic Acid

PubMed Central

Cohen, Jerry D.; Baldi, Bruce G.; Bialek, Krystyna

1985-01-01

A radiochemical synthesis is described for [14C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [14C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles. PMID:16664007

Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels.

PubMed

Herman, Mark A; She, Pengxiang; Peroni, Odile D; Lynch, Christopher J; Kahn, Barbara B

2010-04-09

Whereas the role of adipose tissue in glucose and lipid homeostasis is widely recognized, its role in systemic protein and amino acid metabolism is less well-appreciated. In vitro and ex vivo experiments suggest that adipose tissue can metabolize substantial amounts of branched chain amino acids (BCAAs). However, the role of adipose tissue in regulating BCAA metabolism in vivo is controversial. Interest in the contribution of adipose tissue to BCAA metabolism has been renewed with recent observations demonstrating down-regulation of BCAA oxidation enzymes in adipose tissue in obese and insulin-resistant humans. Using gene set enrichment analysis, we observe alterations in adipose-tissue BCAA enzyme expression caused by adipose-selective genetic alterations in the GLUT4 glucose-transporter expression. We show that the rate of adipose tissue BCAA oxidation per mg of tissue from normal mice is higher than in skeletal muscle. In mice overexpressing GLUT4 specifically in adipose tissue, we observe coordinate down-regulation of BCAA metabolizing enzymes selectively in adipose tissue. This decreases BCAA oxidation rates in adipose tissue, but not in muscle, in association with increased circulating BCAA levels. To confirm the capacity of adipose tissue to modulate circulating BCAA levels in vivo, we demonstrate that transplantation of normal adipose tissue into mice that are globally defective in peripheral BCAA metabolism reduces circulating BCAA levels by 30% (fasting)-50% (fed state). These results demonstrate for the first time the capacity of adipose tissue to catabolize circulating BCAAs in vivo and that coordinate regulation of adipose-tissue BCAA enzymes may modulate circulating BCAA levels.

Rejoining of cut wounds by engineered gelatin-keratin glue.

PubMed

Thirupathi Kumara Raja, S; Thiruselvi, T; Sailakshmi, G; Ganesh, S; Gnanamani, A

2013-08-01

Rejoining of cut tissue ends of a critical site challenges clinicians. The toxicity, antigenicity, low adhesive strength, flexibility, swelling and cost of the currently employed glue demands an alternative. Engineered gelatin-keratin glue (EGK-glue) described in the present study was found to be suitable for wet tissue approximation. EGK-glue was prepared by engineering gelatin with caffeic acid using EDC and conjugating with keratin by periodate oxidation. UV-visible, (1)H NMR and circular dichroism analyses followed by experiments on gelation time, rheology, gel adhesive strength (in vitro), wet tissue approximation (in vivo), H&E staining of tissue sections at scheduled time intervals and tensile strength of the healed skin were carried out to assess the effectiveness of the EGK-glue in comparison with fibrin glue and cyanoacrylate. Results of UV-visible, NMR and CD analyses confirmed the functionalization and secondary structural changes. Increasing concentration of keratin reduces the gelation time (<15s). Lap-shear test demonstrates the maximum adhesive strength of 16.6±1.2kPa. Results of hemocompatibility and cytocompatibility studies suggested the suitability of the glue for clinical applications. Tissue approximation property assessed using the incision wound model (Wistar strain) in comparison with cyanoacrylate and fibrin glue suggested, that EGK-glue explicitly accelerates the rejoining of tissue with a 1.86 fold increase in skin tensile strength after healing. Imparting quinone moiety to gelatin-keratin conjugates through caffeic acid and a weaker oxidizing agent provides an adhesive glue with appreciable strength, and hemocompatible, cytocompatible and biodegradable properties, which, rejoin the cut tissue ends effectively. EGK-glue obtained in the present study finds wide biomedical/clinical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Inbred Strain-Specific Effects of Exercise in Wild Type and Biglycan Deficient Mice

PubMed Central

Wallace, Joseph M.; Golcuk, Kurtulus; Morris, Michael D.; Kohn, David H.

