21 CFR 1271.230 - Process validation.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Process validation. 1271.230 Section 1271.230 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.230 Process validation. (a... validation activities and results must be documented, including the date and signature of the individual(s...

21 CFR 1271.230 - Process validation.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Process validation. 1271.230 Section 1271.230 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.230 Process validation. (a... validation activities and results must be documented, including the date and signature of the individual(s...

21 CFR 1271.230 - Process validation.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Process validation. 1271.230 Section 1271.230 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.230 Process validation. (a... validation activities and results must be documented, including the date and signature of the individual(s...

21 CFR 1271.230 - Process validation.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Process validation. 1271.230 Section 1271.230 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.230 Process validation. (a... validation activities and results must be documented, including the date and signature of the individual(s...

21 CFR 1271.230 - Process validation.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Process validation. 1271.230 Section 1271.230 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.230 Process validation. (a... validation activities and results must be documented, including the date and signature of the individual(s...

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.

MALDI Imaging Mass Spectrometry (MALDI-IMS)—Application of Spatial Proteomics for Ovarian Cancer Classification and Diagnosis

PubMed Central

Gustafsson, Johan O. R.; Oehler, Martin K.; Ruszkiewicz, Andrew; McColl, Shaun R.; Hoffmann, Peter

2011-01-01

MALDI imaging mass spectrometry (MALDI-IMS) allows acquisition of mass data for metabolites, lipids, peptides and proteins directly from tissue sections. IMS is typically performed either as a multiple spot profiling experiment to generate tissue specific mass profiles, or a high resolution imaging experiment where relative spatial abundance for potentially hundreds of analytes across virtually any tissue section can be measured. Crucially, imaging can be achieved without prior knowledge of tissue composition and without the use of antibodies. In effect MALDI-IMS allows generation of molecular data which complement and expand upon the information provided by histology including immuno-histochemistry, making its application valuable to both cancer biomarker research and diagnostics. The current state of MALDI-IMS, key biological applications to ovarian cancer research and practical considerations for analysis of peptides and proteins on ovarian tissue are presented in this review. PMID:21340013

MALDI Imaging Mass Spectrometry (MALDI-IMS)-application of spatial proteomics for ovarian cancer classification and diagnosis.

PubMed

Gustafsson, Johan O R; Oehler, Martin K; Ruszkiewicz, Andrew; McColl, Shaun R; Hoffmann, Peter

2011-01-21

MALDI imaging mass spectrometry (MALDI-IMS) allows acquisition of mass data for metabolites, lipids, peptides and proteins directly from tissue sections. IMS is typically performed either as a multiple spot profiling experiment to generate tissue specific mass profiles, or a high resolution imaging experiment where relative spatial abundance for potentially hundreds of analytes across virtually any tissue section can be measured. Crucially, imaging can be achieved without prior knowledge of tissue composition and without the use of antibodies. In effect MALDI-IMS allows generation of molecular data which complement and expand upon the information provided by histology including immuno-histochemistry, making its application valuable to both cancer biomarker research and diagnostics. The current state of MALDI-IMS, key biological applications to ovarian cancer research and practical considerations for analysis of peptides and proteins on ovarian tissue are presented in this review.

Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections

USGS Publications Warehouse

Bruno, D.W.; Nowak, B.; Elliott, D.G.

2006-01-01

The identification of protozoan and metazoan parasites is traditionally carried out using a series of classical keys based upon the morphology of the whole organism. However, in stained tissue sections prepared for light microscopy, taxonomic features will be missing, thus making parasite identification difficult. This work highlights the characteristic features of representative parasites in tissue sections to aid identification. The parasite examples discussed are derived from species affecting finfish, and predominantly include parasites associated with disease or those commonly observed as incidental findings in disease diagnostic cases. Emphasis is on protozoan and small metazoan parasites (such as Myxosporidia) because these are the organisms most likely to be missed or mis-diagnosed during gross examination. Figures are presented in colour to assist biologists and veterinarians who are required to assess host/parasite interactions by light microscopy.

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.

Structural and molecular interrogation of intact biological systems

PubMed Central

Chung, Kwanghun; Wallace, Jenelle; Kim, Sung-Yon; Kalyanasundaram, Sandhiya; Andalman, Aaron S.; Davidson, Thomas J.; Mirzabekov, Julie J.; Zalocusky, Kelly A.; Mattis, Joanna; Denisin, Aleksandra K.; Pak, Sally; Bernstein, Hannah; Ramakrishnan, Charu; Grosenick, Logan; Gradinaru, Viviana; Deisseroth, Karl

2014-01-01

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease. PMID:23575631

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

Bacterial contamination of amniotic membrane in a tissue bank from Iran.

PubMed

Aghayan, Hamid Reza; Goodarzi, Parisa; Baradaran-Rafii, Alireza; Larijani, Bagher; Moradabadi, Leila; Rahim, Fakher; Arjmand, Babak

2013-09-01

Human Amniotic Membrane (AM) transplantation can promote tissue healing and reduce inflammation, tissue scarring and neovascularization. Homa Peyvand Tamin (HPT) tissue bank has focused on manufacturing human cell and tissue based products including AM. The purpose of this study is to evaluate and identify bacterial contamination of AMs that is produced by HPT for several ophthalmic applications. From July 2006 to April 2011, 122 placentas from cesarean sections were retrieved by HPT after obtaining informed consent from the donors. Besides testing donor's blood sample for viral markers, microbiological evaluation was performed pre and post processing. During tissue processing, decontamination was performed by an antibiotic cocktail including; Gentamicin, Ceftriaxone and Cloxacillin. Of 271 cesarean section AM donors who were screened as potential donors, 122 were accepted for processing and assessed for microbiological contamination. Donors' age were between 21 and 41 years (Mean = 27.61 ± 0.24). More than 92% of mothers were in their first or second gravidity with full term pregnancies. The most prevalent organisms were Staphylococci species (72.53%). After processing, contamination rates markedly decreased by 84.62% (p value = 0.013). According to our results, most of bacterial contaminations were related to donation process and the contamination pattern suggests procurement team as a source. Therefore we recommend that regular training programs should be implemented by tissue banks for procurement staff. These programs should focus on improved donor screening and proper aseptic technique for tissue retrieval. We also suggest that tissue banks should periodically check the rate and types of tissue contaminations. These data help them to find system faults and to update processing methods.

28 CFR 549.80 - Authority to conduct autopsies.

Code of Federal Regulations, 2010 CFR

2010-07-01

... this section, the Warden may order an autopsy or post-mortem operation, including removal of tissue for... person (e.g., coroner, or next-of-kin, or the decedent's consent in the case of tissue removed for...-of-kin that they may telegraph the institution collect with their response. Where permission is not...

28 CFR 549.80 - Authority to conduct autopsies.

Code of Federal Regulations, 2011 CFR

2011-07-01

... this section, the Warden may order an autopsy or post-mortem operation, including removal of tissue for... person (e.g., coroner, or next-of-kin, or the decedent's consent in the case of tissue removed for...-of-kin that they may telegraph the institution collect with their response. Where permission is not...

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.

A review of cutting mechanics and modeling techniques for biological materials.

PubMed

Takabi, Behrouz; Tai, Bruce L

2017-07-01

This paper presents a comprehensive survey on the modeling of tissue cutting, including both soft tissue and bone cutting processes. In order to achieve higher accuracy in tissue cutting, as a critical process in surgical operations, the meticulous modeling of such processes is important in particular for surgical tool development and analysis. This review paper is focused on the mechanical concepts and modeling techniques utilized to simulate tissue cutting such as cutting forces and chip morphology. These models are presented in two major categories, namely soft tissue cutting and bone cutting. Fracture toughness is commonly used to describe tissue cutting while Johnson-Cook material model is often adopted for bone cutting in conjunction with finite element analysis (FEA). In each section, the most recent mathematical and computational models are summarized. The differences and similarities among these models, challenges, novel techniques, and recommendations for future work are discussed along with each section. This review is aimed to provide a broad and in-depth vision of the methods suitable for tissue and bone cutting simulations. Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections.

PubMed

Bolognesi, Maddalena Maria; Manzoni, Marco; Scalia, Carla Rossana; Zannella, Stefano; Bosisio, Francesca Maria; Faretta, Mario; Cattoretti, Giorgio

2017-08-01

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.

Tissue preparation for immunocytochemistry.

PubMed Central

Williams, J H; Mepham, B L; Wright, D H

1997-01-01

AIMS: To investigate the effect of tissue preparation on immunostaining and to establish whether there is a standard tissue preparation schedule that allows optimal demonstration of all antigens. METHODS: Blocks of tonsil were subjected to variations to a standard fixation, processing, and section preparation schedule. The sections were stained with five antibodies-L26 (CD20), UCHL1 (CD45RO), CD3, vimentin, and anti-kappa light chain--using the streptavidinbiotin immunostaining technique. When further investigation was necessary, other tissues and antibodies were used and where weak immunostaining was obtained the use of microwave pretreatment to improve staining was tested. RESULTS: Several factors involved in fixation were found to affect immunoreactivity. These included the duration, pH, and type of fixative used. In tissue processing only temperature and the duration of the dehydration and wax infiltration steps affected immunoreactivity. Of all the factors investigated, the temperature and duration of the section drying had the greatest effect. In contrast, long term storage of cut sections before immunostaining had no effect on the reactivity of the antibodies tested. Antibodies were found to be affected by alterations to tissue preparation by varying degrees, UCHL1 and vimentin being the most susceptible to changes in fixation and L26 to changes in processing. Where weak staining occurred, microwave pretreatment was generally found to eliminate the problem. CONCLUSIONS: There is no standard tissue preparation schedule for the optimal demonstration of all antigens. Factors involved in all aspects of tissue preparation can affect immunoreactivity, so it is important that precise details of the preparation schedule are given when reporting immunocytochemical studies, rather than using the general term "routinely fixed and processed". Images PMID:9215127

Localization of antibody binding sites in ultrathin sections of unembedded frog retinal tissue.

PubMed

Pease, D C; Nir, I; Clark, V; Hall, M

1983-01-01

Much ultrastructural detail is retained in tissue fixed only with aldehydes and subsequently air-dried after suspension in a polyvinyl acetate emulsion. The latter provides an external support only, but permits ultrathin sectioning; thus, an exposure of intracellular contents for potential immunocytochemical reactions is achieved. Sections of unembedded frog retina so prepared have been studied with success. The tissue was incubated first with a rabbit antiserum prepared against gradient purified bovine rod outer segments. Following incubation, reacted sites were labeled with ferritin-conjugated goat anti-rabbit IgG and stained with phosphotungstic acid. Intense labeling of the rod outer segments was clearly achieved, whereas the cone outer segments were without label. Other parts of the retina, including the ellipsoid region of both rods and cones, were also without significant label. These regions provided an intrinsic control for the specificity of the antiserum and established the validity of the general technique.

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.

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.

Rigid spine reinforced polymer microelectrode array probe and method of fabrication

DOEpatents

Tabada, Phillipe; Pannu, Satinderpall S

2014-05-27

A rigid spine-reinforced microelectrode array probe and fabrication method. The probe includes a flexible elongated probe body with conductive lines enclosed within a polymeric material. The conductive lines connect microelectrodes found near an insertion end of the probe to respective leads at a connector end of the probe. The probe also includes a rigid spine, such as made from titanium, fixedly attached to the probe body to structurally reinforce the probe body and enable the typically flexible probe body to penetrate and be inserted into tissue, such as neural tissue. By attaching or otherwise fabricating the rigid spine to connect to only an insertion section of the probe body, an integrally connected cable section of the probe body may remain flexible.

Detection of Myelination Using a Novel Histological Probe

PubMed Central

Xiang, Zhongmin; Nesterov, Evgueni E.; Skoch, Jesse; Lin, Tong; Hyman, Bradley T.; Swager, Timothy M.; Bacskai, Brian J.; Reeves, Steven A.

2005-01-01

Current methods for myelin staining in tissue sections include both histological and immunohistochemical techniques. Fluorescence immunohistochemistry, which uses antibodies against myelin components such as myelin basic protein, is often used because of the convenience for multiple labeling. To facilitate studies on myelin, this paper describes a quick and easy method for direct myelin staining in rodent and human tissues using novel near-infrared myelin (NIM) dyes that are comparable to other well-characterized histochemical reagents. The near-infrared fluorescence spectra of these probes allow fluorescent staining of tissue sections in multiple channels using visible light fluorophores commonly used in immunocytochemistry. These dyes have been used successfully to detect normal myelin structure and myelin loss in a mouse model of demyelination disease. PMID:16046669

A simple model for cell type recognition using 2D-correlation analysis of FTIR images from breast cancer tissue

NASA Astrophysics Data System (ADS)

Ali, Mohamed H.; Rakib, Fazle; Al-Saad, Khalid; Al-Saady, Rafif; Lyng, Fiona M.; Goormaghtigh, Erik

2018-07-01

Breast cancer is the second most common cancer after lung cancer. So far, in clinical practice, most cancer parameters originating from histopathology rely on the visualization by a pathologist of microscopic structures observed in stained tissue sections, including immunohistochemistry markers. Fourier transform infrared spectroscopy (FTIR) spectroscopy provides a biochemical fingerprint of a biopsy sample and, together with advanced data analysis techniques, can accurately classify cell types. Yet, one of the challenges when dealing with FTIR imaging is the slow recording of the data. One cm2 tissue section requires several hours of image recording. We show in the present paper that 2D covariance analysis singles out only a few wavenumbers where both variance and covariance are large. Simple models could be built using 4 wavenumbers to identify the 4 main cell types present in breast cancer tissue sections. Decision trees provide particularly simple models to reach discrimination between the 4 cell types. The robustness of these simple decision-tree models were challenged with FTIR spectral data obtained using different recording conditions. One test set was recorded by transflection on tissue sections in the presence of paraffin while the training set was obtained on dewaxed tissue sections by transmission. Furthermore, the test set was collected with a different brand of FTIR microscope and a different pixel size. Despite the different recording conditions, separating extracellular matrix (ECM) from carcinoma spectra was 100% successful, underlying the robustness of this univariate model and the utility of covariance analysis for revealing efficient wavenumbers. We suggest that 2D covariance maps using the full spectral range could be most useful to select the interesting wavenumbers and achieve very fast data acquisition on quantum cascade laser infrared imaging microscopes.

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.

Washout of heme-containing proteins dramatically improves tetrazolium-based infarct staining.

PubMed

Pitts, Kelly R; Stiko, Ann; Buetow, Bernard; Lott, Fred; Guo, Ping; Virca, Duke; Toombs, Christopher F

2007-01-01

Methods to determine infarct size following ischemia-reperfusion injury include gross staining with triphenyltetrazolium chloride (TTC) and perfusion of colored dyes to demarcate the non-ischemic zone. Infarcted tissue (INF) can typically appear a mottled tan to brownish color, making a border between INF and TTC-positive tissue difficult to discern. Previous work in our lab indicated that following TTC staining, prolonged washing of thick sections dramatically sharpened this boundary. Adult rats underwent 30 min ischemia via LAD ligation and reperfusion/recovery over 24 h. Hearts were then harvested, thick-sectioned, and stained with TTC. Stained sections were stored in PBS at 4 degrees C for up to 3 weeks. Histology on thin sections from infarcted hearts fixed directly after harvest revealed extensive hemorrhage within the INF. However, this hemorrhage is washed out when hearts are stored in PBS for 3 weeks. SDS-PAGE of PBS samples taken at 1, 2, and 3 weeks showed a low molecular weight band appearing over time. Peptide sequencing revealed the presence of several proteins including the heme-containing proteins (HCPs) hemoglobin, cytochrome c, and myoglobin. The loss of HCPs from thick sections to PBS corresponded with the blanching of the previously mottled INF within each section. HPLC analysis of these samples confirmed the loss of HCPs contributes to INF whitening. Further, analysis of infarct size values derived from heart slices with or without HCPs showed a significant decrease in measurement error when values were derived from slices without HCPs. These data suggest that HCPs in the heart tissue contribute to the non-uniform and discolored appearance of the INF, and that washout of these proteins produces an INF more easily distinguished from neighboring non-infarcted tissue. This method greatly reduces the error associated with infarct measurements and improves the analysis of the effects of drug treatments and other interventions designed to impact ischemia reperfusion injury.

DR5 as a reporter system to study auxin response in Populus.

PubMed

Chen, Yiru; Yordanov, Yordan S; Ma, Cathleen; Strauss, Steven; Busov, Victor B

2013-03-01

KEY MESSAGE : Auxin responsive promoter DR5 reporter system is functional in Populus to monitor auxin response in tissues including leaves, roots, and stems. We described the behavior of the DR5::GUS reporter system in stably transformed Populus plants. We found several similarities with Arabidopsis, including sensitivity to native and synthetic auxins, rapid induction after treatment in a variety of tissues, and maximal responses in root tissues. There were also several important differences from Arabidopsis, including slower time to maximum response and lower induction amplitude. Young leaves and stem sections below the apex showed much higher DR5 activity than did older leaves and stems undergoing secondary growth. DR5 activity was highest in cortex, suggesting high levels of auxin concentration and/or sensitivity in this tissue. Our study shows that the DR5 reporter system is a sensitive and facile system for monitoring auxin responses and distribution at cellular resolution in poplar.

DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations.

PubMed

Staffend, Nancy A; Meisel, Robert L

2011-01-01

Fine neuronal morphology, such as dendritic spines, classically has been studied using the Golgi technique; however, Golgi staining is difficult to combine with other histological techniques. With the increasing popularity of fluorescent imaging, a number of fluorescent dyes have been developed that enable the coupling of multiple fluorescent labels in a single preparation. These fluorescent dyes include the lipophilic dialkylcarbocyanine, DiI; traditionally used for anterograde and retrograde neuronal tracing. More recently, DiI labeling has been used in combination with the Gene Gun for "DiOlistic" labeling of neurons in slice preparations. DiI sequesters itself within and diffuses laterally along the neuronal membrane, however once the cell is permeabilized, the DiI begins to leak from the cell membrane. A DiI derivative, Cell Tracker™ CM-DiI, increases dye stability and labeling half-life in permeabilized tissue, however at much greater expense. Here, the DiI and CM-DiI DiOlistic labeling techniques were tested in side-by-side experiments evaluating dye stability within dendritic architecture in medium spiny neurons of the dorsal stratum in both non-permeabilized and permeabilized tissue sections. In tissue sections that were not permeabilized, spine density in DiI labeled sections was higher than in CM-DiI labeling. In contrast, tissue sections that were permeabilized had higher spine densities in CM-DiI labeled neurons. These results suggest that for experiments involving non-permeabilized tissue, traditional DiI will suffice, however for experiments involving permeabilized tissue CM-DiI provides more consistent data. These experiments provide the first quantitative analyses of the impact of methodological permutations on neuronal labeling with DiI.

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.

Evaluation of collagen in connective tissue walls of odontogenic cysts--a histochemical study.

PubMed

Vij, Ruchieka; Vij, Hitesh; Rao, Nirmala N

2011-03-01

The purpose of this study was to evaluate the nature of collagen in the connective tissue walls of odontogenic cysts, like the odontogenic keratocyst (OKC), dentigerous cyst and radicular cyst using picrosirius red stained sections. Furthermore, it was intended to assess if the capsular connective tissue can affect the nature of overlying epithelium, thus emphasizing the role of epithelial-mesenchymal interactions in biological behaviour of the cysts. The material for the study included 51 formalin-fixed paraffin-embedded tissue blocks (15 odontogenic keratocyst, 15 dentigerous cysts, 15 radicular cysts and four normal mucosa and two dental follicular tissue as controls), retrieved from the Department of Oral Pathology and Microbiology, MCODS, Manipal. Tissue blocks were sectioned at 5-μm thickness, stained with picrosirius red stain and observed with polarization and light microscopy. Few sections of OKC and dentigerous cyst exhibited greenish-yellow birefringence in sub-epithelial region, whereas others showed a yellowish-orange birefringence under polarization microscopy. Most radicular cysts had yellowish-orange to orange birefringence. Shift in colour in case OKC and dentigerous cyst was attributed to the presence of inflammation in those sections. These regions also exhibited either a change in phenotype or thickness of overlying epithelium. This technique can be used to study the nature of collagen fibres in odontogenic cyst walls. Further studies with an increased sample size and using various epithelial and mesenchymal markers and ssDNA antibodies should be carried out to confirm the effect of epithelial-mesenchymal interactions on the nature of epithelium of odontogenic cysts. © 2010 John Wiley & Sons A/S.

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

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.

Development and validation of a protocol for optimizing the use of paraffin blocks in molecular epidemiological studies: The example from the HPV-AHEAD study.

PubMed

Mena, Marisa; Lloveras, Belen; Tous, Sara; Bogers, Johannes; Maffini, Fausto; Gangane, Nitin; Kumar, Rekha Vijay; Somanathan, Thara; Lucas, Eric; Anantharaman, Devasena; Gheit, Tarik; Castellsagué, Xavier; Pawlita, Michael; de Sanjosé, Silvia; Alemany, Laia; Tommasino, Massimo

2017-01-01

Worldwide use of formalin-fixed paraffin-embedded blocks (FFPE) is extensive in diagnosis and research. Yet, there is a lack of optimized/standardized protocols to process the blocks and verify the quality and presence of the targeted tissue. In the context of an international study on head and neck cancer (HNC)-HPV-AHEAD, a standardized protocol for optimizing the use of FFPEs in molecular epidemiology was developed and validated. First, a protocol for sectioning the FFPE was developed to prevent cross-contamination and distributed between participating centers. Before processing blocks, all sectioning centers underwent a quality control to guarantee a satisfactory training process. The first and last sections of the FFPEs were used for histopathological assessment. A consensus histopathology evaluation form was developed by an international panel of pathologists and evaluated for four indicators in a pilot analysis in order to validate it: 1) presence/type of tumor tissue, 2) identification of other tissue components that could affect the molecular diagnosis and 3) quality of the tissue. No HPV DNA was found in sections from empty FFPE generated in any histology laboratories of HPV-AHEAD consortium and all centers passed quality assurance for processing after quality control. The pilot analysis to validate the histopathology form included 355 HNC cases. The form was filled by six pathologists and each case was randomly assigned to two of them. Most samples (86%) were considered satisfactory. Presence of >50% of invasive carcinoma was observed in all sections of 66% of cases. Substantial necrosis (>50%) was present in <2% of samples. The concordance for the indicators targeted to validate the histopathology form was very high (kappa > 0.85) between first and last sections and fair to high between pathologists (kappa/pabak 0.21-0.72). The protocol allowed to correctly process without signs of contamination all FFPE of the study. The histopathology evaluation of the cases assured the presence of the targeted tissue, identified the presence of other tissues that could disturb the molecular diagnosis and allowed the assessment of tissue quality.

Comparison between thaw-mounting and use of conductive tape for sample preparation in ToF-SIMS imaging of lipids in Drosophila microRNA-14 model.

PubMed

Le, Minh Uyen Thi; Son, Jin Gyeong; Shon, Hyun Kyoung; Park, Jeong Hyang; Lee, Sung Bae; Lee, Tae Geol

2018-03-30

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging elucidates molecular distributions in tissue sections, providing useful information about the metabolic pathways linked to diseases. However, delocalization of the analytes and inadequate tissue adherence during sample preparation are among some of the unfortunate phenomena associated with this technique due to their role in the reduction of the quality, reliability, and spatial resolution of the ToF-SIMS images. For these reasons, ToF-SIMS imaging requires a more rigorous sample preparation method in order to preserve the natural state of the tissues. The traditional thaw-mounting method is particularly vulnerable to altered distributions of the analytes due to thermal effects, as well as to tissue shrinkage. In the present study, the authors made comparisons of different tissue mounting methods, including the thaw-mounting method. The authors used conductive tape as the tissue-mounting material on the substrate because it does not require heat from the finger for the tissue section to adhere to the substrate and can reduce charge accumulation during data acquisition. With the conductive-tape sampling method, they were able to acquire reproducible tissue sections and high-quality images without redistribution of the molecules. Also, the authors were successful in preserving the natural states and chemical distributions of the different components of fat metabolites such as diacylglycerol and fatty acids by using the tape-supported sampling in microRNA-14 (miR-14) deleted Drosophila models. The method highlighted here shows an improvement in the accuracy of mass spectrometric imaging of tissue samples.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain.

PubMed

Kuipers, Jeroen; Kalicharan, Ruby D; Wolters, Anouk H G; van Ham, Tjakko J; Giepmans, Ben N G

2016-05-25

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

PubMed Central

Kuipers, Jeroen; Kalicharan, Ruby D.; Wolters, Anouk H. G.

2016-01-01

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner. PMID:27285162

21 CFR 589.2000 - Animal proteins prohibited in ruminant feed.

Code of Federal Regulations, 2014 CFR

2014-04-01

... Animal proteins prohibited in ruminant feed. (a) Definitions—(1) Protein derived from mammalian tissues... products may include animal feed. The term includes renderers that also blend animal protein products. (3... in paragraph (e) of this section. (1) Protein blenders, feed manufacturers, and distributors that...

21 CFR 589.2000 - Animal proteins prohibited in ruminant feed.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Animal proteins prohibited in ruminant feed. (a) Definitions—(1) Protein derived from mammalian tissues... products may include animal feed. The term includes renderers that also blend animal protein products. (3... in paragraph (e) of this section. (1) Protein blenders, feed manufacturers, and distributors that...

21 CFR 589.2000 - Animal proteins prohibited in ruminant feed.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Animal proteins prohibited in ruminant feed. (a) Definitions—(1) Protein derived from mammalian tissues... products may include animal feed. The term includes renderers that also blend animal protein products. (3... in paragraph (e) of this section. (1) Protein blenders, feed manufacturers, and distributors that...

21 CFR 589.2000 - Animal proteins prohibited in ruminant feed.

Code of Federal Regulations, 2012 CFR

2012-04-01

... Animal proteins prohibited in ruminant feed. (a) Definitions—(1) Protein derived from mammalian tissues... products may include animal feed. The term includes renderers that also blend animal protein products. (3... in paragraph (e) of this section. (1) Protein blenders, feed manufacturers, and distributors that...

Immunoperoxidase staining for identification of Aspergillus species in routinely processed tissue sections.

PubMed Central

Verweij, P E; Smedts, F; Poot, T; Bult, P; Hoogkamp-Korstanje, J A; Meis, J F

1996-01-01

AIMS: To evaluate the performance of an immunoperoxidase stain using the monoclonal antibody EB-A1 to detect Aspergillus species in formalin fixed, paraffin wax embedded tissue. METHODS: The monoclonal antibody EB-A1 directed against galactomannan was used to detect Aspergillus species in 23 patients with suspected or confirmed invasive aspergillosis. Immunostaining was performed on formalin fixed, paraffin wax embedded tissue using the streptavidin-biotin method and compared with conventional haematoxylin and eosin, periodic acid-Schiff, and Gomori-Grocott stains. Results of immunostaining were semiquantitatively analysed with regard to both intensity of staining and number of positively staining micro-organisms. Tissue sections from 16 patients with confirmed invasive mycoses due to Candida species, Apophysomyces elegans, Rhizopus oryzae, Pseudallescheria boydii and Histoplasma capsulatum were used as controls. RESULTS: In 19 (83%) of 23 cases invasive aspergillosis was confirmed by both histological examination and culture (18 Aspergillus fumigatus and one A flavus). Immunoperoxidase stains were positive in 17 (89%) of 19 cases including one case of disseminated infection due to A flavus. Furthermore, the immunoperoxidase stain was positive in a culture negative tissue section with histological evidence of mycelial development, indicating the presence of Aspergillus species. Some cross-reactivity was observed with the highly related fungus P boydii, although the number of mycelial elements that stained was low. CONCLUSIONS: Immunoperoxidase staining using the monoclonal antibody EB-A1 performs well on routinely processed tissue sections and permits detection and generic identification of Aspergillus species, although it was no better than conventional histopathology in identifying the presence of an infection. An additional advantage is that the immunostain may help to provide an aetiological diagnosis when cultures remain negative. Images PMID:8943743

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Rodriguez-Canales, Jaime; Hanson, Jeffrey C; Hipp, Jason D; Balis, Ulysses J; Tangrea, Michael A; Emmert-Buck, Michael R; Bova, G Steven

2013-01-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This updated chapter 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. Improvement in this area will significantly increase life science quality and productivity. The chapter is divided into introduction, materials, protocols, and notes subheadings. 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 chapter, readers are advised to read through the entire chapter 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.

Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

PubMed

Sarkar, F H; Kupsky, W J; Li, Y W; Sreepathi, P

1994-03-01

Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

Spectral staining of tumor tissue by fiber optic FTIR spectroscopy

NASA Astrophysics Data System (ADS)

Salzer, Reiner; Steiner, Gerald; Kano, Angelique; Richter, Tom; Bergmann, Ralf; Rodig, Heike; Johannsen, Bernd; Kobelke, Jens

2003-07-01

Infrared (IR) optical fiber have aroused great interest in recent years because of their potential in in-vivo spectroscopy. This potential includes the ability to be flexible, small and to guide IR light in a very large range of wavelengths. Two types - silver halide and chalcogenide - infrared transmitting fibers are investigated in the detection of a malignant tumor. As a test sample for all types of fibers we used a thin section of an entire rat brain with glioblastoma. The fibers were connected with a common infrared microscope. Maps across the whole tissue section with more than 200 spectra were recorded by moving the sample with an XY stage. Data evaluation was performed using fuzzy c-means cluster analysis (FCM). The silver halide fibers provided excellent results. The tumor was clearly discernible from healthy tissue. Chalcogenide fibers are not suitable to distinguish tumor from normal tissue because the fiber has a very low transmittance in the important fingerprint region.

Quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor-immune complexity associated with poor prognosis

PubMed Central

Tsujikawa, Takahiro; Kumar, Sushil; Borkar, Rohan N.; Azimi, Vahid; Thibault, Guillaume; Chang, Young Hwan; Balter, Ariel; Kawashima, Rie; Choe, Gina; Sauer, David; El Rassi, Edward; Clayburgh, Daniel R.; Kulesz-Martin, Molly F.; Lutz, Eric R.; Zheng, Lei; Jaffee, Elizabeth M.; Leyshock, Patrick; Margolin, Adam A.; Mori, Motomi; Gray, Joe W.; Flint, Paul W.; Coussens, Lisa M.

2017-01-01

SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment. PMID:28380359

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.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

PubMed Central

Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

2016-01-01

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections.

PubMed

Ellison, Mitchell A; McMahon, Michael B; Bonde, Morris R; Palmer, Cristi L; Luster, Douglas G

2016-01-01

Rust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here. To validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues. This ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

Simulation of ultrasonic pulse propagation, distortion, and attenuation in the human chest wall.

PubMed

Mast, T D; Hinkelman, L M; Metlay, L A; Orr, M J; Waag, R C

1999-12-01

A finite-difference time-domain model for ultrasonic pulse propagation through soft tissue has been extended to incorporate absorption effects as well as longitudinal-wave propagation in cartilage and bone. This extended model has been used to simulate ultrasonic propagation through anatomically detailed representations of chest wall structure. The inhomogeneous chest wall tissue is represented by two-dimensional maps determined by staining chest wall cross sections to distinguish between tissue types, digitally scanning the stained cross sections, and mapping each pixel of the scanned images to fat, muscle, connective tissue, cartilage, or bone. Each pixel of the tissue map is then assigned a sound speed, density, and absorption value determined from published measurements and assumed to be representative of the local tissue type. Computational results for energy level fluctuations and arrival time fluctuations show qualitative agreement with measurements performed on the same specimens, but show significantly less waveform distortion than measurements. Visualization of simulated tissue-ultrasound interactions in the chest wall shows possible mechanisms for image aberration in echocardiography, including effects associated with reflection and diffraction caused by rib structures. A comparison of distortion effects for varying pulse center frequencies shows that, for soft tissue paths through the chest wall, energy level and waveform distortion increase markedly with rising ultrasonic frequency and that arrival-time fluctuations increase to a lesser degree.

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.

A comparative study to evaluate liquid dish washing soap as an alternative to xylene and alcohol in deparaffinization and hematoxylin and eosin staining.

PubMed

Pandey, Pinki; Dixit, Alok; Tanwar, Aparna; Sharma, Anuradha; Mittal, Sanjeev

2014-07-01

Our study presents a new deparaffinizing and hematoxylin and eosin (H and E) staining method that involves the use of easily available, nontoxic and eco-friendly liquid diluted dish washing soap (DWS) by completely eliminating expensive and hazardous xylene and alcohol from deparaffinizing and rehydration prior to staining, staining and from dehydration prior to mounting. The aim was to evaluate and compare the quality of liquid DWS treated xylene and alcohol free (XAF) sections with that of the conventional H and E sections. A total of 100 paraffin embedded tissue blocks from different tissues were included. From each tissue block, one section was stained with conventional H and E (normal sections) and the other with XAF H and E (soapy sections) staining method. Slides were scored using five parameters: Nuclear, cytoplasmic, clarity, uniformity, and crispness of staining. Z-test was used for statistical analysis. Soapy sections scored better for cytoplasmic (90%) and crisp staining (95%) with a statistically significant difference. Whereas for uniformity of staining, normal sections (88%) scored over soapy sections (72%) (Z = 2.82, P < 0.05). For nuclear (90%) and clarity of staining (90%) total scored favored soapy sections, but the difference was not statistically significant. About 84% normal sections stained adequately for diagnosis when compared with 86% in soapy sections (Z = 0.396, P > 0.05). Liquid DWS is a safe and efficient alternative to xylene and alcohol in deparaffinization and routine H and E staining procedure. We are documenting this project that can be used as a model for other histology laboratories.

A comparative study to evaluate liquid dish washing soap as an alternative to xylene and alcohol in deparaffinization and hematoxylin and eosin staining

PubMed Central

Pandey, Pinki; Dixit, Alok; Tanwar, Aparna; Sharma, Anuradha; Mittal, Sanjeev

2014-01-01

Introduction: Our study presents a new deparaffinizing and hematoxylin and eosin (H and E) staining method that involves the use of easily available, nontoxic and eco-friendly liquid diluted dish washing soap (DWS) by completely eliminating expensive and hazardous xylene and alcohol from deparaffinizing and rehydration prior to staining, staining and from dehydration prior to mounting. The aim was to evaluate and compare the quality of liquid DWS treated xylene and alcohol free (XAF) sections with that of the conventional H and E sections. Materials and Methods: A total of 100 paraffin embedded tissue blocks from different tissues were included. From each tissue block, one section was stained with conventional H and E (normal sections) and the other with XAF H and E (soapy sections) staining method. Slides were scored using five parameters: Nuclear, cytoplasmic, clarity, uniformity, and crispness of staining. Z-test was used for statistical analysis. Results: Soapy sections scored better for cytoplasmic (90%) and crisp staining (95%) with a statistically significant difference. Whereas for uniformity of staining, normal sections (88%) scored over soapy sections (72%) (Z = 2.82, P < 0.05). For nuclear (90%) and clarity of staining (90%) total scored favored soapy sections, but the difference was not statistically significant. About 84% normal sections stained adequately for diagnosis when compared with 86% in soapy sections (Z = 0.396, P > 0.05). Conclusion: Liquid DWS is a safe and efficient alternative to xylene and alcohol in deparaffinization and routine H and E staining procedure. We are documenting this project that can be used as a model for other histology laboratories. PMID:25328332

Liquid Microjunction Surface Sampling Probe Electrospray Mass Spectrometry for Detection of Drugs and Metabolites in Thin Tissue Sections

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

Van Berkel, Gary J; Kertesz, Vilmos; Koeplinger, Kenneth A.

2008-01-01

A self-aspirating, liquid micro-junction surface sampling probe/electrospray emitter mass spectrometry system was demonstrated for use in the direct analysis of spotted and dosed drugs and their metabolites in thin tissue sections. Proof-of-principle sampling and analysis directly from tissue without the need for sample preparation was demonstrated first by raster scanning a region on a section of rat liver onto which reserpine was spotted. The mass spectral signal from selected reaction monitoring was used to develop a chemical image of the spotted drug on the tissue. The probe was also used to selectively spot sample areas of sagittal whole mouse bodymore » tissue sections that had been dosed orally (90 mg/kg) with R,S-sulforaphane 3 hrs prior to sacrifice. Sulforaphane and its glutathione and N-acetyl cysteine conjugates were monitored with selected reaction monitoring and detected in the stomach and various other tissues from the dosed mouse. No signal for these species was observed in the tissue from a control mouse. The same dosed tissue section was used to illustrate the possibility of obtaining a line scan across the whole body section. In total these results illustrate the potential for rapid screening of the distribution of drugs and metabolites in tissue sections with the micro-liquid junction surface sampling probe/electrospray mass spectrometry approach.« less

Sarcopenia is linked to treatment toxicity in patients with metastatic colorectal cancer.

PubMed

Barret, Maximilien; Antoun, Sami; Dalban, Cécile; Malka, David; Mansourbakht, Touraj; Zaanan, Aziz; Latko, Ewa; Taieb, Julien

2014-01-01

Chemotherapy toxicity could be linked to decreased skeletal muscle (sarcopenia). We evaluated the effect of sarcopenia on chemotherapy toxicity among metastatic colorectal cancer (mCRC) patients. All consecutive mCRC patients in 3 hospitals were enrolled in this prospective, cross-sectional, multicenter study. Several nutritional indexes and scores were generated. Computed tomography (CT) images were analyzed to evaluate cross-sectional areas of muscle tissue (MT), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT). Toxicities were evaluated in the 2 mo following clinical evaluation. Fifty-one mCRC patients were included in the study. Sarcopenia was observed in 71% of patients (39% of women and 82% of men) whereas only 4% and 18% were considered as underweight using body mass index (BMI) or severely malnourished using the Nutritional Risk Index (NRI), respectively. Grade 3-4 toxicities were observed in 28% of patients. In multivariate analysis including age, sex, BMI, sarcopenia, SAT, and VAT, the only factor associated with Grade 3-4 toxicities was sarcopenia (odds ratio = 13.55; 95% confidence interval [1.08; 169.31], P = 0.043). In mCRC patients undergoing chemotherapy, sarcopenia was much more frequently observed than visible malnutrition. Despite the small number of patients included in our study, we found sarcopenia to be significantly associated with severe chemotherapy toxicity.

Mapping pharmaceuticals in rat brain sections using MALDI imaging mass spectrometry.

PubMed

Hsieh, Yunsheng; Li, Fangbiao; Korfmacher, Walter A

2010-01-01

Matrix-assisted laser desorption/ionization-tandem mass spectrometric method (MALDI-MS/MS) has proven to be a reliable tool for direct measurement of the disposition of small molecules in animal tissue sections. As example, MALDI-MS/MS imaging system was employed for visualizing the spatial distribution of astemizole and its primary metabolite in rat brain tissues. Astemizole is a second-generation antihistamine, a block peripheral H1 receptor, which was introduced to provide comparable therapeutic benefit but was withdrawn in most countries due to toxicity risks. Astemizole was observed to be heterogeneously distributed to most parts of brain tissue slices including cortex, hippocampus, hypothalamic, thalamus, and ventricle regions while its major metabolite, desmethylastemizole, was only found around ventricle sites. We have shown that astemizole alone is likely to be responsible for the central nervous system (CNS) side effects when its exposures became elevated.

Rapid in vivo vertical tissue sectioning by multiphoton tomography

NASA Astrophysics Data System (ADS)

Batista, Ana; Breunig, Hans Georg; König, Karsten

2018-02-01

A conventional tool in the pathological field is histology which involves the analysis of thin sections of tissue in which specific cellular structures are stained with different dyes. The process to obtain these stained tissue sections is time consuming and invasive as it requires tissue removal, fixation, sectioning, and staining. Moreover, imaging of live tissue is not possible. We demonstrate that multiphoton tomography can provide within seconds, non-invasive, label-free, vertical images of live tissue which are in quality similar to conventional light micrographs of histologic stained specimen. In contrast to conventional setups based on laser scanning which image horizontally sections, the vertical in vivo images are directly recorded by combined line scanning and timed adjustments of the height of the focusing optics. In addition, multiphoton tomography provides autofluorescence lifetimes which can be used to determine the metabolic states of cells.

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.

Enhancing regenerative approaches with nanoparticles

PubMed Central

Habibovic, Pamela

2017-01-01

In this review, we discuss recent developments in the field of nanoparticles and their use in tissue regeneration approaches. Owing to their unique chemical properties and flexibility in design, nanoparticles can be used as drug delivery systems, to create novel features within materials or as bioimaging agents, or indeed these properties can be combined to create smart multifunctional structures. This review aims to provide an overview of this research field where the focus will be on nanoparticle-based strategies to stimulate bone regeneration; however, the same principles can be applied for other tissue and organ regeneration strategies. In the first section, nanoparticle-based methods for the delivery of drugs, growth factors and genetic material to promote tissue regeneration are discussed. The second section deals with the addition of nanoparticles to materials to create nanocomposites. Such materials can improve several material properties, including mechanical stability, biocompatibility and biological activity. The third section will deal with the emergence of a relatively new field of research using nanoparticles in advanced cell imaging and stem cell tracking approaches. As the development of nanoparticles continues, incorporation of this technology in the field of regenerative medicine will ultimately lead to new tools that can diagnose, track and stimulate the growth of new tissues and organs. PMID:28404870

Enhancing regenerative approaches with nanoparticles.

PubMed

van Rijt, Sabine; Habibovic, Pamela

2017-04-01

In this review, we discuss recent developments in the field of nanoparticles and their use in tissue regeneration approaches. Owing to their unique chemical properties and flexibility in design, nanoparticles can be used as drug delivery systems, to create novel features within materials or as bioimaging agents, or indeed these properties can be combined to create smart multifunctional structures. This review aims to provide an overview of this research field where the focus will be on nanoparticle-based strategies to stimulate bone regeneration; however, the same principles can be applied for other tissue and organ regeneration strategies. In the first section, nanoparticle-based methods for the delivery of drugs, growth factors and genetic material to promote tissue regeneration are discussed. The second section deals with the addition of nanoparticles to materials to create nanocomposites. Such materials can improve several material properties, including mechanical stability, biocompatibility and biological activity. The third section will deal with the emergence of a relatively new field of research using nanoparticles in advanced cell imaging and stem cell tracking approaches. As the development of nanoparticles continues, incorporation of this technology in the field of regenerative medicine will ultimately lead to new tools that can diagnose, track and stimulate the growth of new tissues and organs. © 2017 The Author(s).

Intratumoral immune cells expressing PD-1/PD-L1 and their prognostic implications in cancer: a meta-analysis.

PubMed

Kim, Younghoon; Wen, Xianyu; Cho, Nam Yun; Kang, Gyeong Hoon

2018-05-01

The prognostic value of immune cells expressing programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) in cancer are controversial, and the potential differential impact of using tissue microarrays and whole tissue sections to assess the positivity of immune cells has not been addressed. The current study included 30 eligible studies with 7251 patients that evaluated the relationship between tumor-infiltrating lymphocytes expressing PD-1/PD-L1 and overall survival and disease-free survival, or progression-free survival. Subgroup analysis was based on the tissue type of cancer and the type of tissue sampling (tissue microarray or whole tissue section). In the meta-analysis, PD-1-positive and PD-L1-positive tumor-infiltrating lymphocytes had a positive effect on disease-free survival or progression-free survival (hazard ratio [HR] 0.732; 95% confidence interval [CI] 0.565, 0.947; and HR 0.727; 95% CI 0.584, 0.905, respectively). PD-L1-positive tumor-infiltrating lymphocytes had a positive impact on overall survival in studies using tissue microarray (HR 0.586; 95% CI 0.476, 0.721), but had a poor impact when only whole tissue sections were considered (HR 1.558; 95% CI 1.232, 1.969). Lung cancer was associated with good overall survival and disease-free survival (HR 0.639; 95% CI 0.491, 0.831; and HR 0.693; 95% CI 0.538, 0.891, respectively) for PD-1-positive tumor-infiltrating lymphocytes, and colorectal cancer showed favorable disease-free survival (HR 0.471; 95% CI 0.308, 0.722) for PD-L1-positive tumor-infiltrating lymphocytes. Immune cells expressing PD-1 and PD-L1 within tumors are associated with the prognosis. However, the correlation may vary among different tumor types and by the type of tissue sampling used for the assessment.

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.

[Magnetic resonance imaging findings on the eyelids of Japanese cadavers for anatomical studies and a comparative examination of their histological pictures].

PubMed

Ide, Atsushi; Wakimasu, Koichi; Shiba, Tomoko; Kodama, Junichi; Shirasawa, Nobuyuki

2009-12-01

To clarify ambiguous areas in interpreting MR images of Japanese eyelids, a histological examination was conducted on cadavers after the MRI for a comparative evaluation. Orbital sections including the unilateral upper and lower palpebrae of two Japanese cadavers (an 87-year-old woman and a 49-year-old man) were examined. Following MRI, the specimens of the same cadavers were examined histologically for a comparative evaluation. In both cadavers, a high signal intensity area with a hazy appearance unlike the orbital fat--fibroadipose tissue rich with connective tissue--was recognized between the orbicularis muscle and orbital septum. The same high signal intensity area that appeared to encase the posterior section of the descending orbital fat was also composed of fibroadipose tissue. Because of the presence of this intervening fibroadipose tissue, the posterior surface of the orbicularis muscle and the orbital septum could not come into contact with each other. Although limited to only two subjects, the current observation proved that fibroadipose tissue exists not only in the superficial layer of the orbital fat but that it further descends to surround the lowest portion of the orbital fat. As already reported, it was mainly the fibroadipose tissue, but not the orbital fat, that descends into the palpebral space.

Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma.

PubMed

Ankle, Madhuri R; Kale, Alka D; Charantimath, Seema

2007-01-01

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Mann-Whitney U test. A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections (p=0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.(p=0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet (p=0.4429) or the H and E-staining techniques (p=0.2717). One per cent crystal violet provides a definite advantage over the H and E-stained sections in selectively staining the mitotic figures.

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

Soft-Tissue Infections and Their Imaging Mimics: From Cellulitis to Necrotizing Fasciitis.

PubMed

Hayeri, Mohammad Reza; Ziai, Pouya; Shehata, Monda L; Teytelboym, Oleg M; Huang, Brady K

2016-10-01

Infection of the musculoskeletal system can be associated with high mortality and morbidity if not promptly and accurately diagnosed. These infections are generally diagnosed and managed clinically; however, clinical and laboratory findings sometimes lack sensitivity and specificity, and a definite diagnosis may not be possible. In uncertain situations, imaging is frequently performed to confirm the diagnosis, evaluate the extent of the disease, and aid in treatment planning. In particular, cross-sectional imaging, including computed tomography and magnetic resonance imaging, provides detailed anatomic information in the evaluation of soft tissues due to their inherent high spatial and contrast resolution. Imaging findings of soft-tissue infections can be nonspecific and can have different appearances depending on the depth and anatomic extent of tissue involvement. Although many imaging features of infectious disease can overlap with noninfectious processes, imaging can help establish the diagnosis when combined with the clinical history and laboratory findings. Radiologists should be familiar with the spectrum of imaging findings of soft-tissue infections to better aid the referring physician in managing these patients. The aim of this article is to review the spectrum of soft-tissue infections using a systematic anatomic compartment approach. We discuss the clinical features of soft-tissue infections, their imaging findings with emphasis on cross-sectional imaging, their potential mimics, and clinical management. © RSNA, 2016.

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.

PubMed

Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R; Changelian, Armen; Laws, Edward R; Santagata, Sandro; Agar, Nathalie Y R; Van Berkel, Gary J

2015-08-01

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 spectrometric detection. Excellent correlation was found between the protein distribution data obtained with this method and 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. 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 with non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis, as expected. This work reveals 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, including AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity, and specificity of this method support the potential of this basic technology, with further advancement, for assisting surgical decision-making. Graphical Abstract Mass spectrometry based profiling of hormones in human pituitary gland and tumor thin tissue sections.

Effect of bar cross-section geometry on stress distribution in overdenture-retaining system simulating horizontal misfit and bone loss.

PubMed

Spazzin, Aloísio Oro; Costa, Ana Rosa; Correr, Américo Bortolazzo; Consani, Rafael Leonardo Xediek; Correr-Sobrinho, Lourenço; dos Santos, Mateus Bertolini Fernandes

2013-08-09

This study evaluated the influence of cross-section geometry of the bar framework on the distribution of static stresses in an overdenture-retaining bar system simulating horizontal misfit and bone loss. Three-dimensional FE models were created including two titanium implants and three cross-section geometries (circular, ovoid or Hader) of bar framework placed in the anterior part of a severely resorbed jaw. One model with 1.4-mm vertical loss of the peri-implant tissue was also created. The models set were exported to mechanical simulation software, where horizontal displacement (10, 50 or 100 μm) was applied simulating the settling of the framework, which suffered shrinkage during the laboratory procedures. The bar material used for the bar framework was a cobalt--chromium alloy. For evaluation of bone loss effect, only the 50-μm horizontal misfit was simulated. Data were qualitatively and quantitatively evaluated using von Mises stress for the mechanical part and maximum principal stress and μ-strain for peri-implant bone tissue given by the software. Stresses were concentrated along the bar and in the join between the bar and cylinder. In the peri-implant bone tissue, the μ-strain was higher in the cervical third. Higher stress levels and μ-strain were found for the models using the Hader bar. The bone loss simulated presented considerable increase on maximum principal stresses and μ-strain in the peri-implant bone tissue. In addition, for the amplification of the horizontal misfit, the higher complexity of the bar cross-section geometry and bone loss increases the levels of static stresses in the peri-implant bone tissue. Copyright © 2013 Elsevier Ltd. All rights reserved.

Conjunctival Expansion Using a Subtenon's Silicone Implant in New Zealand White Rabbits

PubMed Central

Yoon, Ie-na; Lee, Dong-hoon

2007-01-01

Purpose In the field of ophthalmology, the conjunctival autograft is a useful therapeutic material in many cases, but the small size of the autograft is a disadvantage. Therefore, we evaluated the feasibility of taking an expanded sample of conjunctival tissue using a subtenon's silicone implant. Materials and Methods We included a total of nine rabbits; eight rabbits were operative cases, and one was a control. A portion of conjunctival tissue from the control rabbit, which did not undergo surgery, was dissected and examined to determine whether it was histologically different from the experimental group. The surgical procedure was performed on eight rabbits via a subtenon's insertion of a silicone sponge in the left superior-temporal portion; after surgery, we dropped antibiotics into the eyes. We sacrificed a pair of rabbits every three days (on days 3, 6, 9, and 12) after surgery, removed the expanded conjunctival tissues with the silicone sponge implants, and measured their sizes. Results The mean size of the expanded conjunctival tissues was 194.4 mm2. On the third day, we were able to harvest a 223.56 mm2 section of conjunctival tissue, which was the most expanded sample of tissue in the study. On the twelfth day, we removed a 160.38 mm2 section of conjunctival tissue, which was the least expanded sample of tissue. Statistically, there were no significant differences in the mean dimensions of the expanded conjunctival tissues for each time period. Microscopic examinations showed no histological differences between the expanded conjunctival tissues and the normal conjunctival tissues. Conclusion The results reveal that this procedure is a useful method to expand the conjunctiva for grafting and transplantation. PMID:18159586

Investigation of the usefulness of fluorescein sodium fluorescence in stereotactic brain biopsy.

PubMed

Thien, Ady; Han, Julian Xinguang; Kumar, Krishan; Ng, Yew Poh; Rao, Jai Prashanth; Ng, Wai Hoe; King, Nicolas Kon Kam

2018-02-01

Intraoperative frozen section assessment, to confirm acquisition of pathological tissues, is used in stereotactic brain biopsy to minimise sampling errors. Limitations include the dependence on dedicated neuro-oncology pathologists and an increase in operative duration. We investigated the use of intraoperative fluorescein sodium, and compared it to frozen section assessment, for confirming pathological tissue samples in the stereotactic biopsy of gadolinium-contrast-enhancing brain lesions. This prospective observational study consisted of 18 consecutive patients (12 men; median age, 63 years) who underwent stereotactic biopsy of gadolinium-contrast-enhancing brain lesions with intravenous fluorescein sodium administration. Twenty-three specimens were obtained and examined for the presence of fluorescence using a microscope with fluorescence visualisation capability. Positive and negative predictive values were calculated based on the fluorescence status of the biopsy samples with its corresponding intraoperative frozen section and definitive histopathological diagnosis. Nineteen specimens (83%) were fluorescent and four (17%) were non-fluorescent. All 19 fluorescent specimens were confirmed to be lesional on intraoperative frozen section assessment and were suitable for histopathological diagnosis. Three of the non-fluorescent specimens were confirmed to be lesional on intraoperative frozen section assessment. One non-fluorescent specimen was non-diagnostic on frozen section and histological assessments. The positive predictive value was 100% and the negative predictive value was 25%. Fluorescein sodium fluorescence is as accurate as frozen section assessment in confirming sampling of pathological tissue in the stereotactic biopsy of gadolinium-contrast-enhancing brain lesions. Fluorescein sodium fluorescence-guided stereotactic biopsy is a useful addition to the neurosurgical armamentarium.

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.

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

Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

PubMed

Xu, Ruilian; Tang, Jun; Deng, Quantong; He, Wan; Sun, Xiujie; Xia, Ligang; Cheng, Zhiqiang; He, Lisheng; You, Shuyuan; Hu, Jintao; Fu, Yuxiang; Zhu, Jian; Chen, Yixin; Gao, Weina; He, An; Guo, Zhengyu; Lin, Lin; Li, Hua; Hu, Chaofeng; Tian, Ruijun

2018-05-01

Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm 2 and 10 μm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm 2 and 10 μm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

Fixation methods for electron microscopy of human and other liver

PubMed Central

Wisse, Eddie; Braet, Filip; Duimel, Hans; Vreuls, Celien; Koek, Ger; Olde Damink, Steven WM; van den Broek, Maartje AJ; De Geest, Bart; Dejong, Cees HC; Tateno, Chise; Frederik, Peter

2010-01-01

For an electron microscopic study of the liver, expertise and complicated, time-consuming processing of hepatic tissues and cells is needed. The interpretation of electron microscopy (EM) images requires knowledge of the liver fine structure and experience with the numerous artifacts in fixation, embedding, sectioning, contrast staining and microscopic imaging. Hence, the aim of this paper is to present a detailed summary of different methods for the preparation of hepatic cells and tissue, for the purpose of preserving long-standing expertise and to encourage new investigators and clinicians to include EM studies of liver cells and tissue in their projects. PMID:20556830

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.