2010-01-01

Biglycan (bgn)-deficient mice (KO) have defective osteoblasts which lead to changes in the amount and quality of bone. Altered tissue strength in C57BL6/129 (B6;129) KO mice, a property which is independent of tissue quantity, suggests that deficiencies in tissue quality are responsible. However, the response to bgn-deficiency is inbred strain-specific. Mechanical loading influences bone matrix quality in addition to any increase in bone mass or change in bone formation activity. Since many diseases influence the mechanical integrity of bone through altered tissue quality, loading may be a way to prevent and treat extracellular matrix deficiencies. C3H/He (C3H) mice consistently have a less vigorous response to mechanical loading vs. other inbred strains. It was therefore hypothesized that the bones from both wild type (WT) and KO B6;129 mice would be more responsive to exercise than the bones from C3H mice. To test these hypotheses at 11 weeks of age, following 21 consecutive days of exercise, we investigated cross-sectional geometry, mechanical properties, and tissue composition in the tibiae of male mice bred on B6;129 and C3H backgrounds. This study demonstrated inbred strain-specific compositional and mechanical changes following exercise in WT and KO mice, and showed evidence of genotype-specific changes in bone in response to loading in a gene disruption model. This study further shows that exercise can influence bone tissue composition and/or mechanical integrity without changes in bone geometry. Together, these data suggest that exercise may represent a possible means to alter tissue quality and mechanical deficiencies caused by many diseases of bone. PMID:20033775

Immunohistochemical analysis of S6K1 and S6K2 localization in human breast tumors.

PubMed

Filonenko, Valeriy V; Tytarenko, Ruslana; Azatjan, Sergey K; Savinska, Lilya O; Gaydar, Yuriy A; Gout, Ivan T; Usenko, Vasiliy S; Lyzogubov, Valeriy V

2004-12-01

To perform an immunohistochemical analysis of human breast adenomas and adenocarcinomas as well as normal breast tissues in respect of S6 ribosomal protein kinase (S6K) expression and localization in normal and transformed cells. The expression level and localization of S6K have been detected in formalin fixed, paraffin embedded sections of normal human breast tissues, adenomas and adenocarcinomas with different grade of differentiation. Immunohistochemical detection of S6K1 and S6K2 in normal human breast tissues and breast tumors were performed using specific monoclonal and polyclonal antibodies against S6K1 and S6K2 with following semiquantitative analysis. The increase of S6K content in the cytoplasm of epithelial cells in benign and malignant tumors has been detected. Nuclear accumulation of S6K1 and to a greater extend S6K2 have been found in breast adenocarcinomas. About 80% of breast adenocarcinomas cases revealed S6K2 nuclear staining comparing to normal tissues. In 31% of cases more then 50% of cancer cells had strong nuclear staining. Accumulation of S6K1 in the nucleus of neoplastic cells has been demonstrated in 25% of cases. Nuclear localization of S6K in the epithelial cells in normal breast tissues has not been detected. Immunohistochemical analysis of S6K1 and S6K2 expression in normal human breast tissues, benign and malignant breast tumors clearly indicates that both kinases are overexpressed in breast tumors. Semiquantitative analysis of peculiarities of S6K localization in normal tissues and tumors revealed that nucleoplasmic accumulation of S6K (especially S6K2) is a distinguishing feature of cancer cells.

Optical metabolic imaging of live tissue cultures

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Cook, Rebecca S.; Arteaga, Carlos L.; Skala, Melissa C.

2013-02-01

The fluorescence properties, both intensity and fluorescence lifetime, of NADH and FAD, two coenzymes of metabolism, are sensitive, high resolution measures of cellular metabolism. However, often in vivo measurements of tissue are not feasible. In this study, we investigate the stability over time of two-photon auto-fluorescence imaging of NADH and FAD in live-cultured tissues. Our results demonstrate that cultured tissues remain viable for at least several days post excision. Furthermore, the optical redox ratio, NADH fluorescence lifetime, and FAD fluorescence lifetime do not significantly change in the cultured tissues over time. With these findings, we demonstrate the potential of sustained tissue culture techniques for optical metabolic imaging.

High-resolution, 2- and 3-dimensional imaging of uncut, unembedded tissue biopsy samples.

PubMed

Torres, Richard; Vesuna, Sam; Levene, Michael J

2014-03-01

Despite continuing advances in tissue processing automation, traditional embedding, cutting, and staining methods limit our ability for rapid, comprehensive visual examination. These limitations are particularly relevant to biopsies for which immediate therapeutic decisions are most necessary, faster feedback to the patient is desired, and preservation of tissue for ancillary studies is most important. The recent development of improved tissue clearing techniques has made it possible to consider use of multiphoton microscopy (MPM) tools in clinical settings, which could address difficulties of established methods. To demonstrate the potential of MPM of cleared tissue for the evaluation of unembedded and uncut pathology samples. Human prostate, liver, breast, and kidney specimens were fixed and dehydrated by using traditional histologic techniques, with or without incorporation of nucleic acid fluorescent stains into dehydration steps. A benzyl alcohol/benzyl benzoate clearing protocol was substituted for xylene. Multiphoton microscopy was performed on a home-built system. Excellent morphologic detail was achievable with MPM at depths greater than 500 μm. Pseudocoloring produced images analogous to hematoxylin-eosin-stained images. Concurrent second-harmonic generation detection allowed mapping of collagen. Subsequent traditional section staining with hematoxylin-eosin did not reveal any detrimental morphologic effects. Sample immunostains on renal tissue showed preservation of normal reactivity. Complete reconstructions of 1-mm cubic samples elucidated 3-dimensional architectural organization. Multiphoton microscopy on cleared, unembedded, uncut biopsy specimens shows potential as a practical clinical tool with significant advantages over traditional histology while maintaining compatibility with gold standard techniques. Further investigation to address remaining implementation barriers is warranted.

Soft tissue volume augmentation by the use of collagen-based matrices in the dog mandible -- a histological analysis.

PubMed

Thoma, Daniel S; Hämmerle, Christoph H F; Cochran, David L; Jones, Archie A; Görlach, Christoph; Uebersax, Lorenz; Mathes, Stephanie; Graf-Hausner, Ursula; Jung, Ronald E

2011-11-01

The aim was to test, whether or not soft tissue volume augmentation with a specifically designed collagen matrix (CM), leads to ridge width gain in chronic ridge defects similar to those obtained by an autogenous subepithelial connective tissue graft (SCTG). In six dogs, soft tissue volume augmentation was performed by randomly allocating three treatment modalities to chronic ridge defects [CM, SCTG and sham-operated control (Control)]. Dogs were sacrificed at 28 (n = 3) and 84 days (n = 3). Descriptive histology and histomorphometric measurements were performed on non-decalcified sections. SCTG and CM demonstrated favourable tissue integration, and subsequent re-modelling over 84 days. The overall mean amount of newly formed soft tissue (NMT) plus bone (NB) amounted to 3.8 ± 1.2 mm (Control), 6.4 ± 0.9 mm (CM) and 7.2 ± 1.2 mm (SCTG) at 28 days. At 84 days, the mean NMT plus NB reached 2.4 ± 0.9 mm (Control), 5.6 ± 1.5 mm (CM) and 6.0 ± 2.1 mm (SCTG). Statistically significant differences were observed between CM/SCTG and Control at both time-points (p < 0.05). Within the limits of this animal model, the CM performed similar to the SCTG, based on histomorphometric outcomes combining NB and NMT. © 2011 John Wiley & Sons A/S.

A practical approach to the clinical diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrangement using new fluorescence in situ hybridisation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue.

PubMed

Yamaguchi, U; Hasegawa, T; Morimoto, Y; Tateishi, U; Endo, M; Nakatani, F; Kawai, A; Chuman, H; Beppu, Y; Endo, M; Kurotaki, H; Furuta, K

2005-10-01

Over 90% of Ewing's sarcoma/primitive neuroectodermal tumour (ES/PNET) cases have the t(11;22) chromosomal rearrangement, which is also found in other small round cell tumours, including desmoplastic small round cell tumour (DSRCT) and clear cell sarcoma (CCS). Although this rearrangement can be analysed by fluorescence in situ hybridisation (FISH) using routinely formalin fixed, paraffin wax embedded (FFPE) tissues when fresh or frozen tissues are not available, a sensitive and convenient detection method is needed for routine clinical diagnosis. To investigate the usefulness of newly developed probes for detecting EWS rearrangement resulting from chromosomal translocations using FISH and FFPE tissue in the clinical diagnosis of ES/PNET, DSRCT, and CCS. Sixteen ES/PNETs, six DSRCTs, and six CCSs were studied. Three poorly differentiated synovial sarcomas, three alveolar rhabdomyosarcomas, and three neuroblastomas served as negative controls. Interphase FISH analysis was performed on FFPE tissue sections with a commercially available EWSR1 (22q12) dual colour, breakapart rearrangement probe. One fused signal and one split signal of orange and green, demonstrating rearrangement of the EWS gene, was detected in 14 of 16 ES/PNETs, all six DRSCTs, and five of six CCSs, but not in the negative controls. Interphase FISH using this newly developed probe is sensitive and specific for detecting the EWS gene on FFPE tissues and is of value in the routine clinical diagnosis of ES/PNET, DSRCT, and CCS.