Crystal violet stain as a selective stain for the assessment of mitotic figures in oral epithelial dysplasia and oral squamous cell carcinoma.

PubMed

Jadhav, Kiran B; Ahmed Mujib, B R; Gupta, Nidhi

2012-01-01

Assessment of mitotic figures (MFs) is routinely practiced as prognostic indicator in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), but identification of MFs poses a problem in terms of staining characteristics. To evaluate effectiveness of crystal violet stain for staining of MFs and its comparison with hematoxylin and eosin (H and E) stain. Study sample includes archival tissues embedded in paraffin blocks diagnosed as OED (n = 30) and OSCC (n = 30). The control group comprised of tissue specimen from oral mucosa of healthy volunteers (n = 30). Two serial sections of each tissue specimen were stained separately with H and E stain and 1% crystal violet stain. The stained sections were observed under microscope for identification and counting of MFs. Data obtained was statistically analyzed by using the Man-Whitney U test. A significant increase in number of MFs was observed in OED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in crystal violet stained tissue sections when compared with H and E stain. Metaphase is the most commonly observed phase of mitosis in crystal violet stain when compared with H and E stain for all three groups. Crystal violet stain can be considered as selective stain for mitotic figures.

Brain tissues atrophy is not always the best structural biomarker of physiological aging: A multimodal cross-sectional study.

PubMed

Cherubini, Andrea; Caligiuri, Maria Eugenia; Péran, Patrice; Sabatini, Umberto; Cosentino, Carlo; Amato, Francesco

2015-01-01

This study presents a voxel-based multiple regression analysis of different magnetic resonance image modalities, including anatomical T1-weighted, T2* relaxometry, and diffusion tensor imaging. Quantitative parameters sensitive to complementary brain tissue alterations, including morphometric atrophy, mineralization, microstructural damage, and anisotropy loss, were compared in a linear physiological aging model in 140 healthy subjects (range 20-74 years). The performance of different predictors and the identification of the best biomarker of age-induced structural variation were compared without a priori anatomical knowledge. The best quantitative predictors in several brain regions were iron deposition and microstructural damage, rather than macroscopic tissue atrophy. Age variations were best resolved with a combination of markers, suggesting that multiple predictors better capture age-induced tissue alterations. These findings highlight the importance of a combined evaluation of multimodal biomarkers for the study of aging and point to a number of novel applications for the method described.

Diagnosis of filamentous fungi on tissue sections by immunohistochemistry using anti-aspergillus antibody.

PubMed

Challa, Sundaram; Uppin, Shantveer G; Uppin, Megha S; Pamidimukkala, Umabala; Vemu, Lakshmi

2015-06-01

Identification based on histology alone has limitations as Aspergillus species share morphology with other filamentous fungi. Differentiation of Aspergillus species from hyalohyphomycetes and dematiaceous fungi is important as the antifungal susceptibility varies among different species and genera. Given these problems, ancillary techniques are needed to increase specificity. Our aim was to study the utility of immunohistochemistry (IHC) with anti-Aspergillus antibody in the identification of Aspergillus species and to differentiate them from other filamentous fungi. Fifty formalin fixed, paraffin embedded tissue sections including 47 from cases of culture proven filamentous fungi, 3 from colonies of cultures of hyalohyphomycetes, and 11 smears from cultures were subjected to IHC studies using polyclonal rabbit anti-Aspergillus antibody (Abcam, UK) after antigen retrieval. The IHC on tissue sections was positive in 88% cases involving culture proven Aspergillus species. There was no cross reactivity with Mucorales species, Candida species, dematiaceous fungi and hyalohyphomycetes. Hence immunohistochemistry can be used as an ancillary technique for the diagnosis of Aspergillus species. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effect of echo artifacts on characterization of pulsatile tissues in neonatal cranial ultrasonic movies

NASA Astrophysics Data System (ADS)

Fukuzawa, Masayuki; Takahashi, Kazuki; Tabata, Yuki; Kitsunezuka, Yoshiki

2016-04-01

Effect of echo artifacts on characterization of pulsatile tissues has been examined in neonatal cranial ultrasonic movies by characterizing pulsatile intensities with different regions of interest (ROIs). The pulsatile tissue, which is a key point in pediatric diagnosis of brain tissue, was detected from a heartbeat-frequency component in Fourier transform of a time-variation of 64 samples of echo intensity at each pixel in a movie fragment. The averages of pulsatile intensity and power were evaluated in two ROIs: common fan-shape and individual cranial-shape. The area of pulsatile region was also evaluated as the number of pixels where the pulsatile intensity exceeds a proper threshold. The extracranial pulsatile region was found mainly in the sections where mirror image was dominant echo artifact. There was significant difference of pulsatile area between two ROIs especially in the specific sections where mirror image was included, suggesting the suitability of cranial-shape ROI for statistical study on pulsatile tissues in brain. The normalized average of pulsatile power in the cranial-shape ROI exhibited most similar tendency to the normalized pulsatile area which was treated as a conventional measure in spite of its requirement of thresholding. It suggests the potential of pulsatile power as an alternative measure for pulsatile area in further statistical study of pulsatile tissues because it was neither affected by echo artifacts nor threshold.

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.

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.

Hierarchical patch-based co-registration of differently stained histopathology slides

NASA Astrophysics Data System (ADS)

Yigitsoy, Mehmet; Schmidt, Günter

2017-03-01

Over the past decades, digital pathology has emerged as an alternative way of looking at the tissue at subcellular level. It enables multiplexed analysis of different cell types at micron level. Information about cell types can be extracted by staining sections of a tissue block using different markers. However, robust fusion of structural and functional information from different stains is necessary for reproducible multiplexed analysis. Such a fusion can be obtained via image co-registration by establishing spatial correspondences between tissue sections. Spatial correspondences can then be used to transfer various statistics about cell types between sections. However, the multi-modal nature of images and sparse distribution of interesting cell types pose several challenges for the registration of differently stained tissue sections. In this work, we propose a co-registration framework that efficiently addresses such challenges. We present a hierarchical patch-based registration of intensity normalized tissue sections. Preliminary experiments demonstrate the potential of the proposed technique for the fusion of multi-modal information from differently stained digital histopathology sections.

Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

PubMed Central

Andersson, Ann-Catrin; Strömberg, Sara; Bäckvall, Helena; Kampf, Caroline; Uhlen, Mathias; Wester, Kenneth; Pontén, Fredrik

2006-01-01

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome. PMID:16957166

Evidence of specialized tissue in human interatrial septum: histological, immunohistochemical and ultrastructural findings.

PubMed

Mitrofanova, Lubov B; Gorshkov, Andrey N; Lebedev, Dmitry S; Mikhaylov, Evgeny N

2014-01-01

There is a paucity of information on structural organization of muscular bundles in the interatrial septum (IAS). The aim was to investigate histologic and ultrastructural organization of muscular bundles in human IAS, including fossa ovalis (FO) and flap valve. Macroscopic and light microscopy evaluations of IAS were performed from postmortem studies of 40 patients. Twenty three IAS specimens underwent serial transverse sectioning, and 17--longitudinal sectioning. The transverse sections from 10 patients were immunolabeled for HCN4, Caveolin3 and Connexin43. IAS specimens from 6 other patients underwent electron microscopy. In all IAS specimens sections the FO, its rims and the flap valve had muscle fibers consisting of working cardiac myocytes. Besides the typical cardiomyocytes there were unusual cells: tortuous and horseshoe-shaped intertangled myocytes, small and large rounded myocytes with pale cytoplasm. The cells were aggregated in a definite structure in 38 (95%) cases, which was surrounded by fibro-fatty tissue. The height of the structure on transverse sections positively correlated with age (P = 0.03) and AF history (P = 0.045). Immunohistochemistry showed positive staining of the cells for HCN4 and Caveolin3. Electron microscopy identified cells with characteristics similar to electrical conduction cells. Specialized conduction cells in human IAS have been identified, specifically in the FO and its flap valve. The cells are aggregated in a structure, which is surrounded by fibrous and fatty tissue. Further investigations are warranted to explore electrophysiological characteristics of this structure.

Addition of phosphotungstic acid to ethanol for dehydration improves both the ultrastructure and antigenicity of pituitary tissue embedded in LR White acrylic resin.

PubMed

Sakai, Yuko; Hosaka, Masahiro; Hira, Yoshiki; Watanabe, Tsuyoshi

2005-12-01

Although hydrophilic acrylic resins including LR White have been widely utilized as embedding media for immunocytochemical use, the constituents of tissues are often extracted by the resin monomer during the infiltration process of the embedment, resulting in a discernible impairment of the ultrastructure when the tissue is weakly fixed only with aldehydes. To minimize the extraction by the resin monomer, the embedding procedure with LR White resin was reexamined in the present study. Among the treatments tested, a partial dehydration with 70% ethanol containing 2% phosphotungstic acid (PTA) well preserved the ultrastructure of the pituitary tissue without spoiling the antigenicity of LHbeta and other representative markers for the Golgi apparatus. In addition, treatment with 1% tannic acid (TA) prior to the dehydration described above synergistically improved both the ultrastructure and antigenicity of the tissue so that the orientation of the Golgi apparatus could be determined by double immunogold labeling with commercially available anti-GM130 and anti-TGN38 antibodies. The ultrathin sections from the LR White-embedded tissue treated with TA and dehydrated in 70% ethanol containing 2% PTA also enhanced contrast without conventional heavy-metal staining with uranyl acetate and lead citrate. Our findings further suggest that the precipitation of TA and PTA protected the tissue from being extracted during the embedment, probably because an insoluble complex was transiently formed with the constituents of the tissue. This simple modification of the LR White embedment can extend the application of post-embedding immunocytochemistry as an alternative to pre-embedding immunolabeling with frozen ultrathin sections.

3D surface reconstruction and visualization of the Drosophila wing imaginal disc at cellular resolution

NASA Astrophysics Data System (ADS)

Bai, Linge; Widmann, Thomas; Jülicher, Frank; Dahmann, Christian; Breen, David

2013-01-01

Quantifying and visualizing the shape of developing biological tissues provide information about the morphogenetic processes in multicellular organisms. The size and shape of biological tissues depend on the number, size, shape, and arrangement of the constituting cells. To better understand the mechanisms that guide tissues into their final shape, it is important to investigate the cellular arrangement within tissues. Here we present a data processing pipeline to generate 3D volumetric surface models of epithelial tissues, as well as geometric descriptions of the tissues' apical cell cross-sections. The data processing pipeline includes image acquisition, editing, processing and analysis, 2D cell mesh generation, 3D contourbased surface reconstruction, cell mesh projection, followed by geometric calculations and color-based visualization of morphological parameters. In their first utilization we have applied these procedures to construct a 3D volumetric surface model at cellular resolution of the wing imaginal disc of Drosophila melanogaster. The ultimate goal of the reported effort is to produce tools for the creation of detailed 3D geometric models of the individual cells in epithelial tissues. To date, 3D volumetric surface models of the whole wing imaginal disc have been created, and the apicolateral cell boundaries have been identified, allowing for the calculation and visualization of cell parameters, e.g. apical cross-sectional area of cells. The calculation and visualization of morphological parameters show position-dependent patterns of cell shape in the wing imaginal disc. Our procedures should offer a general data processing pipeline for the construction of 3D volumetric surface models of a wide variety of epithelial tissues.

Propidium iodide (PI) stains Nissl bodies and may serve as a quick marker for total neuronal cell count.

PubMed

Niu, Junfei; Li, Chunman; Wu, Haihui; Feng, Xianling; Su, Qingning; Li, Shihe; Zhang, Lihong; Yew, David Tai Wai; Cho, Eric Yu Pang; Sha, Ou

2015-03-01

Propidium iodide (PI) reacts with both DNA and RNA and is a commonly used fluorescent reagent for nucleic acid staining. The aim of the study was to compare the cellular staining patterns of PI with that of Nissl staining in rat nervous tissues and to report a modified staining method that selectively labels Nissl bodies in neurons. Cryosections and paraffin sections of different tissues of normal Sprague-Dawley rats, including trigeminal ganglia, dorsal root ganglia, spinal cord, liver, and small intestine, were stained by either PI or the hematoxylin and eosin method. Some sections were treated with RNase or DNase before the above staining, and some were double stained with PI and a Nissl stain. The sections were observed by light, fluorescence or confocal microscopy. Results showed strong PI signals detected as patterns of granules in the neuronal cytoplasm of all nervous tissues, whereas the staining of neuronal nuclei was weaker. In contrast, nuclei of neuroglial cells were strongly stained by PI, while the cytoplasm was not obviously stained. Pretreatment of the neural tissue with RNase abolished the PI signals. Furthermore, the PI positive granules in neuronal cytoplasm co-localized with Nissl bodies stained by the fluorescent Nissl stain. When the tissue was pretreated with DNase, PI only stained the cytoplasmic granules of neurons, but not that of glial cells. Our results show that PI stains Nissl bodies and may serve as an economical and convenient neuron marker for neuronal cell counting when specific neural markers such as antibodies are not readily available. Copyright © 2015. Published by Elsevier GmbH.

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

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... carcasses may be passed for food after complete removal and condemnation of all affected tissues including...

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... carcasses may be passed for food after complete removal and condemnation of all affected tissues including...

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... carcasses may be passed for food after complete removal and condemnation of all affected tissues including...

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... carcasses may be passed for food after complete removal and condemnation of all affected tissues including...

Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI

PubMed Central

Li, Yiwen; Song, Ying; Zhao, Lian; Gaidosh, Gabriel; Laties, Alan M; Wen, Rong

2009-01-01

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis. PMID:18846097

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

High-resolution matrix-assisted laser desorption ionization–imaging mass spectrometry of lipids in rodent optic nerve tissue

PubMed Central

Anderson, David M. G.; Mills, Daniel; Spraggins, Jeffrey; Lambert, Wendi S.; Calkins, David J.

2013-01-01

Purpose To develop a method for generating high spatial resolution (10 µm) matrix-assisted laser desorption ionization (MALDI) images of lipids in rodent optic nerve tissue. Methods Ice-embedded optic nerve tissue from rats and mice were cryosectioned across the coronal and sagittal axes of the nerve fiber. Sections were thaw mounted on gold-coated MALDI plates and were washed with ammonium acetate to remove biologic salts before being coated in 2,5-dihydroxybenzoic acid by sublimation. MALDI images were generated in positive and negative ion modes at 10 µm spatial resolution. Lipid identification was performed with a high mass resolution Fourier transform ion cyclotron resonance mass spectrometer. Results Several lipid species were observed with high signal intensity in MALDI images of optic nerve tissue. Several lipids were localized to specific structures including in the meninges surrounding the optic nerve and in the central neuronal tissue. Specifically, phosphatidylcholine species were observed throughout the nerve tissue in positive ion mode while sulfatide species were observed in high abundance in the meninges surrounding the optic nerve in negative ion mode. Accurate mass measurements and fragmentation using sustained off-resonance irradiation with a high mass resolution Fourier transform ion cyclotron resonance mass spectrometer instrument allowed for identification of lipid species present in the small structure of the optic nerve directly from tissue sections. Conclusions An optimized sample preparation method provides excellent sensitivity for lipid species present within optic nerve tissue. This allowed the laser spot size and fluence to be reduced to obtain a high spatial resolution of 10 µm. This new imaging modality can now be applied to determine spatial and molecular changes in optic nerve tissue with disease. PMID:23559852

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.

Comparison of renal artery, soft tissue, and nerve damage after irrigated versus nonirrigated radiofrequency ablation.

PubMed

Sakakura, Kenichi; Ladich, Elena; Fuimaono, Kristine; Grunewald, Debby; O'Fallon, Patrick; Spognardi, Anna-Maria; Markham, Peter; Otsuka, Fumiyuki; Yahagi, Kazuyuki; Shen, Kai; Kolodgie, Frank D; Joner, Michael; Virmani, Renu

2015-01-01

The long-term efficacy of radiofrequency ablation of renal autonomic nerves has been proven in nonrandomized studies. However, long-term safety of the renal artery (RA) is of concern. The aim of our study was to determine if cooling during radiofrequency ablation preserved the RA while allowing equivalent nerve damage. A total of 9 swine (18 RAs) were included, and allocated to irrigated radiofrequency (n=6 RAs, temperature setting: 50°C), conventional radiofrequency (n=6 RAs, nonirrigated, temperature setting: 65°C), and high-temperature radiofrequency (n=6 RAs, nonirrigated, temperature setting: 90°C) groups. RAs were harvested at 10 days, serially sectioned from proximal to distal including perirenal tissues and examined after paraffin embedding, and staining with hematoxylin-eosin and Movat pentachrome. RAs and periarterial tissue including nerves were semiquantitatively assessed and scored. A total of 660 histological sections from 18 RAs were histologically examined by light microscopy. Arterial medial injury was significantly less in the irrigated radiofrequency group (depth of medial injury, circumferential involvement, and thinning) than that in the conventional radiofrequency group (P<0.001 for circumference; P=0.003 for thinning). Severe collagen damage such as denatured collagen was also significantly less in the irrigated compared with the conventional radiofrequency group (P<0.001). Nerve damage although not statistically different between the irrigated radiofrequency group and conventional radiofrequency group (P=0.36), there was a trend toward less nerve damage in the irrigated compared with conventional. Compared to conventional radiofrequency, circumferential medial damage in highest-temperature nonirrigated radiofrequency group was significantly greater (P<0.001). Saline irrigation significantly reduces arterial and periarterial tissue damage during radiofrequency ablation, and there is a trend toward less nerve damage. © 2014 American Heart Association, Inc.

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

Successful vaginal birth after caesarean section in patient with Ehler-Danlos syndrome type 2.

PubMed

Maraj, Hemant; Mohajer, Michelle; Bhattacharjee, Deepannita

2011-12-01

We present the case of a 31-year-old woman with Ehler-Danlos syndrome (EDS) type 2. She had a previous caesarean section and went on to have an uncomplicated vaginal birth in her last pregnancy. To our knowledge, this is the first case of a successful vaginal birth after caesarean section in a patient with EDS. EDS is a multisystem disorder involving a genetic defect in collagen and connective-tissue synthesis and structure. It is a heterogeneous group of 11 different inherited disorders. Obstetric complications in these patients include miscarriages, stillbirths, premature rupture of the membranes, preterm labour, uterine prolapse, uterine rupture and severe postpartum haemorrhage. There has been much controversy over the appropriate mode of delivery. Abdominal deliveries are complicated by delayed wound healing and increased perioperative blood loss. Vaginal deliveries may be complicated by tissue friability causing extensive perineal tears, pelvic floor and bladder lesions. Our case highlights that in specific, controlled situations it is possible to have a vaginal delivery even after previous caesarean section in patients with EDS.

Successful vaginal birth after caesarean section in patient with Ehler-Danlos syndrome type 2

PubMed Central

Maraj, Hemant; Mohajer, Michelle; Bhattacharjee, Deepannita

2011-01-01

We present the case of a 31-year-old woman with Ehler-Danlos syndrome (EDS) type 2. She had a previous caesarean section and went on to have an uncomplicated vaginal birth in her last pregnancy. To our knowledge, this is the first case of a successful vaginal birth after caesarean section in a patient with EDS. EDS is a multisystem disorder involving a genetic defect in collagen and connective-tissue synthesis and structure. It is a heterogeneous group of 11 different inherited disorders. Obstetric complications in these patients include miscarriages, stillbirths, premature rupture of the membranes, preterm labour, uterine prolapse, uterine rupture and severe postpartum haemorrhage. There has been much controversy over the appropriate mode of delivery. Abdominal deliveries are complicated by delayed wound healing and increased perioperative blood loss. Vaginal deliveries may be complicated by tissue friability causing extensive perineal tears, pelvic floor and bladder lesions. Our case highlights that in specific, controlled situations it is possible to have a vaginal delivery even after previous caesarean section in patients with EDS. PMID:27579117

HdhQ111 Mice Exhibit Tissue Specific Metabolite Profiles that Include Striatal Lipid Accumulation

PubMed Central

Carroll, Jeffrey B.; Deik, Amy; Fossale, Elisa; Weston, Rory M.; Guide, Jolene R.; Arjomand, Jamshid; Kwak, Seung; Clish, Clary B.; MacDonald, Marcy E.

2015-01-01

The HTT CAG expansion mutation causes Huntington’s Disease and is associated with a wide range of cellular consequences, including altered metabolism. The mutant allele is expressed widely, in all tissues, but the striatum and cortex are especially vulnerable to its effects. To more fully understand this tissue-specificity, early in the disease process, we asked whether the metabolic impact of the mutant CAG expanded allele in heterozygous B6.HdhQ111/+ mice would be common across tissues, or whether tissues would have tissue-specific responses and whether such changes may be affected by diet. Specifically, we cross-sectionally examined steady state metabolite concentrations from a range of tissues (plasma, brown adipose tissue, cerebellum, striatum, liver, white adipose tissue), using an established liquid chromatography-mass spectrometry pipeline, from cohorts of 8 month old mutant and wild-type littermate mice that were fed one of two different high-fat diets. The differential response to diet highlighted a proportion of metabolites in all tissues, ranging from 3% (7/219) in the striatum to 12% (25/212) in white adipose tissue. By contrast, the mutant CAG-expanded allele primarily affected brain metabolites, with 14% (30/219) of metabolites significantly altered, compared to wild-type, in striatum and 11% (25/224) in the cerebellum. In general, diet and the CAG-expanded allele both elicited metabolite changes that were predominantly tissue-specific and non-overlapping, with evidence for mutation-by-diet interaction in peripheral tissues most affected by diet. Machine-learning approaches highlighted the accumulation of diverse lipid species as the most genotype-predictive metabolite changes in the striatum. Validation experiments in cell culture demonstrated that lipid accumulation was also a defining feature of mutant HdhQ111 striatal progenitor cells. Thus, metabolite-level responses to the CAG expansion mutation in vivo were tissue specific and most evident in brain, where the striatum featured signature accumulation of a set of lipids including sphingomyelin, phosphatidylcholine, cholesterol ester and triglyceride species. Importantly, in the presence of the CAG mutation, metabolite changes were unmasked in peripheral tissues by an interaction with dietary fat, implying that the design of studies to discover metabolic changes in HD mutation carriers should include metabolic perturbations. PMID:26295712

Conductive carbon tape used for support and mounting of both whole animal and fragile heat-treated tissue sections for MALDI MS imaging and quantitation.

PubMed

Goodwin, Richard J A; Nilsson, Anna; Borg, Daniel; Langridge-Smith, Pat R R; Harrison, David J; Mackay, C Logan; Iverson, Suzanne L; Andrén, Per E

2012-08-30

Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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.

An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy.

PubMed

Warner, Cherish A; Biedrzycki, Meredith L; Jacobs, Samuel S; Wisser, Randall J; Caplan, Jeffrey L; Sherrier, D Janine

2014-12-01

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process. © 2014 American Society of Plant Biologists. All Rights Reserved.

A guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)(a).

PubMed

Baron, Ellen Jo; Miller, J Michael; Weinstein, Melvin P; Richter, Sandra S; Gilligan, Peter H; Thomson, Richard B; Bourbeau, Paul; Carroll, Karen C; Kehl, Sue C; Dunne, W Michael; Robinson-Dunn, Barbara; Schwartzman, Joseph D; Chapin, Kimberle C; Snyder, James W; Forbes, Betty A; Patel, Robin; Rosenblatt, Jon E; Pritt, Bobbi S

2013-08-01

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

Executive summary: a guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)(a).

PubMed

Baron, Ellen Jo; Miller, J Michael; Weinstein, Melvin P; Richter, Sandra S; Gilligan, Peter H; Thomson, Richard B; Bourbeau, Paul; Carroll, Karen C; Kehl, Sue C; Dunne, W Michael; Robinson-Dunn, Barbara; Schwartzman, Joseph D; Chapin, Kimberle C; Snyder, James W; Forbes, Betty A; Patel, Robin; Rosenblatt, Jon E; Pritt, Bobbi S

2013-08-01

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2013 Recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)a

PubMed Central

Baron, Ellen Jo; Miller, J. Michael; Weinstein, Melvin P.; Richter, Sandra S.; Gilligan, Peter H.; Thomson, Richard B.; Bourbeau, Paul; Carroll, Karen C.; Kehl, Sue C.; Dunne, W. Michael; Robinson-Dunn, Barbara; Schwartzman, Joseph D.; Chapin, Kimberle C.; Snyder, James W.; Forbes, Betty A.; Patel, Robin; Rosenblatt, Jon E.; Pritt, Bobbi S.

2013-01-01

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients. PMID:23845951

21 CFR 892.1540 - Nonfetal ultrasonic monitor.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Nonfetal ultrasonic monitor. 892.1540 Section 892.1540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... tissues in motion. This generic type of device may include signal analysis and display equipment, patient...

21 CFR 892.1540 - Nonfetal ultrasonic monitor.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Nonfetal ultrasonic monitor. 892.1540 Section 892.1540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... tissues in motion. This generic type of device may include signal analysis and display equipment, patient...

21 CFR 892.1540 - Nonfetal ultrasonic monitor.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Nonfetal ultrasonic monitor. 892.1540 Section 892.1540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... tissues in motion. This generic type of device may include signal analysis and display equipment, patient...

Proteomic analysis of laser-captured paraffin-embedded tissues: a molecular portrait of head and neck cancer progression.

PubMed

Patel, Vyomesh; Hood, Brian L; Molinolo, Alfredo A; Lee, Norman H; Conrads, Thomas P; Braisted, John C; Krizman, David B; Veenstra, Timothy D; Gutkind, J Silvio

2008-02-15

Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis. A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

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.

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.

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.

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

Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics.

PubMed

Becker, J Sabine; Matusch, Andreas; Palm, Christoph; Salber, Dagmar; Morton, Kathryn A; Becker, J Susanne

2010-02-01

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been developed and established as an emerging technique in the generation of quantitative images of metal distributions in thin tissue sections of brain samples (such as human, rat and mouse brain), with applications in research related to neurodegenerative disorders. A new analytical protocol is described which includes sample preparation by cryo-cutting of thin tissue sections and matrix-matched laboratory standards, mass spectrometric measurements, data acquisition, and quantitative analysis. Specific examples of the bioimaging of metal distributions in normal rodent brains are provided. Differences to the normal were assessed in a Parkinson's disease and a stroke brain model. Furthermore, changes during normal aging were studied. Powerful analytical techniques are also required for the determination and characterization of metal-containing proteins within a large pool of proteins, e.g., after denaturing or non-denaturing electrophoretic separation of proteins in one-dimensional and two-dimensional gels. LA-ICP-MS can be employed to detect metalloproteins in protein bands or spots separated after gel electrophoresis. MALDI-MS can then be used to identify specific metal-containing proteins in these bands or spots. The combination of these techniques is described in the second section.

Proteomics in Diagnostic Pathology

PubMed Central

Chaurand, Pierre; Sanders, Melinda E.; Jensen, Roy A.; Caprioli, Richard M.

2004-01-01

Direct tissue profiling and imaging mass spectrometry (MS) provide a molecular assessment of numerous expressed proteins within a tissue sample. MALDI MS (matrix-assisted laser desorption ionization) analysis of thin tissue sections results in the visualization of 500 to 1000 individual protein signals in the molecular weight range from 2000 to over 200,000. These signals directly correlate with protein distribution within a specific region of the tissue sample. The systematic investigation of the section allows the construction of ion density maps, or specific molecular images, for virtually every signal detected in the analysis. Ultimately, hundreds of images, each at a specific molecular weight, may be obtained. To date, profiling and imaging MS has been applied to multiple diseased tissues, including human non-small cell lung tumors, gliomas, and breast tumors. Interrogation of the resulting complex MS data sets using modern biocomputational tools has resulted in identification of both disease-state and patient-prognosis specific protein patterns. These studies suggest that such proteomic information will become more and more important in assessing disease progression, prognosis, and drug efficacy. Molecular histology has been known for some time and its value clear in the field of pathology. Imaging mass spectrometry brings a new dimension of molecular data, one focusing on the disease phenotype. The present article reviews the state of the art of the technology and its complementarity with traditional histopathological analyses. PMID:15466373

Computational study of culture conditions and nutrient supply in a hollow membrane sheet bioreactor for large-scale bone tissue engineering.

PubMed

Khademi, Ramin; Mohebbi-Kalhori, Davod; Hadjizadeh, Afra

2014-03-01

Successful bone tissue culture in a large implant is still a challenge. We have previously developed a porous hollow membrane sheet (HMSh) for tissue engineering applications (Afra Hadjizadeh and Davod Mohebbi-Kalhori, J Biomed. Mater. Res. Part A [2]). This study aims to investigate culture conditions and nutrient supply in a bioreactor made of HMSh. For this purpose, hydrodynamic and mass transport behavior in the newly proposed hollow membrane sheet bioreactor including a lumen region and porous membrane (scaffold) for supporting and feeding cells with a grooved section for accommodating gel-cell matrix was numerically studied. A finite element method was used for solving the governing equations in both homogenous and porous media. Furthermore, the cell resistance and waste production have been included in a 3D mathematical model. The influences of different bioreactor design parameters and the scaffold properties which determine the HMSh bioreactor performance and various operating conditions were discussed in detail. The obtained results illustrated that the novel scaffold can be employed in the large-scale applications in bone tissue engineering.

Meningoceles, meningomyeloceles, and encephaloceles: a neuro-dermatopathologic study of 132 cases.

PubMed

Berry, A D; Patterson, J W

1991-06-01

Because there have been few comprehensive histopathologic studies of meningomyeloceles and related malformations, we undertook a systematic study of these lesions. One hundred and thirty two cases were obtained from our surgical pathology files; these included 38 meningoceles, 71 meningomyeloceles, and 23 encephaloceles. Tissue sections were stained with hematoxylin and eosin; special stains included trichrome, alcian blue, Fontana-Masson, Nissl, Holzer, and immunoperoxidase for glial fibrillary acidic protein. Epithelial changes included ulceration, atrophy, or nevoid hyperplasia of the epidermis, and loss of appendages. Mesodermal features included fibrous zones resembling dura, subarachnoid tissue or scar (99% of cases), increased numbers of blood vessels (83%), hypertrophy of arrector pili muscle (42%), lipoma formation (38%), and immature skeletal muscle fibers (5%) that rarely intermingled with neuropil-like matrix. The latter tissue was identified in 71% of cases and included neurons, astrocytes, oligodendroglia, and ependyma. Forty-eight percent of cases included peripheral nerve fibers or roots, and some fibers formed onion bulb or Pacinian corpuscle-like structures. Meningothelial cells were observed in 26% of cases and sometimes formed recognizable whorls. Choroid plexus was noted in 3 cases, one example showing an unusual dystrophic calcification that formed long parallel spicules. Pigmented dendritic cells were observed within zones of fibrous tissue in 10% of cases. These malformations involve complex arrangements of cutaneous, neuroectodermal, and mesodermal elements. Because they may be encountered by dermatopathologists, familiarity with the microscopic features of dysraphic lesions is essential.

Digital 3D reconstructions using histological serial sections of lung tissue including the alveolar capillary network.

PubMed

Grothausmann, Roman; Knudsen, Lars; Ochs, Matthias; Mühlfeld, Christian

2017-02-01

Grothausmann R, Knudsen L, Ochs M, Mühlfeld C. Digital 3D reconstructions using histological serial sections of lung tissue including the alveolar capillary network. Am J Physiol Lung Cell Mol Physiol 312: L243-L257, 2017. First published December 2, 2016; doi:10.1152/ajplung.00326.2016-The alveolar capillary network (ACN) provides an enormously large surface area that is necessary for pulmonary gas exchange. Changes of the ACN during normal or pathological development or in pulmonary diseases are of great functional impact and warrant further analysis. Due to the complexity of the three-dimensional (3D) architecture of the ACN, 2D approaches are limited in providing a comprehensive impression of the characteristics of the normal ACN or the nature of its alterations. Stereological methods offer a quantitative way to assess the ACN in 3D in terms of capillary volume, surface area, or number but lack a 3D visualization to interpret the data. Hence, the necessity to visualize the ACN in 3D and to correlate this with data from the same set of data arises. Such an approach requires a large sample volume combined with a high resolution. Here, we present a technically simple and cost-efficient approach to create 3D representations of lung tissue ranging from bronchioles over alveolar ducts and alveoli up to the ACN from more than 1 mm sample extent to a resolution of less than 1 μm. The method is based on automated image acquisition of serially sectioned epoxy resin-embedded lung tissue fixed by vascular perfusion and subsequent automated digital reconstruction and analysis of the 3D data. This efficient method may help to better understand mechanisms of vascular development and pathology of the lung. Copyright © 2017 the American Physiological Society.

Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

PubMed

Cushway, T R; Lanzetta, M; Cox, G; Trickett, R; Owen, E R

1996-01-01

Fibre-optic technology combined with confocality produces a microscope capable of optical thin sectioning. In this original study, tibial nerves have been stained in a rat model with a vital dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide, and analysed by fibre-optic confocal microscopy to produce detailed images of nerve ultrastructure. Schwann cells, nodes of Ranvier and longitudinal myelinated sheaths enclosing axons were clearly visible. Single axons appeared as brightly staining longitudinal structures. This allowed easy tracing of multiple signal axons within the nerve tissue. An accurate measurement of internodal lengths was easily accomplished. This technique is comparable to current histological techniques, but does not require biopsy, thin sectioning or tissue fixing. This study offers a standard for further in vivo microscopy, including the possibility of monitoring the progression of nerve regeneration following microsurgical neurorraphy.

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

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.

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.

Synchrotron microCT imaging of soft tissue in juvenile zebrafish reveals retinotectal projections

NASA Astrophysics Data System (ADS)

Xin, Xuying; Clark, Darin; Ang, Khai Chung; van Rossum, Damian B.; Copper, Jean; Xiao, Xianghui; La Riviere, Patrick J.; Cheng, Keith C.

2017-02-01

Biomedical research and clinical diagnosis would benefit greatly from full volume determinations of anatomical phenotype. Comprehensive tools for morphological phenotyping are central for the emerging field of phenomics, which requires high-throughput, systematic, accurate, and reproducible data collection from organisms affected by genetic, disease, or environmental variables. Theoretically, complete anatomical phenotyping requires the assessment of every cell type in the whole organism, but this ideal is presently untenable due to the lack of an unbiased 3D imaging method that allows histopathological assessment of any cell type despite optical opacity. Histopathology, the current clinical standard for diagnostic phenotyping, involves the microscopic study of tissue sections to assess qualitative aspects of tissue architecture, disease mechanisms, and physiological state. However, quantitative features of tissue architecture such as cellular composition and cell counting in tissue volumes can only be approximated due to characteristics of tissue sectioning, including incomplete sampling and the constraints of 2D imaging of 5 micron thick tissue slabs. We have used a small, vertebrate organism, the zebrafish, to test the potential of microCT for systematic macroscopic and microscopic morphological phenotyping. While cell resolution is routinely achieved using methods such as light sheet fluorescence microscopy and optical tomography, these methods do not provide the pancellular perspective characteristic of histology, and are constrained by the limited penetration of visible light through pigmented and opaque specimens, as characterizes zebrafish juveniles. Here, we provide an example of neuroanatomy that can be studied by microCT of stained soft tissue at 1.43 micron isotropic voxel resolution. We conclude that synchrotron microCT is a form of 3D imaging that may potentially be adopted towards more reproducible, large-scale, morphological phenotyping of optically opaque tissues. Further development of soft tissue microCT, visualization and quantitative tool development will enhance its utility.

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.

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.

Reduction in Histone H3 Acetylation and Chromatin Remodeling in Corneas of Alloxan-Induced Diabetic Rats.

PubMed

Herencia-Bueno, Karina E; Aldrovani, Marcela; Crivelaro, Roberta M; Thiesen, Roberto; Barros-Sobrinho, Alexandre A F; Claros-Chacaltana, Flor D Y; Padua, Ivan R M; Santos, Daniela M; Laus, José L

2018-05-01

To evaluate acetylation of histone H3, chromatin remodeling, nuclear size and shape, DNA ploidy, and distribution of nucleolus organizing regions (NORs) in corneal epithelial and stromal cells of diabetic and nondiabetic rats. Diabetes was induced by a single intraperitoneal injection of alloxan. All diabetic rats (n = 20) included in the study had 4 weeks of moderate-to-severe hyperglycemia (plasma glucose levels >400 mg/dL). Acetylated histone H3 levels were quantified in corneal tissue using a colorimetric assay. Chromatin remodeling, nuclear sizes (area/perimeter) and shapes (circularity), and DNA ploidies were evaluated from Feulgen-stained tissue sections using video image analysis. Distributions of NORs were studied in tissue sections impregnated with silver ions. Ophthalmic clinical parameters, including corneal sensitivity, were investigated. Twenty nondiabetic rats were used as controls. Acetylation of histone H3 was reduced in the corneas of the diabetic rats. Nuclei in corneal epithelial cells of diabetic rats compacted chromatin, increased in size, modified their shapes, and elevated DNA ploidy. The only nuclear change observed in the corneal stromal cells of diabetic rats was chromatin decompaction. The size of the silver-stained NOR did not differ between the study samples. The corneal sensitivity in diabetic rats was 51.8% lower than that in nondiabetic rats. The results of this study show that alloxan-induced diabetes altered the histone H3 acetylation pattern and compromised the chromatin supraorganization in corneal tissue/cells. Continued research is needed to understand the clinical and morphofunctional significance of changes in corneal cell nuclei of diabetic individuals.

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation.

PubMed

Allen, Joselyn N; Dey, Adwitia; Nissly, Ruth; Fraser, James; Yu, Shan; Balandaram, Gayathri; Peters, Jeffrey M; Hankey-Giblin, Pamela A

2017-04-03

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

Digital pathology: elementary, rapid and reliable automated image analysis.

PubMed

Bouzin, Caroline; Saini, Monika L; Khaing, Kyi-Kyi; Ambroise, Jérôme; Marbaix, Etienne; Grégoire, Vincent; Bol, Vanesa

2016-05-01

Slide digitalization has brought pathology to a new era, including powerful image analysis possibilities. However, while being a powerful prognostic tool, immunostaining automated analysis on digital images is still not implemented worldwide in routine clinical practice. Digitalized biopsy sections from two independent cohorts of patients, immunostained for membrane or nuclear markers, were quantified with two automated methods. The first was based on stained cell counting through tissue segmentation, while the second relied upon stained area proportion within tissue sections. Different steps of image preparation, such as automated tissue detection, folds exclusion and scanning magnification, were also assessed and validated. Quantification of either stained cells or the stained area was found to be correlated highly for all tested markers. Both methods were also correlated with visual scoring performed by a pathologist. For an equivalent reliability, quantification of the stained area is, however, faster and easier to fine-tune and is therefore more compatible with time constraints for prognosis. This work provides an incentive for the implementation of automated immunostaining analysis with a stained area method in routine laboratory practice. © 2015 John Wiley & Sons Ltd.

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.

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.

Role of lenticel morphology, frequency and density on incidence of lenticel breakdown in 'GALA' apples

USDA-ARS?s Scientific Manuscript database

A two-year study was conducted in the arid fruit growing regions of South Africa and eastern Washington State to examine the etiology of lenticel breakdown (LB). The evaluation of lenticel size, including peel imprints and tissue cross-sections, presented no clear relationship between lenticels siz...

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...

Floaters in Mohs micrographic surgery.

PubMed

Alam, Murad; Shah, Anjali D; Ali, Sana; Rauf, Mutahir; Nodzenski, Michael; Ibrahim, Omer

2013-09-01

Floaters are dislodged pieces of tumor tissue than can obscure Mohs micrographic surgery (MMS) frozen sections and confound their interpretation. To understand the common causes of floaters and identify management strategies. An initial virtual consensus conference of Mohs surgeons based on a 60-item questionnaire. Data were validated in interviews with randomly selected Mohs surgeons. Based on retrospective reporting of 230 surgeon-years and 170,404 cases of MMS by 26 surgeons, the mean rate of floaters per tumor treated was 1.8%, and the rate of floaters per tissue block was 0.70%. Not wiping blades between cuts when a stage is separated into subunits can predispose to floaters. There was also strong consensus that basal cell carcinomas, ulcerated tumors, and tissue from the first stage were more likely to yield floaters. There is little consensus on how to manage floaters, with possibilities including taking additional sections, taking an additional stage, or simply noting the floater. Floaters are not rare and can complicate MMS margin assessment. There is significant expert consensus regarding the causes of floaters and the tissue features that may predispose to them. Floaters may be prevented by minimizing their likely causes. There is less consensus on what to do with a floater. © 2013 by the American Society for Dermatologic Surgery, Inc. Published by Wiley Periodicals, Inc.

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.

Independent prognostic value of eosinophil and mast cell infiltration in colorectal cancer tissue.

PubMed

Nielsen, H J; Hansen, U; Christensen, I J; Reimert, C M; Brünner, N; Moesgaard, F

1999-12-01

Overall peritumoural inflammatory cell infiltration is a prognostic variable in solid tumours, but the survival-related impact of the individual cell types within the infiltrate has still not been fully evaluated and compared with the conventional disease classification. In the present study, the prognostic value of individual white cell counts in the peritumoural inflammatory infiltrate in colorectal cancer was assessed. Intra-operative tumour tissue samples from 584 patients undergoing elective surgery for colorectal cancer were included. None of the patients received pre- or post-operative adjuvant chemotherapy. Tissue blocks were cut from the periphery of the tumours and embedded in paraffin. All blocks included both tumour tissue and normal bowel tissue. Serial sections of 4 microm were analysed for tumour tissue inflammatory cell infiltration using a computer- and video-assisted microscope, which allowed semi-automated quantification of cells within a fixed area. Total white cells and individual counts of eosinophils, neutrophils, mast cells, lymphocytes, and plasma cells were evaluated in every tumour specimen. Stratification into four groups with similar numbers of events was used to dichotomize the cell counts with respect to survival. The median observation period was 61 (49-75) months. In a multivariate analysis including Dukes' stage, gender, age, peri-operative blood transfusion, tumour location, and counts of specific inflammatory cells, only advanced Dukes' stage ( p< 0.0001), high age ( p=0.0003), and tumour location in the rectum predicted poor survival, while high counts of eosinophils ( p=0.006) and mast cells ( p=0.02) predicted good survival. Tumour-associated eosinophilia and mastocytosis appear to be independent prognostic variables in colorectal cancer. Future studies should investigate the potential biological role of tumour tissue eosinophils and mast cells in the modulation of tumour growth. Copyright 1999 John Wiley & Sons, Ltd.

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...

Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.

PubMed

Hendijani, Fatemeh

2017-04-01

Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits. © 2017 John Wiley & Sons Ltd.

Discrimination between basal cell carcinoma and hair follicles in skin tissue sections by Raman micro-spectroscopy

NASA Astrophysics Data System (ADS)

Larraona-Puy, M.; Ghita, A.; Zoladek, A.; Perkins, W.; Varma, S.; Leach, I. H.; Koloydenko, A. A.; Williams, H.; Notingher, I.

2011-05-01

Skin cancer is the most common human malignancy and basal cell carcinoma (BCC) represents approximately 80% of the non-melanoma cases. Current methods of treatment require histopathological evaluation of the tissues by qualified personnel. However, this method is subjective and in some cases BCC can be confused with other structures in healthy skin, including hair follicles. In this preliminary study, we investigated the potential of Raman micro-spectroscopy (RMS) to discriminate between hair follicles and BCC in skin tissue sections excised during Mohs micrographic surgery (MMS). Imaging and diagnosis of skin sections was automatically generated using ' a priori'-built spectral model based on LDA. This model had 90 ± 9% sensitivity and 85 ± 9% specificity for discrimination of BCC from dermis and epidermis. The model used selected Raman bands corresponding to the largest spectral differences between the Raman spectra of BCC and the normal skin regions, associated mainly with nucleic acids and collagen type I. Raman spectra corresponding to the epidermis regions of the hair follicles were found to be closer to those of healthy epidermis rather than BCC. Comparison between Raman spectral images and the gold standard haematoxylin and eosin (H&E) histopathology diagnosis showed good agreement. Some hair follicle regions were misclassified as BCC; regions corresponded mainly to the outermost layer of hair follicle (basal cells) which are expected to have higher nucleic acid concentration. This preliminary study shows the ability of RMS to distinguish between BCC and other tissue structures associated to healthy skin which can be confused with BCC due to their similar morphology.

The value of banked samples for oncology drug discovery and development.

PubMed

Shaw, Peter M; Patterson, Scott D

2011-01-01

To gain insights into human biology and pathobiology, ready access to banked human tissue samples that encompass a representative cross section of the population is required. For optimal use, the banked human tissue needs to be appropriately consented, collected, annotated, and stored. If any of these elements are missing, the studies using these samples are compromised. These elements are critical whether the research is for academic or pharmaceutical industry purposes. An additional temporal element that adds enormous value to such banked samples is treatment and outcome information from the people who donated the tissue. To achieve these aims, many different groups have to work effectively together, not least of which are the individuals who donate their tissue with appropriate consent. Such research is unlikely to benefit the donors but others who succumb to the same disease. The development of a large accessible human tissue bank resource (National Cancer Institute's Cancer HUman Biobank [caHUB]) that provides an ongoing supply of human tissue for all working toward the common goal of understanding human health and disease has a number of advantages. These include, but are not limited to, access to a broad cross section of healthy and diseased populations beyond what individual collections may achieve for understanding disease pathobiology, therapeutic target discovery, as well as a source of material for diagnostic assay validation. Models will need to be developed to enable fair access to caHUB under terms that enable appropriate intellectual property protection and ultimate data sharing to ensure that the biobank successfully distributes samples to a broad range of researchers.

The effects of tissue processing on markers for T and B cells from solid tissues.

PubMed

Millard, P R; Rabin, B S; Whiteside, T L; Hubbard, J D

1977-03-01

Suspensions of lymphoid cells from tissues have been used for the determination of the quantitative relationship between the T and B cell populations. The distribution of the lymphocytes within a given tissue, however, cannot be demonstrated once such a suspension has been prepared. Various methods of characterizing lymphocytes within tissues were evaluated. The method of tissue preparation can alter the capability of detecting the lymphocyte markers. Fluorescein-labeled anti-immunoglobulin sera reacted equally well with lymphocytes in tissue regardless of the method of tissue preparation. Complement-coated sheep erythrocytes were less effective in detecting lymphocyte markers in tissue sections than in cell suspensions. Quantitative assays of lymphocytes could be done in suspensions only. Unaltered sheep erythrocytes did not bind to T lymphocytes in tissue. T lymphocytes could be identified in tissue sections, however, by the use of anti-human T cell serum.

Fiber optic-based optical coherence tomography (OCT) for dental applications

NASA Astrophysics Data System (ADS)

Everett, Matthew J.; Colston, Bill W., Jr.; Da Silva, Luiz B.; Otis, Linda L.

1998-09-01

We have developed a hand-held fiber optic based optical coherence tomography (OCT) system for scanning of the oral cavity. We have produced, using this scanning device, in vivo cross-sectional images of hard and soft dental tissues in human volunteers. Clinically relevant anatomical structures, including the gingival margin, periodontal sulcus, and dento- enamel junction, were visible in all the images. The cemento- enamel junction and the alveolar bone were identified in approximately two thirds of the images. These images represent, or our knowledge, the first in vivo OCT images of human dental tissue.

[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.

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.

Detection of Newcastle disease virus RNA by reverse transcription-polymerase chain reaction using formalin-fixed, paraffin-embedded tissue and comparison with immunohistochemistry and in situ hybridization.

PubMed

Wakamatsu, Nobuko; King, Daniel J; Seal, Bruce S; Brown, Corrie C

2007-07-01

The usefulness of reverse transcription-polymerase chain reaction (RT-PCR) from formalin-fixed, paraffin-embedded (FFPE) tissues was examined and compared to the immunohistochemistry (IHC) and in situ hybridization (ISH) assays for detection of Newcastle disease virus (NDV). Spleen and lung tissues were collected from chickens experimentally infected with either of 2 NDV isolates: a low virulent virus (LaSota) and a virulent virus (from the 2002-2003 California outbreak). The tissues were harvested immediately postmortem and fixed in 10% neutral buffered formalin for approximately 52 hours. Also, just before euthanasia, oral and cloacal swabs were collected for virus isolation. RNA was obtained from the FFPE tissues by digestion with proteinase K and subsequent extraction with phenol, chloroform, and isoamyl alcohol. By seminested RT-PCR with primers for the NDV matrix gene, a 232-base pair (bp) product was generated and visualized by electrophoresis. The results of PCR were compared to those of IHC for viral nucleoprotein and ISH for matrix gene (850 bp) on 3-microm sections and to those of virus isolation from swabs. All samples from infected chickens were positive by RT-PCR, including samples that were negative by both IHC and ISH. The RT-PCR positives included tissue from chickens that were no longer shedding virus detectable by virus isolation. The RT-PCR was an effective and sensitive method to detect NDV in FFPE tissues. To the authors' knowledge, this is the first report of NDV detection in FFPE tissues as a diagnostic approach possibly suitable for archival materials.

In situ hybridization and immunofluorescence on resin-embedded tissue to identify the components of Nissl substance.

PubMed

Singhrao, Sim K; Nair-Roberts, Radha G

2010-05-01

It is not clear whether the Nissl substance is present at the axon hillock. To clarify this gap in knowledge, we conducted in situ hybridization (ISH) on mouse brain tissue using 30-microm cryostat and 1-3-microm acrylic resin sections. Cryostat and rehydrated resin sections were exposed to digoxygenin-labeled glutamic acid decarboxylase 1 sense and antisense riboprobes. Consecutive sections from tissue embedded in resin were subjected to the ribosomal protein L26 primary antibody to determine the distribution of the ribo/polysomes. ISH results from the antisense riboprobe in both cryostat and resin-embedded tissue sections demonstrated an abundance of message in the neurons from the substantia nigra pars reticulate. In addition, the resin sections demonstrated hybridization signal in the axon hillock of some neurons. Immunofluorescence labeling of consecutive sections using an antibody to the most abundant ribosomal protein L26 confirmed their distribution in the cell body and the axon hillock of similar neurons. Compared with the 30-microm cryostat sections, the most striking feature of ISH in the thinner resin (2-3 microm) sections was that there was a phenomenal improvement in the overall clarity and spatial resolution. Reexamination of the axon hillock when continuous with the cell body in cryostat sections revealed that the same message was also present, except it was overlooked initially because of overlapping cell populations in thick tissue slices. As ribosomes are a component of Nissl substance, we propose that the axon hillock, like other parts of the neuron, does contain Nissl substance. (c) 2009 Wiley-Liss, Inc.

Could Revision of the Embryology Influence Our Cesarean Delivery Technique: Towards an Optimized Cesarean Delivery for Universal Use

PubMed Central

Stark, Michael; Mynbaev, Ospan; Vassilevski, Yuri; Rozenberg, Patrick

2016-01-01

Until today, there is no standardized Cesarean Section method and many variations exist. The main variations concern the type of abdominal incision, usage of abdominal packs, suturing the uterus in one or two layers, and suturing the peritoneal layers or leaving them open. One of the questions is the optimal location of opening the uterus. Recently, omission of the bladder flap was recommended. The anatomy and histology as results from the embryological knowledge might help to solve this question. The working thesis is that the higher the incision is done, the more damage to muscle tissue can take place contrary to incision in the lower segment, where fibrous tissue prevails. In this perspective, a call for participation in a two-armed prospective study is included, which could result in an optimal, evidence-based Cesarean Section for universal use. PMID:28078171

A Java application for tissue section image analysis.

PubMed

Kamalov, R; Guillaud, M; Haskins, D; Harrison, A; Kemp, R; Chiu, D; Follen, M; MacAulay, C

2005-02-01

The medical industry has taken advantage of Java and Java technologies over the past few years, in large part due to the language's platform-independence and object-oriented structure. As such, Java provides powerful and effective tools for developing tissue section analysis software. The background and execution of this development are discussed in this publication. Object-oriented structure allows for the creation of "Slide", "Unit", and "Cell" objects to simulate the corresponding real-world objects. Different functions may then be created to perform various tasks on these objects, thus facilitating the development of the software package as a whole. At the current time, substantial parts of the initially planned functionality have been implemented. Getafics 1.0 is fully operational and currently supports a variety of research projects; however, there are certain features of the software that currently introduce unnecessary complexity and inefficiency. In the future, we hope to include features that obviate these problems.

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.

Stereological study of rat spleen following acute ethanol treatment.

PubMed

Budec, M; Milićević, Z; Koko, V

2000-05-01

To investigate the acute effect of ethanol (4 g/kg, i.p.) on spleen adult female Wistar rats were treated intraperitoneally with: a) ethanol (4 g/kg body wt), b) naltrexone (5 mg/kg body wt) followed 45 minutes later by ethanol (4 g/kg body wt) and c) naltrexone (5 mg/kg body wt) alone. Untreated and saline-treated rats were used as controls. Twenty hours after the ethanol treatment the animals were sacrificed and the spleens were removed. A piece of tissue from the central part of each organ was fixed in Bouin's solution. Paraffin sections were stained with hematoxylin-eosin and analysed using stereological measurements. The volume densities of the following tissue compartments: red pulp, white pulp (divided in follicles, periarterioral lymphatic sheath and marginal zone) and the connective tissue were determined. Stereological analysis also included parameters of follicles: the areal numerical density (the number of follicles per 1 mm2 of tissue section), the numerical density (the number of follicles per mm3 of tissue) and the mean follicle diameter. The immunoarchitecture of the spleen was preserved following acute ethanol treatment. Unlike other parameters that were unaffected, ethanol evoked a decrease in both volume density of follicle and the mean follicle diameter. Naltrexone pretreatment had no influence on ethanol-induced changes. The data obtained indicate that a single dose of ethanol has a profound effect on rat spleen affecting the follicles, but the mechanism of its action remains to be elucidated.

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.

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.

Chemicals identified in human biological media: a data base. Third annual report, October 1981

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

Cone, M.V.; Baldauf, M.F.; Martin, F.M.

1981-12-01

Part 2 contains the data base in tabular format. There are two sections, the first with records on nondrug substances, and the second with records on drugs. Chemicals in each section are arranged alphabetically by CAS preferred name, CAS registry number, formula, atomic weight, melting point, boiling point, and vapor pressure. Tissues are listed alphabetically with exposure route, analytical method, number of cases, range, and mean - when available in the source document. A variety of information may also be included that is pertinent to the range and mean as well as experimental design, demography, health effects, pathology, morphology, andmore » toxicity. Review articles are included in the data base; however, no data have been extracted from such documents because the original research articles are included.« less

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma.