Ultrasoft microwire neural electrodes improve chronic tissue integration.

PubMed

Du, Zhanhong Jeff; Kolarcik, Christi L; Kozai, Takashi D Y; Luebben, Silvia D; Sapp, Shawn A; Zheng, Xin Sally; Nabity, James A; Cui, X Tracy

2017-04-15

Chronically implanted neural multi-electrode arrays (MEA) are an essential technology for recording electrical signals from neurons and/or modulating neural activity through stimulation. However, current MEAs, regardless of the type, elicit an inflammatory response that ultimately leads to device failure. Traditionally, rigid materials like tungsten and silicon have been employed to interface with the relatively soft neural tissue. The large stiffness mismatch is thought to exacerbate the inflammatory response. In order to minimize the disparity between the device and the brain, we fabricated novel ultrasoft electrodes consisting of elastomers and conducting polymers with mechanical properties much more similar to those of brain tissue than previous neural implants. In this study, these ultrasoft microelectrodes were inserted and released using a stainless steel shuttle with polyethyleneglycol (PEG) glue. The implanted microwires showed functionality in acute neural stimulation. When implanted for 1 or 8weeks, the novel soft implants demonstrated significantly reduced inflammatory tissue response at week 8 compared to tungsten wires of similar dimension and surface chemistry. Furthermore, a higher degree of cell body distortion was found next to the tungsten implants compared to the polymer implants. Our results support the use of these novel ultrasoft electrodes for long term neural implants. One critical challenge to the translation of neural recording/stimulation electrode technology to clinically viable devices for brain computer interface (BCI) or deep brain stimulation (DBS) applications is the chronic degradation of device performance due to the inflammatory tissue reaction. While many hypothesize that soft and flexible devices elicit reduced inflammatory tissue responses, there has yet to be a rigorous comparison between soft and stiff implants. We have developed an ultra-soft microelectrode with Young's modulus lower than 1MPa, closely mimicking the brain tissue modulus. Here, we present a rigorous histological comparison of this novel ultrasoft electrode and conventional stiff electrode with the same size, shape and surface chemistry, implanted in rat brains for 1-week and 8-weeks. Significant improvement was observed for ultrasoft electrodes, including inflammatory tissue reaction, electrode-tissue integration as well as mechanical disturbance to nearby neurons. A full spectrum of new techniques were developed in this study, from insertion shuttle to in situ sectioning of the microelectrode to automated cell shape analysis, all of which should contribute new methods to the field. Finally, we showed the electrical functionality of the ultrasoft electrode, demonstrating the potential of flexible neural implant devices for future research and clinical use. Copyright © 2017. Published by Elsevier Ltd.

Tissue Printing to Visualize Polyphenol Oxidase and Peroxidase in Vegetables, Fruits, and Mushrooms

ERIC Educational Resources Information Center

Melberg, Amanda R.; Flurkey, William H.; Inlow, Jennifer K.

2009-01-01

A simple tissue-printing procedure to determine the tissue location of the endogenous enzymes polyphenol oxidase and peroxidase in a variety of vegetables, fruits, and mushrooms is described. In tissue printing, cell contents from the surface of a cut section of the tissue are transferred to an adsorptive surface, commonly a nitrocellulose…

21 CFR 500.86 - Marker residue and target tissue.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Marker residue and target tissue. 500.86 Section...-Producing Animals § 500.86 Marker residue and target tissue. (a) For each edible tissue, the sponsor shall...) From these data, FDA will select a target tissue and a marker residue and designate the concentration...

21 CFR 500.86 - Marker residue and target tissue.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Marker residue and target tissue. 500.86 Section...-Producing Animals § 500.86 Marker residue and target tissue. (a) For each edible tissue, the sponsor shall...) From these data, FDA will select a target tissue and a marker residue and designate the concentration...