PubMed

Meier, Jeremy D; Xie, Hongtao; Sun, Yang; Sun, Yinghua; Hatami, Nisa; Poirier, Brian; Marcu, Laura; Farwell, D Gregory

2010-06-01

The objectives of this study were to 1) determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract, and 2) evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). Cross-sectional study. University-based medical center. Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700-picosecond pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared with the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than that of normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360 to approximately 660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7 +/- 0.06 ns [not significant] for normal and 1.3 +/- 0.06 ns for tumor, P = 0.0025) and the second-order Laguerre expansion coefficient (LEC-2) (i.e., at 460 nm: 0.135 +/- 0.001 for normal and 0.155 +/- 0.007 for tumor, P < 0.05). These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a noninvasive diagnostic technique for HNSCC. Copyright 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.

Time-resolved laser-induced fluorescence spectroscopy as a diagnostic instrument in head and neck carcinoma

PubMed Central

Meier, Jeremy D.; Xie, Hongtao; Sun, Yang; Sun, Yinghua; Hatami, Nisa; Poirier, Brian; Marcu, Laura; Farwell, D. Gregory

2011-01-01

OBJECTIVE 1) Determine differences in lifetime fluorescence between normal and malignant tissue of the upper aerodigestive tract. 2) Evaluate the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a diagnostic instrument for head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN Cross-sectional study. SETTING University-based medical center. SUBJECTS AND METHODS Nine patients with suspected HNSCC were included. In the operating room, a nitrogen pulse laser (337 nm, 700 ps pulse width) was used to induce tissue autofluorescence of normal tissue and suspected malignant lesions. Spectral intensities and time-domain measurements were obtained and compared to the histopathology at each site. A total of 53 sites were measured. The fluorescence parameters that provided the most discrimination were determined. RESULTS Differences in spectral intensities allowed for discrimination between malignant and normal tissue. The spectral intensity of malignant tissue was lower than the normal tissue, and a shift of peak intensity to a longer wavelength was observed in the normalized spectrum of malignant tissue in the range of 360~660 nm. Multiple time-resolved fluorescence parameters provided the best diagnostic discrimination between normal tissue and carcinoma, including average lifetimes (i.e., at 390 nm: 1.7±0.06 ns for normal and 1.3±0.06 ns for tumor, P=0.0025), and the Laguerre coefficients, LEC-2 (i.e., at 460 nm: 0.135±0.001 for normal and 0.155±0.007 for tumor, P<0.05). CONCLUSION These findings highlight some of the differences in lifetime fluorescence between normal and malignant tissue. TR-LIFS has potential as a non-invasive diagnostic technique for HNSCC. PMID:20493355

The application of polyethylene glycol (PEG) to electron microscopy

PubMed Central

1980-01-01

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis. PMID:7400222

The application of polyethylene glycol (PEG) to electron microscopy.

PubMed

Wolosewick, J J

1980-08-01

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine-coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.

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

Attitudes and Perceptions of Cancer Patients Toward Biospecimen Donation for Cancer Research: A Cross-Sectional Survey Among Chinese Cancer Patients.

PubMed

He, Na; Guo, Yan; He, Min; Qiang, Wanmin; Li, Haixin

2017-08-01

High-quality biospecimen collection from consented patients is crucial for cancer research activities. Patients' attitudes and willingness toward specimen donation influence high-quality biospecimen collection for cancer research activities. We carried out a cross-sectional study among randomly selected patients from 11 cancer departments of Tianjin Medical University Cancer Institute and Hospital between August 2014 and August 2015. A total of 784 patients were included to complete a 30-item self-administered survey. We evaluated the patients' willingness to consider providing leftover samples and additional samples for cancer research purposes. Among 784 patients, 683 (87.1%) and 653 (83.3%) were willing to donate leftover tissue and surplus blood after diagnosis, respectively. Six hundred thirty-one (80.5%) were favorably disposed to consider donating both tissue and blood samples for future cancer research. Female patients showed less willingness to donate biospecimens or related clinical data for research. First-hospitalized or older patients were less willing to provide leftover biospecimens or additional blood samples or even clinical data for research. By contrast, patients with a higher education level were more likely to donate leftover tissues after biopsy or surgery for research activities. Most Chinese cancer patients were willing to consider donating blood and tissue samples for cancer research. Several factors, including age, gender, first hospitalization, and education level, can influence their willingness to donate biospecimens. We need to provide proper education to increase understanding of patients in biobanking activities. This study provides novel empirical data on the likelihood of donating surplus and additional biospecimens and clinical health information among Chinese cancer patients.

A RETROSPECTIVE STUDY OF THE LESIONS ASSOCIATED WITH IRON STORAGE DISEASE IN CAPTIVE EGYPTIAN FRUIT BATS (ROUSETTUS AEGYPTIACUS).

PubMed

Leone, Angelique M; Crawshaw, Graham J; Garner, Michael M; Frasca, Salvatore; Stasiak, Iga; Rose, Karrie; Neal, Dan; Farina, Lisa L

2016-03-01

Egyptian fruit bats (Rousettus aegyptiacus) are one of many species within zoologic collections that frequently develop iron storage disease. The goals of this retrospective multi-institutional study were to determine the tissue distribution of iron storage in captive adult Egyptian fruit bats and the incidence of intercurrent neoplasia and infection, which may be directly or indirectly related to iron overload. Tissue sections from 83 adult Egyptian fruit bats were histologically evaluated by using tissue sections stained with hematoxylin and eosin, trichrome, and Prussian blue techniques. The liver and spleen consistently had the largest amount of iron, but significant amounts of iron were also detected in the pancreas, kidney, skeletal muscle, and lung. Hepatocellular carcinoma (HCC; 11) was the most common neoplasm, followed by cholangiocarcinoma (4). Extrahepatic neoplasms included bronchioloalveolar adenoma (3), pulmonary carcinosarcoma (1), oral sarcoma (1), renal adenocarcinoma (1), transitional cell carcinoma of the urinary bladder (1), mammary gland adenoma (1), and parathyroid adenoma (1). There were also metastatic neoplasms of undetermined primary origin that included three poorly differentiated carcinomas, a poorly differentiated sarcoma, and a neuroendocrine tumor. Bats with hemochromatosis were significantly more likely to have HCC than bats with hemosiderosis (P = 0.032). Cardiomyopathy was identified in 35/77 bats with evaluable heart tissue, but no direct association was found between cardiac damage and the amount of iron observed within the liver or heart. Hepatic abscesses occurred in multiple bats, although a significant association was not observed between hemochromatosis and bacterial infection. To the authors' knowledge, this is the first publication providing evidence of a positive correlation between hemochromatosis and HCC in any species other than humans.

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

Comparison and evaluation of mitotic figures in oral epithelial dysplasia using crystal violet and Feulgen stain.

PubMed

Rao, Roopa S; Patil, Shankargouda; Agarwal, Anveeta

2014-05-01

Routine staining procedures often pose a problem in differentiating a mitotic cell from an apoptotic cell, deteriorating the reliability of histology grading. Although various new methods have been recommended for identifying mitotic figures (MFs) in tissues, the time factor and cost makes them less feasible. Thus, an attempt was made to evaluate the efficacy of crystal violet and Feulgen reaction in identifying MFs and also to see for any variation in the number of MFs in various grades of Epithelial dysplasia. 1. Using crystal violet and Feulgen stain in the identification and counting of MFs on diagnosed cases of epithelial dysplasia and thereby to evaluate their efficacy. 2. To evaluate the variation in the number of MFs in various grades of epithelial dysplasia. The study sample includes retrieval of 30 formalin fixed paraffin embedded tissue sections diagnosed for different grades of epithelial dysplasia (WHO grading system, 2005) from the archives, Department of Oral Pathology, MSRDC, Bengaluru. Ten tissue sections each of mild, moderate and severe epithelial dysplasia were stained with H&E, Feulgen and 1% crystal violet stains and the number of MFs were counted. Five cases of cervical carcinoma were taken as control. Stained sections were compared, and data obtained was statistically analyzed using the Kruskal-Wallis test. A significant increase in the number of MFs (p = 0.02) was observed in Feulgen stained sections as compared to H&E stain. Feulgen stain can be considered as a simple, reliable, cost-effective and reproducible method of staining MFs.

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.

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.

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.

A novel laparoscopic grasper with two parallel jaws capable of extracting the mechanical behaviour of soft tissues.

PubMed

Nazarynasab, Dariush; Farahmand, Farzam; Mirbagheri, Alireza; Afshari, Elnaz

2017-07-01

Data related to force-deformation behaviour of soft tissue plays an important role in medical/surgical applications such as realistically modelling mechanical behaviour of soft tissue as well as minimally invasive surgery (MIS) and medical diagnosis. While the mechanical behaviour of soft tissue is very complex due to its different constitutive components, some issues increase its complexity like behavioural changes between the live and dead tissues. Indeed, an adequate quantitative description of mechanical behaviour of soft tissues requires high quality in vivo experimental data to be obtained and analysed. This paper describes a novel laparoscopic grasper with two parallel jaws capable of obtaining compressive force-deformation data related to mechanical behaviour of soft tissues. This new laparoscopic grasper includes four sections as mechanical hardware, sensory part, electrical/electronical part and data storage part. By considering a unique design for mechanical hardware, data recording conditions will be close to unconfined-compression-test conditions; so obtained data can be properly used in extracting the mechanical behaviour of soft tissues. Also, the other distinguishing feature of this new system is its applicability during different laparoscopic surgeries and subsequently obtaining in vivo data. However, more preclinical examinations are needed to evaluate the practicality of the novel laparoscopic grasper with two parallel jaws.

Tissue expansion and fluid absorption by skin tissue following intradermal injections through hollow microneedles

NASA Astrophysics Data System (ADS)

Shrestha, Pranav; Stoeber, Boris

2017-11-01

Hollow microneedles provide a promising alternative to conventional drug delivery techniques due to improved patient compliance and the dose sparing effect. The dynamics of fluid injected through hollow microneedles into skin, which is a heterogeneous and deformable porous medium, have not been investigated extensively in the past. We have introduced the use of Optical Coherence Tomography (OCT) for real-time visualization of fluid injections into excised porcine tissue. The results from ex-vivo experiments, including cross-sectional tissue images from OCT and pressure/flow-rate measurements, show a transient mode of high flow-rate into the tissue followed by a lower steady-state infusion rate. The injected fluid expands the underlying tissue and causes the external free surface of the skin to rise, forming a characteristic intradermal wheal. We have used OCT to visualize the evolution of tissue and free surface deformation, and advancement of the boundary between regions of expanding and stationary tissue. We will show the effect of different injection parameters such as fluid pressure, viscosity and microneedle retraction on the injected volume. This work has been supported through funding from the Collaborative Health Research Program by the Natural Science and Engineering Research Council of Canada and the Canadian Health Research Institute, and through the Canada Research Chairs program.

Spectroscopic characterization of collagen cross-links in bone

NASA Technical Reports Server (NTRS)

Paschalis, E. P.; Verdelis, K.; Doty, S. B.; Boskey, A. L.; Mendelsohn, R.; Yamauchi, M.

2001-01-01

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.

Molecular diagnosis of skin infections using paraffin-embedded tissue - review and interdisciplinary consensus.

PubMed

Sunderkötter, Cord; Becker, Karsten; Kutzner, Heinz; Meyer, Thomas; Blödorn-Schlicht, Norbert; Reischl, Udo; Nenoff, Pietro; Geißdörfer, Walter; Gräser, Yvonne; Herrmann, Mathias; Kühn, Joachim; Bogdan, Christian

2018-02-01

Nucleic acid amplification techniques (NATs), such as PCR, are highly sensitive and specific methods that have become valuable supplements to culture and serology in the diagnosis of infectious disorders. However, especially when using formalin-fixed and paraffin-embedded tissue, these techniques are associated with both false-negative and false-positive results, a pitfall that is frequently misjudged. Representatives of the German Society of Hygiene and Microbiology (DGHM) and the German Society of Dermatology (DDG) therefore set out to develop a consensus - in the form of a review article - on the appropriate indications for NATs using paraffin-embedded tissue, its contraindications, and the key points to be considered in the pre- and post-analytical phase. Given that fresh, naive tissue is preferably to be used in the workup of a suspected infection, PCR analysis on paraffin sections represents an exception. The latter may be considered if an infection is suspected at a later point in time and fresh tissue has not been preserved or can no longer be obtained. Potential indications include confirmation of histologically suspected infections with Leishmania spp., Bartonella spp., Rickettsia spp., or in case of ecthyma contagiosum. Infections with, for example, mycobacteria or RNA viruses, on the other hand, are not considered useful indications for NATs using paraffin sections. In order to avoid misinterpretation of test results, it is essential that laboratory reports on NATs using paraffin-embedded tissue contain information on the indication/diagnostic circumstances, the required and chosen pre-analytical steps, the limitations of the method, and on diagnostic alternatives. © 2018 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

PubMed Central

Bobik, Krzysztof; Dunlap, John R.; Burch-Smith, Tessa M.

2014-01-01

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality. PMID:25350384

Characterization of some biological specimens using TEM and SEM

NASA Astrophysics Data System (ADS)

Ghosh, Nabarun; Smith, Don W.

2009-05-01

The advent of novel techniques using the Transmission and Scanning Electron Microscopes improved observation on various biological specimens to characterize them. We studied some biological specimens using Transmission and Scanning Electron Microscopes. We followed negative staining technique with Phosphotungstic acid using bacterial culture of Bacillus subtilis. Negative staining is very convenient technique to view the structural morphology of different samples including bacteria, phage viruses and filaments in a cell. We could observe the bacterial cell wall and flagellum very well when trapped the negative stained biofilm from bacterial culture on a TEM grid. We cut ultra thin sections from the fixed root tips of Pisum sativum (Garden pea). Root tips were pre fixed with osmium tetroxide and post fixed with uranium acetate and placed in the BEEM capsule for block making. The ultrathin sections on the grid under TEM showed the granular chromatin in the nucleus. The protein bodies and large vacuoles with the storage materials were conspicuous. We followed fixation, critical point drying and sputter coating with gold to view the tissues with SEM after placing on stubs. SEM view of the leaf surface of a dangerous weed Tragia hispida showed the surface trichomes. These trichomes when break on touching releases poisonous content causing skin irritation. The cultured tissue from in vitro culture of Albizia lebbeck, a tree revealed the regenerative structures including leaf buds and stomata on the tissue surface. SEM and TEM allow investigating the minute details characteristic morphological features that can be used for classroom teaching.

Protective effects of melatonin and quercetin on experimental lung injury induced by carbon tetrachloride in rats.

PubMed

Taslidere, Elif; Esrefoglu, Mukaddes; Elbe, Hulya; Cetin, Asli; Ates, Burhan

2014-03-01

Exposure to carbon tetrachloride (CCl4), a well-known toxicant, causes tissue damage by inducing oxidative stress via formation of free radicals. The fundamental structure of the organs of rats and humans is similar, so administration of CCl4 to rats is an accepted experimental model to produce oxidative damage to various tissues including pulmonary tissue. In this study, we evaluated the protective capacity of melatonin and quercetin against CCl4-induced oxidative lung damage in rats. Rats were divided into five groups each containing seven rats as follows: Control group, Olive oil group CCl4 group, CCl4+Melatonin, and CCl4+Quercetin group. The tissue samples were processed by routine histological and biochemical procedures. Sections were stained with Hematoxylin-eosin and Masson's trichrome. Histopathologic damage score was calculated. Malondialdehyde (MDA) and glutathione (GSH) levels and catalase (CAT) activities were assayed. The lung sections of control groups showed normal histological characteristics. Fibrosis, interstitial hemorrhage, epithelial desquamation in bronchiole and alveoli, intra-alveolar edema, leukocyte, and macrophage infiltration were observed in lung sections of rats exposed to CCl4 alone. The findings were reduced in the treatments groups. The MDA level in the CCI4 group were significantly higher than in the other groups (p < .001), and the CAT and GSH levels in the CCI4+Mel and CCI4+Quer groups were significantly higher than in the CCI4 group (p < .05). In conclusion, we suggest that agents with antioxidant properties such as melatonin and quercetin may have positive effects in the treatment of pulmonary diseases characterized by especially edema, inflammation, and fibrosis.

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.

Comparison of Carbon Dioxide Laser With Surgical Blade for Removal of Epulis Fissuratum. A Randomized Clinical Trial

PubMed Central

Karimi, Abbas; Sobouti, Farhad; Torabi, Sara; Bakhshandehfard, Ali; Amirian, Armaghan; Shariati, Mahsa; Morshedi, Ehsan; Barati, Maryam

2016-01-01

Introduction: Epulis fissuratum is often formed as a result of a poor fitting denture. The conventional treatment for this fibrous hyperplastic tissue is to excise it using a scalpel and to close the wound by a continuous or an interrupted suture. The increased utilization of lasers in dentistry also includes the utilization of carbon dioxide (CO2) lasers in place of surgical scalpels in soft tissue surgeries. The objective of this study is to assess the feasibility of utilizing CO2 laser in place of scalpel in surgical treatment of epulis fissuratum. Methods: In this clinical trial research (IRCT code: IRCT2016071124969N2), 19 patients were selected with nearly symmetrical epulis fissuratums in the anterior part of the jaws. The hyperplastic tissue was evenly divided into two sections in each patient. One section was randomly selected and cut by CO2 laser and the other section by a surgical scalpel. The wound created by the scalpel was closed by appropriate number of interrupted sutures. Surgery duration and bleeding as well as vestibular depth, re-epithelialization and edema in both sections were noted and recorded after 7 and 14 days postoperatively. Results: The time of surgery and the amount of bleeding during surgery in the laser section was less and the vestibular depth was more than surgical scalpel section (P < 0.05). Surgical scalpel wound at day seventh healed significantly better than the section treated by the CO2 laser (P < 0.05). Wound in both sections healed similarly on day 14 and no statistical difference was observed. Edema presence was also equal in both sides after 7th and 14th following the surgery. Conclusion: According to the results it could be concluded that the use of CO2 laser may result in less surgery time, less bleeding during surgery, more vestibular depth, better re-epithelialization of the wound and less need for suturing. CO2 laser may be a clinically preferred method for surgical treatment of epulis fissuratum. PMID:28144443

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 ...

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.

Do enteric neurons make hypocretin?

PubMed

Baumann, Christian R; Clark, Erika L; Pedersen, Nigel P; Hecht, Jonathan L; Scammell, Thomas E

2008-04-10

Hypocretins (orexins) are wake-promoting neuropeptides produced by hypothalamic neurons. These hypocretin-producing cells are lost in people with narcolepsy, possibly due to an autoimmune attack. Prior studies described hypocretin neurons in the enteric nervous system, and these cells could be an additional target of an autoimmune process. We sought to determine whether enteric hypocretin neurons are lost in narcoleptic subjects. Even though we tried several methods (including whole mounts, sectioned tissue, pre-treatment of mice with colchicine, and the use of various primary antisera), we could not identify hypocretin-producing cells in enteric nervous tissue collected from mice or normal human subjects. These results raise doubts about whether enteric neurons produce hypocretin.

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.

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.

Brown Adipose Tissue and Its Relationship to Bone Structure in Pediatric Patients

PubMed Central

Ponrartana, Skorn; Aggabao, Patricia C.; Hu, Houchun H.; Aldrovandi, Grace M.; Wren, Tishya A. L.

2012-01-01

Context: Emerging evidence suggests a possible link between brown adipose tissue (BAT) and bone metabolism. Objective: The objective of this study was to examine the relationships between BAT and bone cross-sectional dimensions in children and adolescents. Design: This was a cross-sectional study. Setting: The study was conducted at a pediatric referral center. Patients: Patients included 40 children and teenagers (21 males and 19 females) successfully treated for pediatric malignancies. Interventions: There were no interventions. Main Outcome Measures: The volume of BAT was determined by fluorodeoxyglucose-positron emission tomography/computed tomography. Measures of the cross-sectional area and cortical bone area and measures of thigh musculature and sc fat were determined at the midshaft of the femur. Results: Regardless of sex, there were significant correlations seen between BAT volume and the cross-sectional dimensions of the bone (r values between 0.68 and 0.77; all P ≤ 0 .001). Multiple regression analyses indicated that the volume of BAT predicted femoral cross-sectional area and cortical bone area, even after accounting for height, weight, and gender. The addition of muscle as an independent variable increased the predictive power of the model but significantly decreased the contribution of BAT. Conclusions: The volume of BAT is positively associated with the amount of bone and the cross-sectional size of the femur in children and adolescents. This relation between BAT and bone structure could, at least in part, be mediated by muscle. PMID:22593587

Automated MALDI Matrix Coating System for Multiple Tissue Samples for Imaging Mass Spectrometry

NASA Astrophysics Data System (ADS)

Mounfield, William P.; Garrett, Timothy J.

2012-03-01

Uniform matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is key for reproducible analyte ion signals. Current methods often result in nonhomogenous matrix deposition, and take time and effort to produce acceptable ion signals. Here we describe a fully-automated method for matrix deposition using an enclosed spray chamber and spray nozzle for matrix solution delivery. A commercial air-atomizing spray nozzle was modified and combined with solenoid controlled valves and a Programmable Logic Controller (PLC) to control and deliver the matrix solution. A spray chamber was employed to contain the nozzle, sample, and atomized matrix solution stream, and to prevent any interference from outside conditions as well as allow complete control of the sample environment. A gravity cup was filled with MALDI matrix solutions, including DHB in chloroform/methanol (50:50) at concentrations up to 60 mg/mL. Various samples (including rat brain tissue sections) were prepared using two deposition methods (spray chamber, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed a uniform coating of matrix crystals across the sample. Overall, the mass spectral images gathered from tissues coated using the spray chamber system were of better quality and more reproducible than from tissue specimens prepared by the inkjet deposition method.

Automated MALDI matrix coating system for multiple tissue samples for imaging mass spectrometry.

PubMed

Mounfield, William P; Garrett, Timothy J

2012-03-01

Uniform matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is key for reproducible analyte ion signals. Current methods often result in nonhomogenous matrix deposition, and take time and effort to produce acceptable ion signals. Here we describe a fully-automated method for matrix deposition using an enclosed spray chamber and spray nozzle for matrix solution delivery. A commercial air-atomizing spray nozzle was modified and combined with solenoid controlled valves and a Programmable Logic Controller (PLC) to control and deliver the matrix solution. A spray chamber was employed to contain the nozzle, sample, and atomized matrix solution stream, and to prevent any interference from outside conditions as well as allow complete control of the sample environment. A gravity cup was filled with MALDI matrix solutions, including DHB in chloroform/methanol (50:50) at concentrations up to 60 mg/mL. Various samples (including rat brain tissue sections) were prepared using two deposition methods (spray chamber, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed a uniform coating of matrix crystals across the sample. Overall, the mass spectral images gathered from tissues coated using the spray chamber system were of better quality and more reproducible than from tissue specimens prepared by the inkjet deposition method.

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.

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.

Using synchrotron X-ray phase-contrast micro-computed tomography to study tissue damage by laser irradiation.

PubMed

Robinson, Alan M; Stock, Stuart R; Soriano, Carmen; Xiao, Xianghui; Richter, Claus-Peter

2016-11-01

The aim of this study was to determine if X-ray micro-computed tomography could be used to locate and characterize tissue damage caused by laser irradiation and to describe its advantages over classical histology for this application. A surgical CO 2 laser, operated in single pulse mode (100 milliseconds) at different power settings, was used to ablate different types of cadaveric animal tissues. Tissue samples were then harvested and imaged with synchrotron X-ray phase-contrast and micro-computed tomography to generate stacks of virtual sections of the tissues. Subsequently, Fiji (ImageJ) software was used to locate tissue damage, then to quantify volumes of laser ablation cones and thermal coagulation damage from 3D renderings of tissue image stacks. Visual comparisons of tissue structures in X-ray images with those visible by classic light microscopy histology were made. We demonstrated that micro-computed tomography could be used to rapidly identify areas of surgical laser ablation, vacuolization, carbonization, and thermally coagulated tissue. Quantification and comparison of the ablation crater, which represents the volume of ablated tissue, and the thermal coagulation zone volumes were performed faster than we could by classical histology. We demonstrated that these procedures can be performed on fresh hydrated and non-sectioned plastic embedded tissue. We demonstrated that the application of non-destructive micro-computed tomography to the visualization and analysis of laser induced tissue damage without tissue sectioning is possible. This will improve evaluation of new surgical lasers and their corresponding effect on tissues. Lasers Surg. Med. 48:866-877, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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.

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.

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.

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.

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.

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.

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

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.

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.

Tumor tissue characterization using polarization-sensitive second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Tokarz, Danielle; Cisek, Richard; Golaraei, Ahmad; Krouglov, Serguei; Navab, Roya; Niu, Carolyn; Sakashita, Shingo; Yasufuku, Kazuhiro; Tsao, Ming-Sound; Asa, Sylvia L.; Barzda, Virginijus; Wilson, Brian C.

2015-06-01

Changes in the ultrastructure of collagen in various tumor and non-tumor human tissues including lung, pancreas and thyroid were investigated ex vivo by a polarization-sensitive second harmonic generation (SHG) microscopy technique referred to as polarization-in, polarization-out (PIPO) SHG. This involves measuring the orientation of the linear polarization of outgoing SHG as a function of the linear polarization orientation of incident laser radiation. From the PIPO SHG data, the second-order nonlinear optical susceptibility tensor component ratio, χ(2) ZZZ'/χ(2) ZXX', for each pixel of the SHG image was obtained and presented as color-coded maps. Further, the orientation of collagen fibers in the tissue was deduced. Since the χ(2) ZZZ'/χ(2) ZXX' values represent the organization of collagen in the tissue, theses maps revealed areas of altered collagen structure (not simply concentration) within tissue sections. Statistically-significant differences in χ(2) ZZZ'/χ(2) ZXX' were found between tumor and non-tumor tissues, which varied from organ to organ. Hence, PIPO SHG microscopy could potentially be used to aid pathologists in diagnosing cancer. Additionally, PIPO SHG microscopy could aid in characterizing the structure of collagen in other collagen-related biological processes such as wound repair.

Sensory Innervation of the Nonspecialized Connective Tissues in the Low Back of the Rat

PubMed Central

Corey, Sarah M.; Vizzard, Margaret A.; Badger, Gary J.; Langevin, Helene M.

2011-01-01

Chronic musculoskeletal pain, including low back pain, is a worldwide debilitating condition; however, the mechanisms that underlie its development remain poorly understood. Pathological neuroplastic changes in the sensory innervation of connective tissue may contribute to the development of nonspecific chronic low back pain. Progress in understanding such potentially important abnormalities is hampered by limited knowledge of connective tissue's normal sensory innervation. The goal of this study was to evaluate and quantify the sensory nerve fibers terminating within the nonspecialized connective tissues in the low back of the rat. With 3-dimensional reconstructions of thick (30–80 μm) tissue sections we have for the first time conclusively identified sensory nerve fiber terminations within the collagen matrix of connective tissue in the low back. Using dye labeling techniques with Fast Blue, presumptive dorsal root ganglia cells that innervate the low back were identified. Of the Fast Blue-labeled cells, 60–88% also expressed calcitonin gene-related peptide (CGRP) immunoreactivity. Based on the immunolabeling with CGRP and the approximate size of these nerve fibers (≤2 μm) we hypothesize that they are Aδ or C fibers and thus may play a role in the development of chronic pain. PMID:21411968

Mast Cells Synthesize, Store, and Release Nerve Growth Factor

NASA Astrophysics Data System (ADS)

Leon, A.; Buriani, A.; dal Toso, R.; Fabris, M.; Romanello, S.; Aloe, L.; Levi-Montalcini, R.

1994-04-01

Mast cells and nerve growth factor (NGF) have both been reported to be involved in neuroimmune interactions and tissue inflammation. In many peripheral tissues, mast cells interact with the innervating fibers. Changes in the behaviors of both of these elements occur after tissue injury/inflammation. As such conditions are typically associated with rapid mast cell activation and NGF accumulation in inflammatory exudates, we hypothesized that mast cells may be capable of producing NGF. Here we report that (i) NGF mRNA is expressed in adult rat peritoneal mast cells; (ii) anti-NGF antibodies clearly stain vesicular compartments of purified mast cells and mast cells in histological sections of adult rodent mesenchymal tissues; and (iii) medium conditioned by peritoneal mast cells contains biologically active NGF. Mast cells thus represent a newly recognized source of NGF. The known actions of NGF on peripheral nerve fibers and immune cells suggest that mast cell-derived NGF may control adaptive/reactive responses of the nervous and immune systems toward noxious tissue perturbations. Conversely, alterations in normal mast cell behaviors may provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states, including those of an autoimmune nature.

Myositis Ossificans.

PubMed

Walczak, Brian E; Johnson, Christopher N; Howe, B Matthew

2015-10-01

Myositis ossificans is a self-limiting, benign ossifying lesion that can affect any type of soft tissue, including subcutaneous fat, tendons, and nerves. It is most commonly found in muscle as a solitary lesion. Ossifying soft-tissue lesions historically have been inconsistently classified. Fundamentally, myositis ossificans can be categorized into nonhereditary and hereditary types, with the latter being a distinct entity with a separate pathophysiology and treatment approach. The etiology of myositis ossificans is variable; however, clinical presentation generally is characterized by an ossifying soft-tissue mass. Advanced cross-sectional imaging alone can be nonspecific and may appear to be similar to more sinister etiologies. Therefore, the evaluation of a suspicious soft-tissue mass often necessitates multiple imaging modalities for accurate diagnosis. When imaging is indeterminate, biopsy may be required for a histologic diagnosis. However, histopathology varies based on stage of evolution. The treatment of myositis ossificans is complex and is often made in a multidisciplinary fashion because accurate diagnosis is fundamental to a successful outcome. Copyright 2015 by the American Academy of Orthopaedic Surgeons.

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...

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...

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.

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

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

Rapid demonstration of Legionella pneumophila in unembedded tissue. An adaptation of the Giménez stain.

PubMed

Greer, P W; Chandler, F W; Hicklin, M D

1980-06-01

The Giménez stain, originally developed for demonstrating rickettsiae, readily stained the Legionnaires' disease bacterium (Legionella pneumophila) in frozen tissue sections and smears of fresh or formalin-fixed lung tissue from patients who had confirmed Legionnaires' disease. With the Giménez procedure, the bacterium stained bright red against a blue-green background. The tissue Gram procedures also stained L. pneumophila in frozen sections and smears, but the staining reaction was weak, and these stains were neither as sensitive nor a consistent as the Giménez procedure.

Periodontal tissue regeneration by combined implantation of adipose tissue-derived stem cells and platelet-rich plasma in a canine model.

PubMed

Tobita, Morikuni; Uysal, Cagri A; Guo, Xin; Hyakusoku, Hiko; Mizuno, Hiroshi

2013-12-01

One goal of periodontal therapy is to regenerate periodontal tissues. Stem cells, growth factors and scaffolds and biomaterials are vital for the restoration of the architecture and function of complex tissues. Adipose tissue-derived stem cells (ASCs) are an ideal population of stem cells for practical regenerative medicine. In addition, platelet-rich plasma (PRP) can be useful for its ability to stimulate tissue regeneration. PRP contains various growth factors and may be useful as a cell carrier in stem cell therapies. The purpose of this study was to determine whether a mixture of ASCs and PRP promoted periodontal tissue regeneration in a canine model. Autologous ASCs and PRP were implanted into areas with periodontal tissue defects. Periodontal tissue defects that received PRP alone or non-implantation were also examined. Histologic, immunohistologic and x-ray studies were performed 1 or 2 months after implantation. The amount of newly formed bone and the scale of newly formed cementum in the region of the periodontal tissue defect were analyzed on tissue sections. The areas of newly formed bone and cementum were greater 2 months after implantation of ASCs and PRP than at 1 month after implantation, and the radiopacity in the region of the periodontal tissue defect increased markedly by 2 months after implantation. The ASCs and PRP group exhibited periodontal tissue with the correct architecture, including alveolar bone, cementum-like structures and periodontal ligament-like structures, by 2 months after implantation. These findings suggest that a combination of autologous ASCs and PRP promotes periodontal tissue regeneration that develops the appropriate architecture for this complex tissue. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Prevalence of Soft Tissue Calcifications in CBCT Images of Mandibular Region.

PubMed

Khojastepour, Leila; Haghnegahdar, Abdolaziz; Sayar, Hamed

2017-06-01

Most of the soft tissue calcifications within the head and neck region might not be accompanied by clinical symptoms but may indicate some pathological conditions. The aim of this research was to determine the prevalence of soft tissue calcifications in cone beam computed tomography (CBCT) images of mandibular region. In this cross sectional study the CBCT images of 602 patients including 294 men and 308 women with mean age 41.38±15.18 years were evaluated regarding the presence, anatomical location; type (single or multiple) and size of soft tissue calcification in mandibular region. All CBCT images were acquired by NewTom VGi scanner. Odds ratio and chi-square tests were used for data analysis and p < 0.05 was considered to be statistically significant. 156 out of 602 patients had at least one soft tissue calcification in their mandibular region (25.9%. of studied population with mean age 51.7±18.03 years). Men showed significantly higher rate of soft tissue calcification than women (30.3% vs. 21.8%). Soft tissue calcification was predominantly seen at posterior region of the mandible (88%) and most of them were single (60.7%). The prevalence of soft tissue calcification increased with age. Most of the detected soft tissue calcifications were smaller than 3mm (90%). Soft tissue calcifications in mandibular area were a relatively common finding especially in posterior region and more likely to happen in men and in older age group.

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.

The Mosasaur Tooth Attachment Apparatus as Paradigm for the Evolution of the Gnathostome Periodontium

PubMed Central

Luan, Xianghong; Walker, Cameron; Dangaria, Smit; Ito, Yoshihiro; Druzinsky, Robert; Jarosius, Kristina; Lesot, Herve; Rieppel, Olivier

2009-01-01

Vertebrate teeth are attached to jaws by a variety of mechanisms, including acrodont, pleurodont, and thecodont modes of attachment. Recent studies have suggested that various modes of attachment exist within each sub-category. Especially squamates feature a broad diversity of modes of attachment. Here we have investigated tooth attachment tissues in the late cretaceous mosasaur Clidastes and compared mosasaur tooth attachment with modes of attachment found in other extant reptiles. Using histologic analysis of ultrathin ground sections, four distinct mineralized tissues that anchor mosasaur teeth to the jaw were identified: (i) an acellular cementum layer at the interface between root and cellular cementum, (ii) a massive cone consisting of trabecular cellular cementum, (iii) the mineralized periodontal ligament containing mineralized Sharpey’s fibers, and (iv) the interdental ridges connecting adjacent teeth. The complex, multilayered attachment apparatus in mosasaurs was compared with attachment tissues in extant reptiles, including Iguana and Caiman. Based on our comparative analysis we postulate the presence of a quadruple-layer tissue architecture underlying reptilian tooth attachment, comprised of acellular cementum, cellular cementum, mineralized periodontal ligament, and interdental ridge (alveolar bone). We propose that the mineralization status of the periodontal ligament is a dynamic feature in vertebrate evolution subject to functional adaptation. PMID:19469852

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.

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.

Customized Fabrication of Osteochondral Tissue for Articular Joint Surface Repair

DTIC Science & Technology

2015-09-01

Kilroy EJ, Tuan RS. (2015) Projection Stereolithographic Fabrication of Human Adipose Stem Cell-incorporated Biodegradable Scaffolds for Cartilage...necessary to include the publications already specified above in this section. Nothing to Report (1) Live Cell-scaffold Printing using Biodegradable ...and PCL scaffolds using PSL (Figure 1,2). Solid PDLLA-PEG scaffold (30%) possesses a compressive modulus up to 800kPa and is biodegradable in

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

On the importance of mathematical methods for analysis of MALDI-imaging mass spectrometry data.

PubMed

Trede, Dennis; Kobarg, Jan Hendrik; Oetjen, Janina; Thiele, Herbert; Maass, Peter; Alexandrov, Theodore

2012-03-21

In the last decade, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS), also called as MALDI-imaging, has proven its potential in proteomics and was successfully applied to various types of biomedical problems, in particular to histopathological label-free analysis of tissue sections. In histopathology, MALDI-imaging is used as a general analytic tool revealing the functional proteomic structure of tissue sections, and as a discovery tool for detecting new biomarkers discriminating a region annotated by an experienced histologist, in particular, for cancer studies. A typical MALDI-imaging data set contains 10⁸ to 10⁹ intensity values occupying more than 1 GB. Analysis and interpretation of such huge amount of data is a mathematically, statistically and computationally challenging problem. In this paper we overview some computational methods for analysis of MALDI-imaging data sets. We discuss the importance of data preprocessing, which typically includes normalization, baseline removal and peak picking, and hightlight the importance of image denoising when visualizing IMS data.

On the Importance of Mathematical Methods for Analysis of MALDI-Imaging Mass Spectrometry Data.

PubMed

Trede, Dennis; Kobarg, Jan Hendrik; Oetjen, Janina; Thiele, Herbert; Maass, Peter; Alexandrov, Theodore

2012-03-01

In the last decade, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS), also called as MALDI-imaging, has proven its potential in proteomics and was successfully applied to various types of biomedical problems, in particular to histopathological label-free analysis of tissue sections. In histopathology, MALDI-imaging is used as a general analytic tool revealing the functional proteomic structure of tissue sections, and as a discovery tool for detecting new biomarkers discriminating a region annotated by an experienced histologist, in particular, for cancer studies. A typical MALDI-imaging data set contains 108 to 109 intensity values occupying more than 1 GB. Analysis and interpretation of such huge amount of data is a mathematically, statistically and computationally challenging problem. In this paper we overview some computational methods for analysis of MALDI-imaging data sets. We discuss the importance of data preprocessing, which typically includes normalization, baseline removal and peak picking, and hightlight the importance of image denoising when visualizing IMS data.

Pyrylium Salts as Reactive Matrices for MALDI-MS Imaging of Biologically Active Primary Amines

NASA Astrophysics Data System (ADS)

Shariatgorji, Mohammadreza; Nilsson, Anna; Källback, Patrik; Karlsson, Oskar; Zhang, Xiaoqun; Svenningsson, Per; Andren, Per E.

2015-06-01

Many neuroactive substances, including endogenous biomolecules, environmental compounds, and pharmaceuticals possess primary amine functional groups. Among these are catecholamine neurotransmitters (e.g., dopamine), many substituted phenethylamines (e.g., amphetamine), as well as amino acids and neuropeptides. In most cases, mass spectrometric (ESI and MALDI) analyses of trace amounts of such compounds are challenging because of their poor ionization properties. We present a method for chemical derivatization of primary amines by reaction with pyrylium salts that facilitates their detection by MALDI-MS and enables the imaging of primary amines in brain tissue sections. A screen of pyrylium salts revealed that the 2,4-diphenyl-pyranylium ion efficiently derivatizes primary amines and can be used as a reactive MALDI-MS matrix that induces both derivatization and desorption. MALDI-MS imaging with such matrix was used to map the localization of dopamine and amphetamine in brain tissue sections and to quantitatively map the distribution of the neurotoxin β- N-methylamino-L-alanine.

Comprehensive Review of Applicable Supercritical Fluid Extraction Research

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

Harvey, Scott; Wright, Cherylyn W.; Wright, Bob W.

2001-09-10

This comprehensive supercritical fluid extraction (SFE) literature review is divided into three major sections. The first section describes the electronic literature search details including the abstract service used and the different topics searched. This section also contains an overview of the seven search topics that yielded relevant references along with a brief synopsis of the most significant literature citations. These seven groupings are (1) chemical warfare agents; (2) explosives; (3) hazardous chemicals; (4) poisons, toxins and mycotoxins; (5) toxic (lethal) chemical and toxicants; (6) pesticides in soil; and (7) pesticides from plant and animal tissues. The second section contains tablesmore » of each of these groupings. Each of the seven tables contains entries for individual literature citations listed along with the specific compounds or compound classes that are addressed. The third section refers to the abstracts used in the literature search.« less

DRAQ5 and Eosin (‘D&E’) as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues

PubMed Central

Elfer, Katherine N.; Sholl, Andrew B.; Wang, Mei; Tulman, David B.; Mandava, Sree H.; Lee, Benjamin R.; Brown, J. Quincy

2016-01-01

Real-time on-site histopathology review of biopsy tissues at the point-of-procedure has great potential for significant clinical value and improved patient care. For instance, on-site review can aid in rapid screening of diagnostic biopsies to reduce false-negative results, or in quantitative assessment of biospecimen quality to increase the efficacy of downstream laboratory and histopathology analysis. However, the only currently available rapid pathology method, frozen section analysis (FSA), is too time- and labor-intensive for use in screening large quantities of biopsy tissues and is too destructive for maximum tissue conservation in multiple small needle core biopsies. In this work we demonstrate the spectrally-compatible combination of the nuclear stain DRAQ5 and the anionic counterstain eosin as a dual-component fluorescent staining analog to hematoxylin and eosin intended for use on fresh, unsectioned tissues. Combined with optical sectioning fluorescence microscopy and pseudo-coloring algorithms, DRAQ5 and eosin (“D&E”) enables very fast, non-destructive psuedohistological imaging of tissues at the point-of-acquisition with minimal tissue handling and processing. D&E was validated against H&E on a one-to-one basis on formalin-fixed paraffin-embedded and frozen section tissues of various human organs using standard epi-fluorescence microscopy, demonstrating high fidelity of the staining mechanism as an H&E analog. The method was then applied to fresh, whole 18G renal needle core biopsies and large needle core prostate biospecimen biopsies using fluorescence structured illumination optical sectioning microscopy. We demonstrate the ability to obtain high-resolution histology-like images of unsectioned, fresh tissues similar to subsequent H&E staining of the tissue. The application of D&E does not interfere with subsequent standard-of-care H&E staining and imaging, preserving the integrity of the tissue for thorough downstream analysis. These results indicate that this dual-stain pseudocoloring method could provide a real-time histology-like image at the time of acquisition and valuable objective tissue analysis for the clinician at the time of service. PMID:27788264

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.

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.

BOOK REVIEW Handbook of Physics in Medicine and Biology Handbook of Physics in Medicine and Biology

NASA Astrophysics Data System (ADS)

Tabakov, Slavik

2010-11-01

This is a multi-author handbook (66 authors) aiming to describe various applications of physics to medicine and biology, from anatomy and physiology to medical equipment. This unusual reference book has 44 chapters organized in seven sections: 1. Anatomical physics; 2. Physics of perception; 3. Biomechanics; 4. Electrical physics; 5. Diagnostic physics; 6. Physics of accessory medicine; 7. Physics of bioengineering. Each chapter has separate page numbering, which is inconvenient but understandable with the number of authors. Similarly there is some variation in the emphasis of chapters: for some the emphasis is more technical and for others clinical. Each chapter has a separate list of references. The handbook includes hundreds of diagrams, images and tables, making it a useful tool for both medical physicists/engineers and other medical/biology specialists. The first section (about 40 pages) includes five chapters on physics of the cell membrane; protein signaling; cell biology and biophysics of the cell membrane; cellular thermodynamics; action potential transmission and volume conduction. The physics of these is well explained and illustrated with clear diagrams and formulae, so it could be a suitable reference for physicists/engineers. The chapters on cellular thermodynamics and action potential transmission have a very good balance of technical/clinical content. The second section (about 85 pages) includes six chapters on medical decision making; senses; somatic senses: touch and pain; hearing; vision; electroreception. Again these are well illustrated and a suitable reference for physicists/engineers. The chapter on hearing stands out with good balance and treatment of material, but some other chapters contain less physics and are close to typical physiological explanations. One could query the inclusion of the chapter on medical decision making, which also needs more detail. The third section (about 80 pages) includes eight chapters on biomechanics; artificial muscle; cardiovascular system; control of cardiac output and arterial blood pressure regulation; fluid dynamics of the cardiovascular system; fluid dynamics; modeling and simulation of the cardiovascular system to determine work using bond graphs; anatomy and physics of respiration. The diagrams and data in this section could be used as reference material, but some chapters (such as that on the cardiovascular system) again take the form of physiological explanations. The best chapters in this section are on fluid dynamics and modeling. The fourth section (about 30 pages) includes two chapters on electrodes and recording of bioelectrical signals: theory and practice. Both chapters deal with electrodes and are well written and illustrated reference materials. This section could have been larger but the equipment associated with bioelectrical signals (such as ECG and EEG) is described in the next section. The fifth section (about 210 pages) includes 19 chapters on medical sensing and imaging; electrocardiogram: electrical information retrieval and diagnostics from the beating heart; electroencephalography: basic concepts and brain applications; bioelectric impedance analysis; x-ray and computed tomography; confocal microscopy; magnetic resonance imaging; positron emission tomography; in vivo fluorescence imaging and spectroscopy; optical coherence tomography; ultrasonic imaging; near-field imaging; atomic force microscopy; scanning ion conductance microscopy; quantitative thermographic imaging; intracoronary thermography; schlieren imaging: optical techniques to visualize thermal interactions with biological tissues; helium ion microscopy; electron microscopy: SEM/TEM. This is by far the largest section covering various methods and medical equipment and the variation in emphasis/quality is more prominent. The chapters on ECG and EEG are again more physiological with less physics, but the chapter on bioelectric impedance analysis is a good interdisciplinary article. The imaging chapters also vary in style and quality: while those on MRI and ultrasound provide a suitable introduction to the methods, the chapters on x-ray and PET need more detail. However this section includes some methods/equipment rarely featured in medical physics/engineering books (such as OCT or HIM). From this point of view the last eight chapters in the section will be a very useful reference for various specialists. The sixth section (about 30 pages) includes three chapters on lab-on-a-chip; the biophysics of DNA microarrays; nuclear medicine. While the first two could provide an interesting reference, the chapter on nuclear medicine needs much more detail. The last (seventh) section (15 pages) has only one chapter on biophysics of regenerative medicine, which is a good introduction, emphasizing biochemical factors important for improving/replacing tissues or tissue structures. The book ends with an index covering about 1400 terms. The handbook will be useful for the preparation of teaching materials and for undergraduate students, but should be complemented with more detailed/specific reference materials such as the Encyclopedia of Medical Devices and Instrumentation, the Encyclopedia of Medical Physics Emitel, or others. Parts of the handbook would be less suitable for more demanding readers (such as trainee medical physicists or radiologists, for example). In conclusion, the Handbook of Physics in Medicine and Biology includes materials that are rarely combined together, which strengthens its interdisciplinary approach and makes it an additional reference for a departmental library.

Do enteric neurons make hypocretin? ☆

PubMed Central

Baumann, Christian R.; Clark, Erika L.; Pedersen, Nigel P.; Hecht, Jonathan L.; Scammell, Thomas E.

2008-01-01

Hypocretins (orexins) are wake-promoting neuropeptides produced by hypothalamic neurons. These hypocretin-producing cells are lost in people with narcolepsy, possibly due to an autoimmune attack. Prior studies described hypocretin neurons in the enteric nervous system, and these cells could be an additional target of an autoimmune process. We sought to determine whether enteric hypocretin neurons are lost in narcoleptic subjects. Even though we tried several methods (including whole mounts, sectioned tissue, pre-treatment of mice with colchicine, and the use of various primary antisera), we could not identify hypocretin-producing cells in enteric nervous tissue collected from mice or normal human subjects. These results raise doubts about whether enteric neurons produce hypocretin. PMID:18191238

Applications of multiphoton microscopy in the field of colorectal cancer

NASA Astrophysics Data System (ADS)

Wang, Shu; Li, Lianhuang; Zhu, Xiaoqin; Zheng, Liqin; Zhuo, Shuangmu; Chen, Jianxin

2018-06-01

Multiphoton microscopy (MPM) is a powerful tool for visualizing cellular and subcellular details within living tissue by its unique advantages of being label-free, its intrinsic optical sectioning ability, near-infrared excitation for deep penetration depth into tissue, reduced photobleaching and phototoxicity in the out-of-focus regions, and being capable of providing quantitative information. In this review, we focus on applications of MPM in the field of colorectal cancer, including monitoring cancer progression, detecting tumor metastasis and microenvironment, evaluating the cancer therapy response, and visualizing and ablating pre-invasive cancer cells. We also present one of the major challenges and the future research direction to exploit a colorectal multiphoton endoscope.

Malignant lymphoma in a West Indian manatee (Trichechus manatus).

PubMed

Hammer, Anne S; Klausen, Bjarne; Knold, Steffen; Dietz, Hans H; Dutoit, Stephen J Hamilton

2005-10-01

We identified a malignant lymphoma infiltrating the lung, liver, kidney, mesenteric lymph nodes, and eye as the cause of death in a male West Indian manatee (Trichechus manatus). Diagnosis was based on gross, histopathologic, and immunohistochemical studies. Tissue samples from ten organs were included in a tissue microarray and sections from this array were subjected to immunohistochemical staining. The cytoplasm of the neoplastic lymphocytes identified in six organs was positive for CD3, a marker for T-cell differentiation. The neoplastic cells were negative for CD79alpha, a marker for B-cell differentiation. The cause of this neoplasm was not determined. This is the first report of malignant lymphoma in the mammal order Sirenia.

Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to nanodroplets.

PubMed

Zhu, Ying; Dou, Maowei; Piehowski, Paul D; Liang, Yiran; Wang, Fangjun; Chu, Rosalie K; Chrisler, Will; Smith, Jordan N; Schwarz, Kaitlynn C; Shen, Yufeng; Shukla, Anil K; Moore, Ronald J; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-06-24

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution due to the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 µm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-µm-thick rat brain cortex tissue sections with diameters of 50, 100, and 200 µm, respectively. We also analyzed 100-µm-diameter sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ~1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Histologic evaluation of autogenous connective tissue and acellular dermal matrix grafts in humans.

PubMed

Cummings, Lewis C; Kaldahl, Wayne B; Allen, Edward P

2005-02-01

The clinical success of root coverage with autogenous connective tissue (CT) or acellular dermal matrix (ADM) has been well documented. However, limited histological results of CT grafts have been reported, and a case report of a human block section has been published documenting an ADM graft. The purpose of this study is to document the histological results of CT grafts, ADM grafts, and coronally advanced flaps to cover denuded roots in humans. This study included four patients previously treatment planned for extractions of three or more anterior teeth. Three teeth in each patient were selected and randomly designated to receive either a CT or ADM graft beneath a coronally advanced flap (tests) or coronally advanced flap alone (control). Six months postoperatively block section extractions were performed and the teeth processed for histologic evaluation with hematoxylin-eosin and Verhoeff's stains. Histologically, both the CT and ADM were well incorporated within the recipient tissues. New fibroblasts, vascular elements, and collagen were present throughout the ADM, while retention of the transplanted elastic fibers was apparent. No effect on the keratinization or connective tissue organization of the overlying alveolar mucosa was evident with either graft. For both materials, areas of cemental deposition were present within the root notches, the alveolar bone was essentially unaffected, and the attachments to the root surfaces were similar. Although CT and ADM have a slightly different histological appearance, both can successfully be used to cover denuded roots with similar attachments and no adverse healing.

Evaluation of the biological response of wear debris.

PubMed

Chang, Bong-Soon; Brown, Phillip Rand; Sieber, Ann; Valdevit, Antonio; Tateno, Kei; Kostuik, John Philip

2004-01-01

An animal study was conducted to evaluate the biological response to titanium particles from an artificial intervertebral disc in terms of serology and histologic changes. To determine the biological response to wear debris in the retroperitoneal and epidural space. Few wear studies exist about mechanical artificial discs. Twenty-three New Zealand white rabbits were used for two approaches of the lumbar spine. In a retroperitoneal group (10 rabbits), lateral flank approach at the L2-L3 area was used. In an epidural group (13 rabbits), a dorsal laminotomy of L2 was performed. The wear debris was obtained from mechanical test cycling of the implantable intervertebral disc. At 4 and 12 weeks postoperatively, five or six animals from each group were killed. The tissues, including deposition site, regional lymph nodes and major organs, were evaluated with hematoxylin and eosin staining. At death all rabbits were found to be healthy. Blood results from the predeath samples were found to be consistent with the preoperative blood work values. Scar tissue was minimal with good healing. All organs were found to be normal in appearance. On histopathology sections, adverse reactions such as fibrosis, granuloma formation or necrosis were not found in any tissues. Free particles were found sparingly in all tissue sections with minimal cellular response. No remarkable difference was found according to groups or time intervals. Smaller particles were found to be engulfed in macrophages without adverse biological consequences. Titanium particles traveled from the sites of deposition but elicited no to minimal biological response.

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.

Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with end-stage equine recurrent uveitis.

PubMed

Pearce, Jacqueline W; Galle, Laurence E; Kleiboeker, Steve B; Turk, James R; Schommer, Susan K; Dubielizig, Richard R; Mitchell, William J; Moore, Cecil P; Giuliano, Elizabeth A

2007-11-01

Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.

The Isolation of Pure Populations of Neurons by Laser Capture Microdissection: Methods and Application in Neuroscience.

PubMed

Morris, Renée; Mehta, Prachi

2018-01-01

In mammals, the central nervous system (CNS) is constituted of various cellular elements, posing a challenge to isolating specific cell types to investigate their expression profile. As a result, tissue homogenization is not amenable to analyses of motor neurons profiling as these represent less than 10% of the total spinal cord cell population. One way to tackle the problem of tissue heterogeneity and obtain meaningful genomic, proteomic, and transcriptomic profiling is to use laser capture microdissection technology (LCM). In this chapter, we describe protocols for the capture of isolated populations of motor neurons from spinal cord tissue sections and for downstream transcriptomic analysis of motor neurons with RT-PCR. We have also included a protocol for the immunological confirmation that the captured neurons are indeed motor neurons. Although focused on spinal cord motor neurons, these protocols can be easily optimized for the isolation of any CNS neurons.

Generalized Fokker-Planck theory for electron and photon transport in biological tissues: application to radiotherapy.

PubMed

Olbrant, Edgar; Frank, Martin

2010-12-01

In this paper, we study a deterministic method for particle transport in biological tissues. The method is specifically developed for dose calculations in cancer therapy and for radiological imaging. Generalized Fokker-Planck (GFP) theory [Leakeas and Larsen, Nucl. Sci. Eng. 137 (2001), pp. 236-250] has been developed to improve the Fokker-Planck (FP) equation in cases where scattering is forward-peaked and where there is a sufficient amount of large-angle scattering. We compare grid-based numerical solutions to FP and GFP in realistic medical applications. First, electron dose calculations in heterogeneous parts of the human body are performed. Therefore, accurate electron scattering cross sections are included and their incorporation into our model is extensively described. Second, we solve GFP approximations of the radiative transport equation to investigate reflectance and transmittance of light in biological tissues. All results are compared with either Monte Carlo or discrete-ordinates transport solutions.