21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

Code of Federal Regulations, 2010 CFR

2010-04-01

... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All donors...

21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

Code of Federal Regulations, 2013 CFR

2013-04-01

... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All donors...

21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

Code of Federal Regulations, 2014 CFR

2014-04-01

... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All donors...

21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

Code of Federal Regulations, 2012 CFR

2012-04-01

... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All donors...

Azimuthally invariant Mueller-matrix mapping of biological optically anisotropic network

NASA Astrophysics Data System (ADS)

Ushenko, Yu. O.; Vanchuliak, O.; Bodnar, G. B.; Ushenko, V. O.; Grytsyuk, M.; Pavlyukovich, N.; Pavlyukovich, O. V.; Antonyuk, O.

2017-09-01

A new technique of Mueller-matrix mapping of polycrystalline structure of histological sections of biological tissues is suggested. The algorithms of reconstruction of distribution of parameters of linear and circular dichroism of histological sections liver tissue of mice with different degrees of severity of diabetes are found. The interconnections between such distributions and parameters of linear and circular dichroism of liver of mice tissue histological sections are defined. The comparative investigations of coordinate distributions of parameters of amplitude anisotropy formed by Liver tissue with varying severity of diabetes (10 days and 24 days) are performed. The values and ranges of change of the statistical (moments of the 1st - 4th order) parameters of coordinate distributions of the value of linear and circular dichroism are defined. The objective criteria of cause of the degree of severity of the diabetes differentiation are determined.

CdSe/CdS semiconductor quantum rods as robust fluorescent probes for paraffin-embedded tissue imaging.

PubMed

Zacheo, Antonella; Quarta, Alessandra; Mangoni, Antonella; Pompa, Pier Paolo; Mastria, Rosanna; Capogrossi, Maurizio C; Rinaldi, Ross; Pellegrino, Teresa

2011-09-01

Immunofluorescence techniques on formalin fixed paraffin-embedded sections allow for the evaluation of the expression and spatial distribution of specific markers in patient tissue specimens or for monitoring the fate of labeled cells after in vivo injection. This technique suffers however from the auto-fluorescence background signal of the embedded tissue that eventually confounds the analysis. Here we show that rod-like semiconductor nanocrystals (QRs), intramuscularly injected in living mice, could be clearly detected by confocal microscopy in formalin fixed paraffin-embedded tissue sections. Despite the low amount of QRs amount injected (25 picomoles), these were clearly visible after 24 h in the muscle sections and their fluorescence signal was stronger than that of CdSe/ZnS quantum dots (QDs) similarly functionalized and in the case of QRs only, the signal lasted even after 21 days after the injection. © 2011 IEEE

Ex vivo applications of multiphoton microscopy in urology

NASA Astrophysics Data System (ADS)

Jain, Manu; Mukherjee, Sushmita

2016-03-01

Background: Routine urological surgery frequently requires rapid on-site histopathological tissue evaluation either during biopsy or intra-operative procedure. However, resected tissue needs to undergo processing, which is not only time consuming but may also create artifacts hindering real-time tissue assessment. Likewise, pathologist often relies on several ancillary methods, in addition to H&E to arrive at a definitive diagnosis. Although, helpful these techniques are tedious and time consuming and often show overlapping results. Therefore, there is a need for an imaging tool that can rapidly assess tissue in real-time at cellular level. Multiphoton microscopy (MPM) is one such technique that can generate histology-quality images from fresh and fixed tissue solely based on their intrinsic autofluorescence emission, without the need for tissue processing or staining. Design: Fresh tissue sections (neoplastic and non-neoplastic) from biopsy and surgical specimens of bladder and kidney were obtained. Unstained deparaffinized slides from biopsy of medical kidney disease and oncocytic renal neoplasms were also obtained. MPM images were acquired using with an Olympus FluoView FV1000MPE system. After imaging, fresh tissues were submitted for routine histopathology. Results: Based on the architectural and cellular details of the tissue, MPM could characterize normal components of bladder and kidney. Neoplastic tissue could be differentiated from non-neoplastic tissue and could be further classified as per histopathological convention. Some of the tumors had unique MPM signatures not otherwise seen on H&E sections. Various subtypes of glomerular lesions were identified as well as renal oncocytic neoplasms were differentiated on unstained deparaffinized slides. Conclusions: We envision MPM to become an integral part of regular diagnostic workflow for rapid assessment of tissue. MPM can be used to evaluate the adequacy of biopsies and triage tissues for ancillary studies. It can also be used as an adjunct to frozen section analysis for intra-operative margin assessment. Further, it can play an important role for pathologist for guiding specimen grossing, selecting tissue for tumor banking and as a rapid ancillary diagnostic tool.