The morphology and histopathology of Nephridiacanthus major (Acanthocephala: Oligacanthorhynchidae) from hedgehogs in Iran.

PubMed

Heckmann, Richard A; Amin, Omar M; Halajian, Ali; El-Naggar, Atif M

2013-02-01

The morphology of Nephridiacanthus major (Bremser 1811 in Westrumb 1821) Golvan, 1962 collected from the long-eared hedgehog Hemiechinus auritus (Gmelin 1770) and the Eastern European hedgehog Erinaceus concolor Martin, 1838 (Erinaceidae) is described using SEM for the first time. This acanthocephalan was previously described from hedgehogs in Europe, Asia, and Africa. Measurements of specimens from Iran, Bulgaria, Germany, Central Asia, Morocco, and Egypt show considerable variations in the size of the trunk, proboscis, proboscis hooks and receptacle, and eggs. The SEM studies add new perspectives to its morphology. Features observed for the first time include the near terminal position and shape of the female gonopore and orifice, among others. Histopathological studies for this species are reported for the first time. Tissue sections show extensive damage near the proboscis with hemorrhaging and formation of collagenous connective tissue, compression of the intestinal mucosa, obstruction of intestinal lumen, and extensive necrosis of host epithelial tissue.

MicroRNAs and the metabolic hallmarks of aging.

PubMed

Victoria, Berta; Nunez Lopez, Yury O; Masternak, Michal M

2017-11-05

Aging, the natural process of growing older, is characterized by a progressive deterioration of physiological homeostasis at the cellular, tissue, and organismal level. Metabolically, the aging process is characterized by extensive changes in body composition, multi-tissue/multi-organ insulin resistance, and physiological declines in multiple signaling pathways including growth hormone, insulin/insulin-like growth factor 1, and sex steroids regulation. With this review, we intend to consolidate published information about microRNAs that regulate critical metabolic processes relevant to aging. In certain occasions we uncover relationships likely relevant to aging, which has not been directly described before, such as the miR-451/AMPK axis. We have also included a provocative section highlighting the potential role in aging of a new designation of miRNAs, namely fecal miRNAs, recently discovered to regulate intestinal microbiota in mammals. Copyright © 2016. Published by Elsevier B.V.

Application of SEM and EDX in studying biomineralization in plant tissues.

PubMed

He, Honghua; Kirilak, Yaowanuj

2014-01-01

This chapter describes protocols using formalin-acetic acid-alcohol (FAA) to fix plant tissues for studying biomineralization by means of scanning electron microscopy (SEM) and qualitative energy-dispersive X-ray microanalysis (EDX). Specimen preparation protocols for SEM and EDX mainly include fixation, dehydration, critical point drying (CPD), mounting, and coating. Gold-coated specimens are used for SEM imaging, while gold- and carbon-coated specimens are prepared for qualitative X-ray microanalyses separately to obtain complementary information on the elemental compositions of biominerals. During the specimen preparation procedure for SEM, some biominerals may be dislodged or scattered, making it difficult to determine their accurate locations, and light microscopy is used to complement SEM studies. Specimen preparation protocols for light microscopy generally include fixation, dehydration, infiltration and embedding with resin, microtome sectioning, and staining. In addition, microwave processing methods are adopted here to speed up the specimen preparation process for both SEM and light microscopy.

Optical properties of cells with melanin

NASA Astrophysics Data System (ADS)

Rohde, Barukh; Coats, Israel; Krueger, James; Gareau, Dan

2014-02-01

The optical properties of pigmented lesions have been studied using diffuse reflectance spectroscopy in a noninvasive configuration on optically thick samples such as skin in vivo. However, it is difficult to un-mix the effects of absorption and scattering with diffuse reflectance spectroscopy techniques due to the complex anatomical distributions of absorbing and scattering biomolecules. We present a device and technique that enables absorption and scattering measurements of tissue volumes much smaller than the optical mean-free path. Because these measurements are taken on fresh-frozen sections, they are direct measurements of the optical properties of tissue, albeit in a different hydration state than in vivo tissue. Our results on lesions from 20 patients including melanomas and nevi show the absorption spectrum of melanin in melanocytes and basal keratinocytes. Our samples consisted of fresh frozen sections that were unstained. Fitting the spectrum as an exponential decay between 500 and 1100 nm [mua = A*exp(-B*(lambda-C)) + D], we report on the fit parameters of and their variation due to biological heterogeneity as A = 4.20e4 +/- 1.57e5 [1/cm], B = 4.57e-3 +/- 1.62e-3 [1/nm], C = 210 +/- 510 [nm] , D = 613 +/- 534 [1/cm]. The variability in these results is likely due to highly heterogeneous distributions of eumelanin and pheomelanin.

Simulation of a fast diffuse optical tomography system based on radiative transfer equation

NASA Astrophysics Data System (ADS)

Motevalli, S. M.; Payani, A.

2016-12-01

Studies show that near-infrared (NIR) light (light with wavelength between 700nm and 1300nm) undergoes two interactions, absorption and scattering, when it penetrates a tissue. Since scattering is the predominant interaction, the calculation of light distribution in the tissue and the image reconstruction of absorption and scattering coefficients are very complicated. Some analytical and numerical methods, such as radiative transport equation and Monte Carlo method, have been used for the simulation of light penetration in tissue. Recently, some investigators in the world have tried to develop a diffuse optical tomography system. In these systems, NIR light penetrates the tissue and passes through the tissue. Then, light exiting the tissue is measured by NIR detectors placed around the tissue. These data are collected from all the detectors and transferred to the computational parts (including hardware and software), which make a cross-sectional image of the tissue after performing some computational processes. In this paper, the results of the simulation of an optical diffuse tomography system are presented. This simulation involves two stages: a) Simulation of the forward problem (or light penetration in the tissue), which is performed by solving the diffusion approximation equation in the stationary state using FEM. b) Simulation of the inverse problem (or image reconstruction), which is performed by the optimization algorithm called Broyden quasi-Newton. This method of image reconstruction is faster compared to the other Newton-based optimization algorithms, such as the Levenberg-Marquardt one.

Spatiotemporal modeling of laser tissue soldering using photothermal nanocomposites.

PubMed

Mushaben, Madaline; Urie, Russell; Flake, Tanner; Jaffe, Michael; Rege, Kaushal; Heys, Jeffrey

2018-02-01

Laser tissue soldering using photothermal solders is a technology that facilitates rapid sealing using heat-induced changes in the tissue and the solder material. The solder material is made of gold nanorods embedded in a protein matrix patch that can be placed over the tissue rupture site and heated with a laser. Although laser tissue soldering is an attractive approach for surgical repair, potential photothermal damage can limit the success of this approach. Development of predictive mathematical models of photothermal effects including cell death, can lead to more efficient approaches in laser-based tissue repair. We describe an experimental and modeling investigation into photothermal solder patches for sealing porcine and mouse cadaver intestine sections using near-infrared laser irradiation. Spatiotemporal changes in temperature were determined at the surface as well as various depths below the patch. A mathematical model, based on the finite element method, predicts the spatiotemporal temperature distribution in the patch and surrounding tissue, as well as concomitant cell death in the tissue is described. For both the porcine and mouse intestine systems, the model predicts temperatures that are quantitatively similar to the experimental measurements with the model predictions of temperature increase often being within a just a few degrees of experimental measurements. This mathematical model can be employed to identify optimal conditions for minimizing healthy cell death while still achieving a strong seal of the ruptured tissue using laser soldering. Lasers Surg. Med. 50:143-152, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

Comparison of immunohistochemistry and polymerase chain reaction for detection of West Nile virus in naturally infected dead birds.

PubMed

Sandhu, Tejbir; Sidhu, Dalbinder; Dhillon, Major; Fang, Ying

2010-10-04

Credible vector-borne disease surveillance programs, especially in developing countries with limited resources, must include diagnostic tests that are efficient, inexpensive and simple and safe to administer while maintaining high levels of sensitivity and specificity. Since immunohistochemistry (IHC) includes most of these features, its sensitivity, specificity, predictive positive value (PPV) and predictive negative value (PNV) for West Nile virus (WNv) screening were compared to those of the gold standard, RT-PCR testing of kidney tissue in dead birds. IHC and RT-PCR were performed for WNv antigen on 41 dead birds (belonging to five orders) collected from the northwest region of the Riverside County of California. Fixed tissue sections were screened by IHC using polyclonal antibodies, and frozen kidney tissues were tested with RT-PCR. Kidney screening with IHC showed sensitivity, specificity, PPV and NPV of 95.45%, 73.68%, 80.77% and 93.33%, respectively. Based on WNv screening of kidney tissue, IHC and RT-PCR were in agreement with 95.45% (21/22) for positive dead birds and were in 100% (22/22) agreement when multi-organ screening by IHC was performed. The present study showed that IHC is as equally effective as RT-PCR in screening for WNv in dead birds. Therefore, IHC can effectively serve as a competent screening technique for those disease surveillance agencies that lack expensive RT-PCR technology while promoting safer biohazardous conditions, except at the initial stage of tissue collection.

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.

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.

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.

Relationships between root respiration rate and root morphology, chemistry and anatomy in Larix gmelinii and Fraxinus mandshurica.

PubMed

Jia, Shuxia; McLaughlin, Neil B; Gu, Jiacun; Li, Xingpeng; Wang, Zhengquan

2013-06-01

Tree roots are highly heterogeneous in form and function. Previous studies revealed that fine root respiration was related to root morphology, tissue nitrogen (N) concentration and temperature, and varied with both soil depth and season. The underlying mechanisms governing the relationship between root respiration and root morphology, chemistry and anatomy along the root branch order have not been addressed. Here, we examined these relationships of the first- to fifth-order roots for near surface roots (0-10 cm) of 22-year-old larch (Larix gmelinii L.) and ash (Fraxinus mandshurica L.) plantations. Root respiration rate at 18 °C was measured by gas phase O2 electrodes across the first five branching order roots (the distal roots numbered as first order) at three times of the year. Root parameters of root diameter, specific root length (SRL), tissue N concentration, total non-structural carbohydrates (starch and soluble sugar) concentration (TNC), cortical thickness and stele diameter were also measured concurrently. With increasing root order, root diameter, TNC and the ratio of root TNC to tissue N concentration increased, while the SRL, tissue N concentration and cortical proportion decreased. Root respiration rate also monotonically decreased with increasing root order in both species. Cortical tissue (including exodermis, cortical parenchyma and endodermis) was present in the first three order roots, and cross sections of the cortex for the first-order root accounted for 68% (larch) and 86% (ash) of the total cross section of the root. Root respiration was closely related to root traits such as diameter, SRL, tissue N concentration, root TNC : tissue N ratio and stele-to-root diameter proportion among the first five orders, which explained up to 81-94% of variation in the rate of root respiration for larch and up to 83-93% for ash. These results suggest that the systematic variations of root respiration rate within tree fine root system are possibly due to the changes of tissue N concentration and anatomical structure along root branch orders in both tree species, which provide deeper understanding in the mechanism of how root traits affect root respiration in woody plants.

Method for procuring specific populations of viable human prostate cells for research.

PubMed

Fischer, A H; Philips, A; Taysavang, P; McKenney, J K; Amin, M B

2001-04-01

A wider range of research can be conducted on viable tissue samples than on fixed or frozen samples. A major obstacle to studying viable prostate tissue samples is the inability to accurately identify cancer on direct examination of unembedded tissue. We used a dissecting microscope to identify cancer in unfixed prostate tissue samples stained on the cut surface with 0.5% aqueous toluidine blue. We measured the diagnostic accuracy of this technique in 25 consecutive prostatectomies, determined the viability of procured samples, and estimated the effect on final pathologic assessment. Both surfaces of a 3- to 5-mm thick cross-section taken midway between base and apex of the prostate were examined. A 4-mm punch biopsy was directed to one benign and one malignant area when clearly present. The dissecting microscope allowed clearcut recognition of carcinoma in 17 of the 25 cross-sections, and carcinoma was confirmed in all 17 (100%). In 8 of 25 cases, no procurement was attempted because no carcinoma was evident in the one cross-section studied. Twenty of 25 cross-sections were adequate for benign tissue procurement; five of the cross-sections were not suitable for procurement because of the presence of extensive carcinoma or atrophy. Seventeen of the 20 were accurately diagnosed as benign (85%); one showed pseudohyperplastic adenocarcinoma, one showed focal high-grade prostatic intraepithelial neoplasia, and one showed urothelial carcinoma in situ. Prostatic epithelium obtained with the technique remains viable and can be separated from stroma. The dissecting microscope technique appears to facilitate rather than interfere with accurate pathologic assessment: extraprostatic extension or positive margins were correctly identified during tissue procurement in three cases. The procedure takes only about 30 minutes.

Micro-CT scouting for transmission electron microscopy of human tissue specimens

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

Morales, A. G.; Stempinski, E. S.; XIAO, X.

Transmission electron microscopy (TEM) provides sub-nanometre-scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. Here, we describe micro-CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench-top micro-CT scanner with 10 m resolution was used to determine the location of patches of themore » mucous membrane in osmium-stained human nasal scraping samples. Furthermore, once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra-thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation. Lay description Electron microscopy provides very high levels of detail in a small area, and thus the question of where to look in an opaque sample, such as a stained tissue specimen, needs to be answered by sectioning the sample in small steps and examining the sections under a light microscope, until the region of interest is found. The search process can be lengthy and labor intensive, especially for a study involving a large number of samples. Small areas of interest can be missed in the process if not enough regions are examined. We also describe a method to directly locate the region of interest within a whole sample using micro-CT imaging, bypassing the need of blindly sectioning. Micro-CT enables locating the region within 3D space; this information provides a guide for sectioning the sample to expose that precise location for high resolution electron microscopy imaging. In a human tissue specimen study, this method considerably reduced the time and labor of the search process.« less

Micro-CT scouting for transmission electron microscopy of human tissue specimens

DOE PAGES

Morales, A. G.; Stempinski, E. S.; XIAO, X.; ...

2016-02-08

Transmission electron microscopy (TEM) provides sub-nanometre-scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. Here, we describe micro-CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench-top micro-CT scanner with 10 m resolution was used to determine the location of patches of themore » mucous membrane in osmium-stained human nasal scraping samples. Furthermore, once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra-thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation. Lay description Electron microscopy provides very high levels of detail in a small area, and thus the question of where to look in an opaque sample, such as a stained tissue specimen, needs to be answered by sectioning the sample in small steps and examining the sections under a light microscope, until the region of interest is found. The search process can be lengthy and labor intensive, especially for a study involving a large number of samples. Small areas of interest can be missed in the process if not enough regions are examined. We also describe a method to directly locate the region of interest within a whole sample using micro-CT imaging, bypassing the need of blindly sectioning. Micro-CT enables locating the region within 3D space; this information provides a guide for sectioning the sample to expose that precise location for high resolution electron microscopy imaging. In a human tissue specimen study, this method considerably reduced the time and labor of the search process.« less

38 CFR 1.460 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... with an Organ Procurement Organization, eye bank or tissue bank with written, detailed responsibilities...-being of the user. Eye bank and tissue bank. The term “eye bank and tissue bank” means an “establishment... bank, and/or tissue bank as defined in this section. Records. The term “records” means any information...

38 CFR 1.460 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... with an Organ Procurement Organization, eye bank or tissue bank with written, detailed responsibilities...-being of the user. Eye bank and tissue bank. The term “eye bank and tissue bank” means an “establishment... bank, and/or tissue bank as defined in this section. Records. The term “records” means any information...

38 CFR 1.460 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... with an Organ Procurement Organization, eye bank or tissue bank with written, detailed responsibilities...-being of the user. Eye bank and tissue bank. The term “eye bank and tissue bank” means an “establishment... bank, and/or tissue bank as defined in this section. Records. The term “records” means any information...

38 CFR 1.460 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... with an Organ Procurement Organization, eye bank or tissue bank with written, detailed responsibilities...-being of the user. Eye bank and tissue bank. The term “eye bank and tissue bank” means an “establishment... bank, and/or tissue bank as defined in this section. Records. The term “records” means any information...

Alternative to xylene as a clearing agent in histopathology

PubMed Central

Alwahaibi, Nasar; Aljaradi, Shaima; Alazri, Horiyah

2018-01-01

INTRODUCTION: Clearing is an essential step in processing tissue for light microscopy. Xylene is the clearing agent used most commonly worldwide. Xylene is toxic and therefore a threat to personnel working in histopathology laboratories. We evaluated a safer alternative clearing agent for use in the histopathology laboratory. MATERIALS AND METHODS: We used 230 formalin-fixed, paraffin-embedded tissue blocks from 19 different tissues. Half of the specimens were processed using xylene and half were processed using UltraClear™. Tissues were evaluated for eight parameters: sectioning, nuclear staining, cytoplasmic staining, overall cell morphology, clarity of staining, uniformity of staining, quality of immunohistochemistry (IHC), and cost. RESULTS: Both UltraClear™ and xylene processed sections scored 100% for IHC. Sections processed using UltraClear™ were easy to cut (81.7%) as were xylene processed sections (96.5%). UltraClear™ processed sections showed 67%, 60.9%, 52.2%, 63.5%, and 67% for nuclear staining, cytoplasmic staining, cell morphology, clarity of staining, and uniformity of staining, respectively. UltraClear™ is twice as expensive as xylene. We found that tissues processed using UltraClear™ were easy to cut and worked well for both hematoxylin and eosin and IHC staining. CONCLUSION: UltraClear™ is less toxic, less flammable, friendlier to the environment, and easy to handle, but it is two times expensive than xylene. The findings of this study recommend the use of UltraClear™ solution as a routine clearing agent in histopathology laboratories. However, further studies are required. PMID:29692586

DALMATIAN: An Algorithm for Automatic Cell Detection and Counting in 3D.

PubMed

Shuvaev, Sergey A; Lazutkin, Alexander A; Kedrov, Alexander V; Anokhin, Konstantin V; Enikolopov, Grigori N; Koulakov, Alexei A

2017-01-01

Current 3D imaging methods, including optical projection tomography, light-sheet microscopy, block-face imaging, and serial two photon tomography enable visualization of large samples of biological tissue. Large volumes of data obtained at high resolution require development of automatic image processing techniques, such as algorithms for automatic cell detection or, more generally, point-like object detection. Current approaches to automated cell detection suffer from difficulties originating from detection of particular cell types, cell populations of different brightness, non-uniformly stained, and overlapping cells. In this study, we present a set of algorithms for robust automatic cell detection in 3D. Our algorithms are suitable for, but not limited to, whole brain regions and individual brain sections. We used watershed procedure to split regional maxima representing overlapping cells. We developed a bootstrap Gaussian fit procedure to evaluate the statistical significance of detected cells. We compared cell detection quality of our algorithm and other software using 42 samples, representing 6 staining and imaging techniques. The results provided by our algorithm matched manual expert quantification with signal-to-noise dependent confidence, including samples with cells of different brightness, non-uniformly stained, and overlapping cells for whole brain regions and individual tissue sections. Our algorithm provided the best cell detection quality among tested free and commercial software.

A new laser reflectance system capable of measuring changing cross-sectional area of soft tissues during tensile testing.

PubMed

Pokhai, Gabriel G; Oliver, Michele L; Gordon, Karen D

2009-09-01

Determination of the biomechanical properties of soft tissues such as tendons and ligaments is dependent on the accurate measurement of their cross-sectional area (CSA). Measurement methods, which involve contact with the specimen, are problematic because soft tissues are easily deformed. Noncontact measurement methods are preferable in this regard, but may experience difficulty in dealing with the complex cross-sectional shapes and glistening surfaces seen in soft tissues. Additionally, existing CSA measurement systems are separated from the materials testing machine, resulting in the inability to measure CSA during testing. Furthermore, CSA measurements are usually made in a different orientation, and with a different preload, prior to testing. To overcome these problems, a noncontact laser reflectance system (LRS) was developed. Designed to fit in an Instron 8872 servohydraulic test machine, the system measures CSA by orbiting a laser transducer in a circular path around a soft tissue specimen held by tissue clamps. CSA measurements can be conducted before and during tensile testing. The system was validated using machined metallic specimens of various shapes and sizes, as well as different sizes of bovine tendons. The metallic specimens could be measured to within 4% accuracy, and the tendons to within an average error of 4.3%. Statistical analyses showed no significant differences between the measurements of the LRS and those of the casting method, an established measurement technique. The LRS was successfully used to measure the changing CSA of bovine tendons during uniaxial tensile testing. The LRS developed in this work represents a simple, quick, and accurate way of reconstructing complex cross-sectional profiles and calculating cross-sectional areas. In addition, the LRS represents the first system capable of automatically measuring changing CSA of soft tissues during tensile testing, facilitating the calculation of more accurate biomechanical properties.

Raman spectroscopy imaging reveals interplay between atherosclerosis and medial calcification in the human aorta

PubMed Central

You, Amanda Y. F.; Bergholt, Mads S.; St-Pierre, Jean-Philippe; Kit-Anan, Worrapong; Pence, Isaac J.; Chester, Adrian H.; Yacoub, Magdi H.; Bertazzo, Sergio; Stevens, Molly M.

2017-01-01

Medial calcification in the human aorta accumulates during aging and is known to be aggravated in several diseases. Atherosclerosis, another major cause of cardiovascular calcification, shares some common aggravators. However, the mechanisms of cardiovascular calcification remain poorly understood. To elucidate the relationship between medial aortic calcification and atherosclerosis, we characterized the cross-sectional distributions of the predominant minerals in aortic tissue, apatite and whitlockite, and the associated extracellular matrix. We also compared the cellular changes between atherosclerotic and nonatherosclerotic human aortic tissues. This was achieved through the development of Raman spectroscopy imaging methods that adapted algorithms to distinguish between the major biomolecules present within these tissues. We present a relationship between apatite, cholesterol, and triglyceride in atherosclerosis, with the relative amount of all molecules concurrently increased in the atherosclerotic plaque. Further, the increase in apatite was disproportionately large in relation to whitlockite in the aortic media directly underlying a plaque, indicating that apatite is more pathologically significant in atherosclerosis-aggravated medial calcification. We also discovered a reduction of β-carotene in the whole aortic intima, including a plaque in atherosclerotic aortic tissues compared to nonatherosclerotic tissues. This unprecedented biomolecular characterization of the aortic tissue furthers our understanding of pathological and physiological cardiovascular calcification events in humans. PMID:29226241

Acridine orange--its use in the specific staining of DNA in mammalian tissue sections.

PubMed

Dutt, M K

1981-01-01

This paper reports on a new method for the use of acridine orange (AO) in an aqueous solution at pH 4.5 for staining DNA of rat tissue sections from which RNA has been extracted selectively with cold phosphoric acid. Not only this, AO can also be used as dye-SO2 reagent, prepared with NHCl and potassium metabisulphite, for staining DNA-aldehyde molecules of acid-hydrolysed tissue sections. AO samples, manufactured by the National Aniline Division as well as by G. T. Gurr have been used with equal success. Studies of stained sections under light microscope reveal the presence of specifically stained yellowish-orange nuclei. Those sections under fluorescent microscope with proper exciter and barrier filters reveal nuclei of maroon colour. The in situ absorption spectra of nuclei stained with AO-SO2 following acid-hydrolysis of tissue sections as well as those of nuclei stained with an aqueous solution of the dye following extraction of RNA have been presented herein. The mode of binding in the former case has been considered to be due to binding of the teritary amino group of the dye molecules with the DNA-aldehyde molecules and in the latter case to be due to electrostatic binding between the positively charged dye molecules with negatively charged phosphate groups of DNA. Implications of all these findings have been discussed.

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.

The pars intermedia: an anatomic basis for a coordinated vascular response to female genital arousal.

PubMed

Shih, Cheryl; Cold, Christopher J; Yang, Claire C

2013-06-01

The pars intermedia is an area of the vulva that has been inconsistently described in the literature. We conducted anatomic studies to better describe the tissues and vascular structures of the pars intermedia and proposed a functional rationale of the pars intermedia in the female sexual response. Nine cadaveric vulvectomy specimens were used. Each was serially sectioned and stained with hematoxylin and eosin and Masson's trichrome. Histologic ultrastructural description of the pars intermedia. The pars intermedia contains veins traveling longitudinally in the angle of the clitoris, supported by collagen-rich stromal tissues. These veins drain the different vascular compartments of the vulva, including the clitoris, the bulbs, and labia minora; also, the interconnecting veins link the different vascular compartments. The pars intermedia is not composed of erectile tissue, distinguishing it from the erectile tissues of the corpora cavernosa of the clitoris as well as the corpus spongiosum of the clitoral (vestibular) bulbs. The venous communications of the pars intermedia, linking the erectile tissues with the other vascular compartments of the vulva, appear to provide the anatomic basis for a coordinated vascular response during female sexual arousal. © 2012 International Society for Sexual Medicine.

Disaccharides Protect Antigens from Drying-Induced Damage in Routinely Processed Tissue Sections

PubMed Central

Boi, Giovanna; Scalia, Carla Rossana; Gendusa, Rossella; Ronchi, Susanna; Cattoretti, Giorgio

2015-01-01

Drying of the tissue section, partial or total, during immunostaining negatively affects both the staining of tissue antigens and the ability to remove previously deposited antibody layers, particularly during sequential rounds of de-staining and re-staining for multiple antigens. The cause is a progressive loss of the protein-associated water up to the removal of the non-freezable water, a step which abolishes the immunoavailability of the epitope. In order to describe and prevent these adverse effects, we tested, among other substances, sugars, which are known to protect unicellular organisms from freezing and dehydration, and stabilize drugs and reagents in solid state form in medical devices. Disaccharides (lactose, sucrose) prevented the air drying-induced antigen masking and protected tissue-bound antigens and antibodies from air drying-induced damage. Complete removal of the bound antibody layers by chemical stripping was permitted if lactose was present during air drying. Lactose, sucrose and other disaccharides prevent air drying artifacts, allow homogeneous, consistent staining and the reuse of formalin-fixed, paraffin-embedded tissue sections for repeated immunostaining rounds by guaranteeing constant staining quality in suboptimal hydration conditions. PMID:26487185

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.

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.