Neutron organ dose and the influence of adipose tissue

NASA Astrophysics Data System (ADS)

Simpkins, Robert Wayne

Neutron fluence to dose conversion coefficients have been assessed considering the influences of human adipose tissue. Monte Carlo code MCNP4C was used to simulate broad parallel beam monoenergetic neutrons ranging in energy from thermal to 10 MeV. Simulated Irradiations were conducted for standard irradiation geometries. The targets were on gender specific mathematical anthropomorphic phantoms modified to approximate human adipose tissue distributions. Dosimetric analysis compared adipose tissue influence against reference anthropomorphic phantom characteristics. Adipose Male and Post-Menopausal Female Phantoms were derived introducing interstitial adipose tissue to account for 22 and 27 kg additional body mass, respectively, each demonstrating a Body Mass Index (BMI) of 30. An Adipose Female Phantom was derived introducing specific subcutaneous adipose tissue accounting for 15 kg of additional body mass demonstrating a BMI of 26. Neutron dose was shielded in the superficial tissues; giving rise to secondary photons which dominated the effective dose for Incident energies less than 100 keV. Adipose tissue impact on the effective dose was a 25% reduction at the anterior-posterior incidence ranging to a 10% increase at the lateral incidences. Organ dose impacts were more distinctive; symmetrically situated organs demonstrated a 15% reduction at the anterior-posterior Incidence ranging to a 2% increase at the lateral incidences. Abdominal or asymmetrically situated organs demonstrated a 50% reduction at the anterior-posterior incidence ranging to a 25% increase at the lateral incidences.

Motion artifact and background noise suppression on optical microangiography frames using a naïve Bayes mask.

PubMed

Reif, Roberto; Baran, Utku; Wang, Ruikang K

2014-07-01

Optical coherence tomography (OCT) is a technique that allows for the three-dimensional (3D) imaging of small volumes of tissue (a few millimeters) with high resolution (∼10  μm). Optical microangiography (OMAG) is a method of processing OCT data, which allows for the extraction of the tissue vasculature with capillary resolution from the OCT images. Cross-sectional B-frame OMAG images present the location of the patent blood vessels; however, the signal-to-noise-ratio of these images can be affected by several factors such as the quality of the OCT system and the tissue motion artifact. This background noise can appear in the en face projection view image. In this work we propose to develop a binary mask that can be applied on the cross-sectional B-frame OMAG images, which will reduce the background noise while leaving the signal from the blood vessels intact. The mask is created by using a naïve Bayes (NB) classification algorithm trained with a gold standard image which is manually segmented by an expert. The masked OMAG images present better contrast for binarizing the image and quantifying the result without the influence of noise. The results are compared with a previously developed frequency rejection filter (FRF) method which is applied on the en face projection view image. It is demonstrated that both the NB and FRF methods provide similar vessel length fractions. The advantage of the NB method is that the results are applicable in 3D and that its use is not limited to periodic motion artifacts.

Expression of proteinase inhibitor II proteins during floral development in Solanum americanum.

PubMed

Sin, Suk-Fong; Chye, Mee-Len

2004-10-01

The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.

AutoStitcher: An Automated Program for Efficient and Robust Reconstruction of Digitized Whole Histological Sections from Tissue Fragments

NASA Astrophysics Data System (ADS)

Penzias, Gregory; Janowczyk, Andrew; Singanamalli, Asha; Rusu, Mirabela; Shih, Natalie; Feldman, Michael; Stricker, Phillip D.; Delprado, Warick; Tiwari, Sarita; Böhm, Maret; Haynes, Anne-Maree; Ponsky, Lee; Viswanath, Satish; Madabhushi, Anant

2016-07-01

In applications involving large tissue specimens that have been sectioned into smaller tissue fragments, manual reconstruction of a “pseudo whole-mount” histological section (PWMHS) can facilitate (a) pathological disease annotation, and (b) image registration and correlation with radiological images. We have previously presented a program called HistoStitcher, which allows for more efficient manual reconstruction than general purpose image editing tools (such as Photoshop). However HistoStitcher is still manual and hence can be laborious and subjective, especially when doing large cohort studies. In this work we present AutoStitcher, a novel automated algorithm for reconstructing PWMHSs from digitized tissue fragments. AutoStitcher reconstructs (“stitches”) a PWMHS from a set of 4 fragments by optimizing a novel cost function that is domain-inspired to ensure (i) alignment of similar tissue regions, and (ii) contiguity of the prostate boundary. The algorithm achieves computational efficiency by performing reconstruction in a multi-resolution hierarchy. Automated PWMHS reconstruction results (via AutoStitcher) were quantitatively and qualitatively compared to manual reconstructions obtained via HistoStitcher for 113 prostate pathology sections. Distances between corresponding fiducials placed on each of the automated and manual reconstruction results were between 2.7%-3.2%, reflecting their excellent visual similarity.

Overlapping MALDI-Mass Spectrometry Imaging for In-Parallel MS and MS/MS Data Acquisition without Sacrificing Spatial Resolution

NASA Astrophysics Data System (ADS)

Hansen, Rebecca L.; Lee, Young Jin

2017-09-01

Metabolomics experiments require chemical identifications, often through MS/MS analysis. In mass spectrometry imaging (MSI), this necessitates running several serial tissue sections or using a multiplex data acquisition method. We have previously developed a multiplex MSI method to obtain MS and MS/MS data in a single experiment to acquire more chemical information in less data acquisition time. In this method, each raster step is composed of several spiral steps and each spiral step is used for a separate scan event (e.g., MS or MS/MS). One main limitation of this method is the loss of spatial resolution as the number of spiral steps increases, limiting its applicability for high-spatial resolution MSI. In this work, we demonstrate multiplex MS imaging is possible without sacrificing spatial resolution by the use of overlapping spiral steps, instead of spatially separated spiral steps as used in the previous work. Significant amounts of matrix and analytes are still left after multiple spectral acquisitions, especially with nanoparticle matrices, so that high quality MS and MS/MS data can be obtained on virtually the same tissue spot. This method was then applied to visualize metabolites and acquire their MS/MS spectra in maize leaf cross-sections at 10 μm spatial resolution. [Figure not available: see fulltext.

MALDI imaging facilitates new topical drug development process by determining quantitative skin distribution profiles.

PubMed

Bonnel, David; Legouffe, Raphaël; Eriksson, André H; Mortensen, Rasmus W; Pamelard, Fabien; Stauber, Jonathan; Nielsen, Kim T

2018-04-01

Generation of skin distribution profiles and reliable determination of drug molecule concentration in the target region are crucial during the development process of topical products for treatment of skin diseases like psoriasis and atopic dermatitis. Imaging techniques like mass spectrometric imaging (MSI) offer sufficient spatial resolution to generate meaningful distribution profiles of a drug molecule across a skin section. In this study, we use matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to generate quantitative skin distribution profiles based on tissue extinction coefficient (TEC) determinations of four different molecules in cross sections of human skin explants after topical administration. The four drug molecules: roflumilast, tofacitinib, ruxolitinib, and LEO 29102 have different physicochemical properties. In addition, tofacitinib was administrated in two different formulations. The study reveals that with MALDI-MSI, we were able to observe differences in penetration profiles for both the four drug molecules and the two formulations and thereby demonstrate its applicability as a screening tool when developing a topical drug product. Furthermore, the study reveals that the sensitivity of the MALDI-MSI techniques appears to be inversely correlated to the drug molecules' ability to bind to the surrounding tissues, which can be estimated by their Log D values. Graphical abstract.

Hyperspectral imaging fluorescence excitation scanning for detecting colorectal cancer: pilot study

NASA Astrophysics Data System (ADS)

Leavesley, Silas J.; Wheeler, Mikayla; Lopez, Carmen; Baker, Thomas; Favreau, Peter F.; Rich, Thomas C.; Rider, Paul F.; Boudreaux, Carole W.

2016-03-01

Optical spectroscopy and hyperspectral imaging have shown the theoretical potential to discriminate between cancerous and non-cancerous tissue with high sensitivity and specificity. To date, these techniques have not been able to be effectively translated to endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents a new technology that may be well-suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The objective of this pilot study was to evaluate the changes in the fluorescence excitation spectrum of resected specimen pairs of colorectal adenocarcinoma and normal colorectal mucosa. Patients being treated for colorectal adenocarcinoma were enrolled. Representative adenocarcinoma and normal colonic mucosa specimens were collected from each case. Specimens were flash frozen in liquid nitrogen. Adenocarcinoma was confirmed by histologic evaluation of H&E permanent sections. Hyperspectral image data of the fluorescence excitation of adenocarcinoma and surrounding normal tissue were acquired using a custom microscope configuration previously developed in our lab. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation spectral range of 390-450 nm. We conclude that fluorescence excitation-scanning hyperspectral imaging may offer an alternative approach for differentiating adenocarcinoma and surrounding normal mucosa of the colon. Future work will focus on expanding the number of specimen pairs analyzed and will utilize fresh tissues where possible, as flash freezing and reconstituting tissues may have altered the autofluorescence properties.

Pharmacokinetic and pharmacometabolomic study of pirfenidone in normal mouse tissues using high mass resolution MALDI-FTICR-mass spectrometry imaging.

PubMed

Sun, Na; Fernandez, Isis E; Wei, Mian; Wu, Yin; Aichler, Michaela; Eickelberg, Oliver; Walch, Axel

2016-02-01

Given the importance of pirfenidone as the first worldwide-approved drug for idiopathic pulmonary fibrosis treatment, its pharmacodynamic properties and the metabolic response to pirfenidone treatment have not been fully elucidated. The aim of the present study was to get molecular insights of pirfenidone-related pharmacometabolomic response using MALDI-FTICR-MSI. Quantitative MALDI-FTICR-MSI was carried out for determining the pharmacokinetic properties of pirfenidone and its related metabolites 5-hydroxymethyl pirfenidone and 5-carboxy pirfenidone in lung, liver and kidney. To monitor the effect of pirfenidone administration on endogenous cell metabolism, additional in situ endogenous metabolite imaging was performed in lung tissue sections. While pirfenidone is highly abundant and delocalized across the whole micro-regions of lung, kidney and liver, 5-hydroxymethyl pirfenidone and 5-carboxy pirfenidone demonstrate heterogeneous distribution patterns in lung and kidney. In situ endogenous metabolite imaging study of lung tissue indicates no significant effects of pirfenidone on metabolic pathways. Remarkably, we found 129 discriminative m/z values which represent clear differences between control and treated lungs, the majority of which are currently unknown. PCA analysis and heatmap view can accurately distinguish control and treated groups. This is the first pharmacokinetic study to investigate the tissue distribution of orally administered pirfenidone and its related metabolites simultaneously in organs without labeling. The combination of pharmametabolome with histological features provides detailed mapping of drug effects on metabolism as response of healthy lung tissue to pirfenidone treatment.

Antibody-secreting cells in respiratory tract tissues in the absence of eosinophils as supportive partners.

PubMed

Sealy, Robert E; Surman, Sherri L; Vogel, Peter; Hurwitz, Julia L

2016-11-01

Antibody-secreting cells (ASCs) in respiratory tract tissues provide a first line of defense against invading pathogens. These cells often secrete IgA that is efficiently transcytosed across epithelial barriers into the airway lumen where pathogens can be blocked at their point of entry. Previous literature has reported that in the bone marrow, eosinophils are required for the maintenance of ASCs, and that eosinophils co-localize with ASCs as nearest neighbors. To determine if these rules similarly apply to the maintenance of ASCs in respiratory tract tissues, we evaluated virus-specific responses 1 month and 4 months following an intranasal virus infection of eosinophil-null (∆dblGATA-1) mice. Results showed that ASCs were fractionally reduced, but were nonetheless observed in respiratory tract tissues in the absence of eosinophils. Virus-specific antibodies were similarly observed in the airways of eosinophil-deficient mice. Respiratory tract ASCs were also present in mice lacking neutrophils (Mcl1 ∆M ). The staining of tissue sections from the upper respiratory tract of wild-type mice following viral infections demonstrated that virus-specific ASCs were most frequently situated adjacent to epithelial cells rather than eosinophils or neutrophils. Taken together, these data emphasize that rules for cell maintenance are not absolute and that ASCs can survive in the respiratory tract without eosinophils or neutrophils as their nearest neighbors. © The Japanese Society for Immunology. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.