Prevalence of Soft Tissue Calcifications in CBCT Images of Mandibular Region

PubMed Central

Khojastepour, Leila; Haghnegahdar, Abdolaziz; Sayar, Hamed

2017-01-01

Statement of the Problem: Most of the soft tissue calcifications within the head and neck region might not be accompanied by clinical symptoms but may indicate some pathological conditions. Purpose: The aim of this research was to determine the prevalence of soft tissue calcifications in cone beam computed tomography (CBCT) images of mandibular region. Materials and Method: In this cross sectional study the CBCT images of 602 patients including 294 men and 308 women with mean age 41.38±15.18 years were evaluated regarding the presence, anatomical location; type (single or multiple) and size of soft tissue calcification in mandibular region. All CBCT images were acquired by NewTom VGi scanner. Odds ratio and chi-square tests were used for data analysis and p< 0.05 was considered to be statistically significant. Results: 156 out of 602 patients had at least one soft tissue calcification in their mandibular region (25.9%. of studied population with mean age 51.7±18.03 years). Men showed significantly higher rate of soft tissue calcification than women (30.3% vs. 21.8%). Soft tissue calcification was predominantly seen at posterior region of the mandible (88%) and most of them were single (60.7%). The prevalence of soft tissue calcification increased with age. Most of the detected soft tissue calcifications were smaller than 3mm (90%). Conclusion: Soft tissue calcifications in mandibular area were a relatively common finding especially in posterior region and more likely to happen in men and in older age group. PMID:28620632

Characterization of human breast cancer by scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Chen, Di; Malyarenko, Eugene; Seviaryn, Fedar; Yuan, Ye; Sherman, Mark; Bandyopadhyay, Sudeshna; Gierach, Gretchen; Greenway, Christopher W.; Maeva, Elena; Strumban, Emil; Duric, Neb; Maev, Roman

2013-03-01

Objectives: The purpose of this study was to characterize human breast cancer tissues by the measurement of microacoustic properties. Methods: We investigated eight breast cancer patients using acoustic microscopy. For each patient, seven blocks of tumor tissue were collected from seven different positions around a tumor mass. Frozen sections (10 micrometer, μm) of human breast cancer tissues without staining and fixation were examined in a scanning acoustic microscope with focused transducers at 80 and 200 MHz. Hematoxylin and Eosin (H and E) stained sections from the same frozen breast cancer tissues were imaged by optical microscopy for comparison. Results: The results of acoustic imaging showed that acoustic attenuation and sound speed in cancer cell-rich tissue regions were significantly decreased compared with the surrounding tissue regions, where most components are normal cells/tissues, such as fibroblasts, connective tissue and lymphocytes. Our observation also showed that the ultrasonic properties were influenced by arrangements of cells and tissue patterns. Conclusions: Our data demonstrate that attenuation and sound speed imaging can provide biomechanical information of the tumor and normal tissues. The results also demonstrate the potential of acoustic microscopy as an auxiliary method for operative detection and localization of cancer affected regions.

Cross-sectional evaluation of the prevalence and factors associated with soft tissue scarring after the removal of miniscrews.

PubMed

Jung, Sung-ah; Choi, Yoon Jeong; Lee, Dong-Won; Kim, Kyung-Ho; Chung, Chooryung J

2015-05-01

To investigate the prevalence of distinguishable soft tissue scarring after the removal of temporary anchorage devices (TADs) such as orthodontic miniscrews and to analyze the factors associated with scar formation. The prevalence of soft tissue scarring in 66 patients (202 miniscrew removal sites) was clinically investigated at least 1 year after miniscrew removal. To determine the clinical factors associated with soft tissue scar formation, miniscrew stability; host factors including age, gender, and gingival biotype; and miniscrew-related factors such as insertion site, vertical position, and insertion period were evaluated. The prevalence of a distinguishable scar remaining at least 1 year after miniscrew removal was 44.6%. Patients with flat gingiva showed a significantly higher prevalence of soft tissue scar formation than did those with pronounced scalloped gingiva (P < .05). Maxillary buccal removal sites showed a significantly higher prevalence of soft tissue scar formation than did those in the mandible or palatal slope (P < .05). Miniscrew sites at the alveolar mucosa showed a significantly lower prevalence of soft tissue scar formation than did those in the mucogingival junction or the attached gingiva (P < .01). The prevalence of distinguishable scarring after miniscrew removal was fairly high. On the basis of our results, patients with flat gingiva and buccal interdental gingival insertion sites are more susceptible to scar formation.

Common cellular events occur during wound healing and organ regeneration in the sea cucumber Holothuria glaberrima.

PubMed

San Miguel-Ruiz, José E; García-Arrarás, José E

2007-10-18

All animals possess some type of tissue repair mechanism. In some species, the capacity to repair tissues is limited to the healing of wounds. Other species, such as echinoderms, posses a striking repair capability that can include the replacement of entire organs. It has been reported that some mechanisms, namely extracellular matrix remodeling, appear to occur in most repair processes. However, it remains unclear to what extent the process of organ regeneration, particularly in animals where loss and regeneration of complex structures is a programmed natural event, is similar to wound healing. We have now used the sea cucumber Holothuria glaberrima to address this question. Animals were lesioned by making a 3-5 mm transverse incision between one of the longitudinal muscle pairs along the bodywall. Lesioned tissues included muscle, nerve, water canal and dermis. Animals were allowed to heal for up to four weeks (2, 6, 12, 20, and 28 days post-injury) before sacrificed. Tissues were sectioned in a cryostat and changes in cellular and tissue elements during repair were evaluated using classical dyes, immmuohistochemistry and phalloidin labeling. In addition, the temporal and spatial distribution of cell proliferation in the animals was assayed using BrdU incorporation. We found that cellular events associated with wound healing in H. glaberrima correspond to those previously shown to occur during intestinal regeneration. These include: (1) an increase in the number of spherule-containing cells, (2) remodeling of the extracellular matrix, (3) formation of spindle-like structures that signal dedifferentiation of muscle cells in the area flanking the lesion site and (4) intense cellular division occurring mainly in the coelomic epithelium after the first week of regeneration. Our data indicate that H. glaberrima employs analogous cellular mechanisms during wound healing and organ regeneration. Thus, it is possible that regenerative limitations in some organisms are due either to the absence of particular mechanisms associated with repair or the inability of activating the repair process in some tissues or stages.

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.

Comparison of fixation and processing methods for hairless guinea pig skin following sulfur mustard exposure. (Reannouncement with new availability information)

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

Bryant, M.A.; Braue Jr, E.H.

1992-12-31

Ten anesthetized hairless guinea pigs Crl:IAF(HA)BR were exposed to 10 pi of neat sulfur mustard (HD) in a vapor cup on their skin for 7 min. At 24 h postexposure, the guinea pigs were euthanatized and skin sections taken for histologic evaluation. The skin was fixed using either 10% neutral buffered formalin (NBF), McDowell Trump fixative (4CF-IG), Zenker`s formol-saline (Helly`s fluid), or Zenker`s fluid. Fixed skin sections were cut in half: one half was embedded in paraffin and the other half in plastic (glycol methacrylate). Paraffin-embedded tissue was stained with hematoxylin and eosin; plastic-embedded tissue was stained with Lee`s methylenemore » blue basic fuchsin. Skin was also frozen unfixed, sectioned by cryostat, and stained with pinacyanole. HD-exposed skin was evaluated histologically for the presence of epidermal and follicular necrosis, microblister formation, epidermitis, and intracellular edema to determine the optimal fixation and embedding method for lesion preservation. The percentage of histologic sections with lesions varied little between fixatives and was similar for both paraffin and plastic embedding material. Plastic-embedded sections were thinner, allowing better histologic evaluation, but were more difficult to stain. Plastic embedding material did not infiltrate tissue fixed in Zenker`s fluid or Zenker`s formol-saline. Frozen tissue sections were prepared in the least processing time and lesion preservation was comparable to fixed tissue. It was concluded that standard histologic processing using formalin fixation and paraffin embedding is adequate for routine histopathological evaluation of HD skin lesions in the hairless guinea pig.... Sulfur mustard, Vesicating agents, Pathology, Hairless guinea pig model, Fixation.« less

Microscopic fluorescence spectral analysis of basal cell carcinomas

NASA Astrophysics Data System (ADS)

He, Qingli; Lui, Harvey; Zloty, David; Cowan, Bryce; Warshawski, Larry; McLean, David I.; Zeng, Haishan

2007-05-01

Background and Objectives. Laser-induced autofluorescence (LIAF) is a promising tool for cancer diagnosis. This method is based on the differences in autofluorescence spectra between normal and cancerous tissues, but the underlined mechanisms are not well understood. The objective of this research is to study the microscopic origins and intrinsic fluorescence properties of basal cell carcinoma (BCC) for better understanding of the mechanism of in vivo fluorescence detection and margin delineation of BCCs on skin patients. A home-made micro- spectrophotometer (MSP) system was used to image the fluorophore distribution and to measure the fluorescence spectra of various microscopic structures and regions on frozen tissue sections. Materials and Methods. BCC tissue samples were obtained from 14 patients undergoing surgical resections. After surgical removal, each tissue sample was immediately embedded in OCT medium and snap-frozen in liquid nitrogen. The frozen tissue block was then cut into 16-μm thickness sections using a cryostat microtome and placed on microscopic glass slides. The sections for fluorescence study were kept unstained and unfixed, and then analyzed by the MSP system. The adjacent tissue sections were H&E stained for histopathological examination and also served to help identify various microstructures on the adjacent unstained sections. The MSP system has all the functions of a conventional microscope, plus the ability of performing spectral analysis on selected micro-areas of a microscopic sample. For tissue fluorescence analysis, 442nm He-Cd laser light is used to illuminate and excite the unstained tissue sections. A 473-nm long pass filter was inserted behind the microscope objective to block the transmitted laser light while passing longer wavelength fluorescence signal. The fluorescence image of the sample can be viewed through the eyepieces and also recorded by a CCD camera. An optical fiber is mounted onto the image plane of the photograph port of the microscope to collect light from a specific micro area of the sample. The collected light is transmitted via the fiber to a disperserve type CCD spectrometer for spectral analysis. Results. The measurement results showed significant spectral differences between normal and cancerous tissues. For normal tissue regions, the spectral results agreed with our previous findings on autofluorescence of normal skin sections. For the cancerous regions, the epidermis showed very weak fluorescence signal, while the stratum corneum exhibited fluorescence emissions peaking at about 510 nm. In the dermis, the basal cell island and a band of surrounding areas showed very weak fluorescence signal, while distal dermis above and below the basal cell island showed greater fluorescence signal but with different spectral shapes. The very weak autofluorescence from the basal cell island and its surrounding area may be attributed to their degenerative properties that limited the production of collagens. Conclusions. The obtained microscopic results very well explain the in vivo fluorescence properties of BCC lesions in that they have decreased fluorescence intensity compared to the surrounding normal skin. The intrinsic spectra of various microstructures and the microscopic fluorescence images (corresponding fluorophore distribution in tissue) obtained in this study will be used for further theoretical modeling of in vivo fluorescence spectroscopy and imaging of skin cancers.

Quantitative Analysis of Immunohistochemistry in Melanoma Tumors

PubMed Central

Lilyquist, Jenna; White, Kirsten Anne Meyer; Lee, Rebecca J.; Philips, Genevieve K.; Hughes, Christopher R.; Torres, Salina M.

2017-01-01

Abstract Identification of positive staining is often qualitative and subjective. This is particularly troublesome in pigmented melanoma lesions, because melanin is difficult to distinguish from the brown stain resulting from immunohistochemistry (IHC) using horse radish peroxidase developed with 3,3′-Diaminobenzidine (HRP-DAB). We sought to identify and quantify positive staining, particularly in melanoma lesions. We visualized G-protein coupled estrogen receptor (GPER) expression developed with HRP-DAB and counterstained with Azure B (stains melanin) in melanoma tissue sections (n = 3). Matched sections (n = 3), along with 22 unmatched sections, were stained only with Azure B as a control. Breast tissue (n = 1) was used as a positive HRP-DAB control. Images of the stained tissues were generated using a Nuance Spectral Imaging Camera. Analysis of the images was performed using the Nuance Spectral Imaging software and SlideBook. Data was analyzed using a Kruskal–Wallis one way analysis of variance (ANOVA). We showed that a pigmented melanoma tissue doubly stained with anti-GPER HRP-DAB and Azure B can be unmixed using spectra derived from a matched, Azure B-only section, and an anti-GPER HRP-DAB control. We unmixed each of the melanoma lesions using each of the Azure B spectra, evaluated the mean intensity of positive staining, and examined the distribution of the mean intensities (P = .73; Kruskal–Wallis). These results suggest that this method does not require a matched Azure B-only stained control tissue for every melanoma lesion, allowing precious tissues to be conserved for other studies. Importantly, this quantification method reduces the subjectivity of protein expression analysis, and provides a valuable tool for accurate evaluation, particularly for pigmented tissues. PMID:28403073

Zooming in: high resolution 3D reconstruction of differently stained histological whole slide images

NASA Astrophysics Data System (ADS)

Lotz, Johannes; Berger, Judith; Müller, Benedikt; Breuhahn, Kai; Grabe, Niels; Heldmann, Stefan; Homeyer, André; Lahrmann, Bernd; Laue, Hendrik; Olesch, Janine; Schwier, Michael; Sedlaczek, Oliver; Warth, Arne

2014-03-01

Much insight into metabolic interactions, tissue growth, and tissue organization can be gained by analyzing differently stained histological serial sections. One opportunity unavailable to classic histology is three-dimensional (3D) examination and computer aided analysis of tissue samples. In this case, registration is needed to reestablish spatial correspondence between adjacent slides that is lost during the sectioning process. Furthermore, the sectioning introduces various distortions like cuts, folding, tearing, and local deformations to the tissue, which need to be corrected in order to exploit the additional information arising from the analysis of neighboring slide images. In this paper we present a novel image registration based method for reconstructing a 3D tissue block implementing a zooming strategy around a user-defined point of interest. We efficiently align consecutive slides at increasingly fine resolution up to cell level. We use a two-step approach, where after a macroscopic, coarse alignment of the slides as preprocessing, a nonlinear, elastic registration is performed to correct local, non-uniform deformations. Being driven by the optimization of the normalized gradient field (NGF) distance measure, our method is suitable for differently stained and thus multi-modal slides. We applied our method to ultra thin serial sections (2 μm) of a human lung tumor. In total 170 slides, stained alternately with four different stains, have been registered. Thorough visual inspection of virtual cuts through the reconstructed block perpendicular to the cutting plane shows accurate alignment of vessels and other tissue structures. This observation is confirmed by a quantitative analysis. Using nonlinear image registration, our method is able to correct locally varying deformations in tissue structures and exceeds the limitations of globally linear transformations.

What We Should Know Before Using Tissue Engineering Techniques to Repair Injured Tendons: A Developmental Biology Perspective

PubMed Central

Liu, Chia-Feng; Aschbacher-Smith, Lindsey; Barthelery, Nicolas J.; Dyment, Nathaniel; Butler, David

2011-01-01

Tendons connect muscles to bones, and serve as the transmitters of force that allow all the movements of the body. Tenocytes are the basic cellular units of tendons, and produce the collagens that form the hierarchical fiber system of the tendon. Tendon injuries are common, and difficult to repair, particularly in the case of the insertion of tendon into bone. Successful attempts at cell-based repair therapies will require an understanding of the normal development of tendon tissues, including their differentiated regions such as the fibrous mid-section and fibrocartilaginous insertion site. Many genes are known to be involved in the formation of tendon. However, their functional roles in tendon development have not been fully characterized. Tissue engineers have attempted to generate functional tendon tissue in vitro. However, a lack of knowledge of normal tendon development has hampered these efforts. Here we review studies focusing on the developmental mechanisms of tendon development, and discuss the potential applications of a molecular understanding of tendon development to the treatment of tendon injuries. PMID:21314435

42 CFR 486.322 - Condition: Relationships with hospitals, critical access hospitals, and tissue banks.

Code of Federal Regulations, 2013 CFR

2013-10-01

... access hospitals, and tissue banks. 486.322 Section 486.322 Public Health CENTERS FOR MEDICARE & MEDICAID... Measures § 486.322 Condition: Relationships with hospitals, critical access hospitals, and tissue banks. (a... hospital and critical access hospital staff. (c) Standard: Cooperation with tissue banks. (1) The OPO must...

42 CFR 486.322 - Condition: Relationships with hospitals, critical access hospitals, and tissue banks.

Code of Federal Regulations, 2014 CFR

2014-10-01

... access hospitals, and tissue banks. 486.322 Section 486.322 Public Health CENTERS FOR MEDICARE & MEDICAID... Measures § 486.322 Condition: Relationships with hospitals, critical access hospitals, and tissue banks. (a... hospital and critical access hospital staff. (c) Standard: Cooperation with tissue banks. (1) The OPO must...

42 CFR 486.322 - Condition: Relationships with hospitals, critical access hospitals, and tissue banks.

Code of Federal Regulations, 2010 CFR

2010-10-01

... access hospitals, and tissue banks. 486.322 Section 486.322 Public Health CENTERS FOR MEDICARE & MEDICAID... Measures § 486.322 Condition: Relationships with hospitals, critical access hospitals, and tissue banks. (a... hospital and critical access hospital staff. (c) Standard: Cooperation with tissue banks. (1) The OPO must...

42 CFR 486.322 - Condition: Relationships with hospitals, critical access hospitals, and tissue banks.

Code of Federal Regulations, 2012 CFR

2012-10-01

... access hospitals, and tissue banks. 486.322 Section 486.322 Public Health CENTERS FOR MEDICARE & MEDICAID... Measures § 486.322 Condition: Relationships with hospitals, critical access hospitals, and tissue banks. (a... hospital and critical access hospital staff. (c) Standard: Cooperation with tissue banks. (1) The OPO must...

42 CFR 486.322 - Condition: Relationships with hospitals, critical access hospitals, and tissue banks.

Code of Federal Regulations, 2011 CFR

2011-10-01

... access hospitals, and tissue banks. 486.322 Section 486.322 Public Health CENTERS FOR MEDICARE & MEDICAID... Measures § 486.322 Condition: Relationships with hospitals, critical access hospitals, and tissue banks. (a... hospital and critical access hospital staff. (c) Standard: Cooperation with tissue banks. (1) The OPO must...

9 CFR 71.21 - Tissue and blood testing at slaughter.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Tissue and blood testing at slaughter... GENERAL PROVISIONS § 71.21 Tissue and blood testing at slaughter. (a) Any person moving livestock or... this section 9 within their facility for blood and tissue sample collection; 9 FSIS also has equipment...

9 CFR 71.21 - Tissue and blood testing at slaughter.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Tissue and blood testing at slaughter... GENERAL PROVISIONS § 71.21 Tissue and blood testing at slaughter. (a) Any person moving livestock or... in accordance with paragraph (b) of this section 9 within their facility for blood and tissue sample...

9 CFR 71.21 - Tissue and blood testing at slaughter.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Tissue and blood testing at slaughter... GENERAL PROVISIONS § 71.21 Tissue and blood testing at slaughter. (a) Any person moving livestock or... in accordance with paragraph (b) of this section 9 within their facility for blood and tissue sample...

9 CFR 71.21 - Tissue and blood testing at slaughter.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Tissue and blood testing at slaughter... GENERAL PROVISIONS § 71.21 Tissue and blood testing at slaughter. (a) Any person moving livestock or... in accordance with paragraph (b) of this section 9 within their facility for blood and tissue sample...

21 CFR 500.86 - Marker residue and target tissue.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS GENERAL Regulation of Carcinogenic Compounds Used in Food-Producing Animals § 500.86 Marker residue and target tissue. (a) For each edible tissue, the sponsor shall... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Marker residue and target tissue. 500.86 Section...

21 CFR 500.86 - Marker residue and target tissue.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS GENERAL Regulation of Carcinogenic Compounds Used in Food-Producing Animals § 500.86 Marker residue and target tissue. (a) For each edible tissue, the sponsor shall... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Marker residue and target tissue. 500.86 Section...

21 CFR 500.86 - Marker residue and target tissue.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS GENERAL Regulation of Carcinogenic Compounds Used in Food-Producing Animals § 500.86 Marker residue and target tissue. (a) For each edible tissue, the sponsor shall... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Marker residue and target tissue. 500.86 Section...

40 CFR 430.120 - Applicability; description of the tissue, filter, non-woven, and paperboard from purchased pulp...

Code of Federal Regulations, 2011 CFR

2011-07-01

... tissue, filter, non-woven, and paperboard from purchased pulp subcategory. 430.120 Section 430.120... PULP, PAPER, AND PAPERBOARD POINT SOURCE CATEGORY Tissue, Filter, Non-Woven, and Paperboard From Purchased Pulp Subcategory § 430.120 Applicability; description of the tissue, filter, non-woven, and...

40 CFR 430.120 - Applicability; description of the tissue, filter, non-woven, and paperboard from purchased pulp...

Code of Federal Regulations, 2014 CFR

2014-07-01

... tissue, filter, non-woven, and paperboard from purchased pulp subcategory. 430.120 Section 430.120... (CONTINUED) THE PULP, PAPER, AND PAPERBOARD POINT SOURCE CATEGORY Tissue, Filter, Non-Woven, and Paperboard From Purchased Pulp Subcategory § 430.120 Applicability; description of the tissue, filter, non-woven...

40 CFR 430.120 - Applicability; description of the tissue, filter, non-woven, and paperboard from purchased pulp...

Code of Federal Regulations, 2012 CFR

2012-07-01

... tissue, filter, non-woven, and paperboard from purchased pulp subcategory. 430.120 Section 430.120... (CONTINUED) THE PULP, PAPER, AND PAPERBOARD POINT SOURCE CATEGORY Tissue, Filter, Non-Woven, and Paperboard From Purchased Pulp Subcategory § 430.120 Applicability; description of the tissue, filter, non-woven...

40 CFR 430.120 - Applicability; description of the tissue, filter, non-woven, and paperboard from purchased pulp...

Code of Federal Regulations, 2013 CFR

2013-07-01

... tissue, filter, non-woven, and paperboard from purchased pulp subcategory. 430.120 Section 430.120... (CONTINUED) THE PULP, PAPER, AND PAPERBOARD POINT SOURCE CATEGORY Tissue, Filter, Non-Woven, and Paperboard From Purchased Pulp Subcategory § 430.120 Applicability; description of the tissue, filter, non-woven...

40 CFR 430.120 - Applicability; description of the tissue, filter, non-woven, and paperboard from purchased pulp...

Code of Federal Regulations, 2010 CFR

2010-07-01

... tissue, filter, non-woven, and paperboard from purchased pulp subcategory. 430.120 Section 430.120... PULP, PAPER, AND PAPERBOARD POINT SOURCE CATEGORY Tissue, Filter, Non-Woven, and Paperboard From Purchased Pulp Subcategory § 430.120 Applicability; description of the tissue, filter, non-woven, and...

21 CFR 1270.43 - Retention, recall, and destruction of human tissue.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Retention, recall, and destruction of human tissue. 1270.43 Section 1270.43 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN TISSUE INTENDED FOR TRANSPLANTATION Inspection of Tissue Establishments § 1270.43 Retention...

21 CFR 1270.43 - Retention, recall, and destruction of human tissue.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Retention, recall, and destruction of human tissue. 1270.43 Section 1270.43 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN TISSUE INTENDED FOR TRANSPLANTATION Inspection of Tissue Establishments § 1270.43 Retention...

21 CFR 1270.43 - Retention, recall, and destruction of human tissue.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Retention, recall, and destruction of human tissue. 1270.43 Section 1270.43 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN TISSUE INTENDED FOR TRANSPLANTATION Inspection of Tissue Establishments § 1270.43 Retention...

21 CFR 1270.43 - Retention, recall, and destruction of human tissue.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Retention, recall, and destruction of human tissue. 1270.43 Section 1270.43 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN TISSUE INTENDED FOR TRANSPLANTATION Inspection of Tissue Establishments § 1270.43 Retention...

21 CFR 1270.43 - Retention, recall, and destruction of human tissue.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Retention, recall, and destruction of human tissue. 1270.43 Section 1270.43 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... HUMAN TISSUE INTENDED FOR TRANSPLANTATION Inspection of Tissue Establishments § 1270.43 Retention...

Frequency dependence of tissue attenuation measured by acoustic microscopy.

PubMed

Daft, C M; Briggs, G A; O'Brien, W D

1989-05-01

Broadband scanning acoustic microscopy (SAM) has been used to investigate the mechanical properties of sections of tissue with a resolution of around 8 microns. The work reported here extends these results by reporting the frequency dependence of the attenuation coefficient from 100-500 MHz. A discussion of the theory of the measurements is presented. The scanning laser acoustic microscope (SLAM) is used to characterize similar tissue sections at 100 MHz. The data obtained with the two forms of acoustic microscopy are compared with results from the literature.

A new technique for Gram staining paraffin-embedded tissue.

PubMed Central

Engbaek, K; Johansen, K S; Jensen, M E

1979-01-01

Five techniques for Gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Brown and Hopp's method was the most satisfactory for distinguishing Gram-positive and Gram-negative bacteria. However, this method cannot be recommended as the preparations were frequently overstained, and the Gram-negative bacteria were stained indistinctly. A modification of Brown and Hopps' method was developed which stains larger numbers of Gram-negative bacteria and differentiates well between different cell types and connective tissue, and there is no risk of overstaining. PMID:86548

Fully automated three-dimensional microscopy system

NASA Astrophysics Data System (ADS)

Kerschmann, Russell L.

2000-04-01

Tissue-scale structures such as vessel networks are imaged at micron resolution with the Virtual Tissue System (VT System). VT System imaging of cubic millimeters of tissue and other material extends the capabilities of conventional volumetric techniques such as confocal microscopy, and allows for the first time the integrated 2D and 3D analysis of important tissue structural relationships. The VT System eliminates the need for glass slide-mounted tissue sections and instead captures images directly from the surface of a block containing a sample. Tissues are en bloc stained with fluorochrome compounds, embedded in an optically conditioned polymer that suppresses image signals form dep within the block , and serially sectioned for imaging. Thousands of fully registered 2D images are automatically captured digitally to completely convert tissue samples into blocks of high-resolution information. The resulting multi gigabyte data sets constitute the raw material for precision visualization and analysis. Cellular function may be seen in a larger anatomical context. VT System technology makes tissue metrics, accurate cell enumeration and cell cycle analyses possible while preserving full histologic setting.

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

Small sarcocysts can be a feature of experimental infections with Sarcocystis neurona merozoites.

PubMed

Marsh, Antoinette E; Chaney, Sarah B; Howe, Daniel K; Saville, William J; Reed, Stephen M

2017-10-15

Several reports indicate the presence of small tissue cysts associated with Sarcocystis neurona infections. Several failed attempts to develop tissue cysts in potential intermediate host using in vitro derived parasites originally isolated from horses with equine protozoal myeloencephalitis suggest that the experimental methods to achieve bradyzoites with those isolates was not possible. Those prior studies reported the lack of detectable sarcocysts based on histology and in vivo feeding trials. A recent report of successful production and detection of small sarcocysts triggered us to review archived tissues from earlier experimental infection studies. The retrospective review sought to determine if small sized sarcocysts were not detected due to their relatively smaller size and infrequency as compared to larger sized sarcocysts produced with other isolates in these experimental inoculation trials. Tissues from two prior in vivo inoculation studies, involving in vitro-produced parasites inoculated into laboratory-reared cats and raccoons, were re-examined by immunohistochemistry staining to more easily detect the tissue cysts. In the experimental cat study no small tissue cysts were seen, consistent with the original publication results. However, in the experimental raccoon study, one raccoon inoculated with an EPM-derived isolate, SN-UCD1, had small sarcocysts not reported in the original publication. This retrospective study suggests that much closer scrutiny of tissues, including the use of immunohistochemistry on tissue sections is required to detect the smaller S. neurona sarcocysts associated with the experimental inoculations of the isolates originally derived from horses with EPM. Published by Elsevier B.V.

Extravascular transport in normal and tumor tissues.

PubMed

Jain, R K; Gerlowski, L E

1986-01-01

The transport characteristics of the normal and tumor tissue extravascular space provide the basis for the determination of the optimal dosage and schedule regimes of various pharmacological agents in detection and treatment of cancer. In order for the drug to reach the cellular space where most therapeutic action takes place, several transport steps must first occur: (1) tissue perfusion; (2) permeation across the capillary wall; (3) transport through interstitial space; and (4) transport across the cell membrane. Any of these steps including intracellular events such as metabolism can be the rate-limiting step to uptake of the drug, and these rate-limiting steps may be different in normal and tumor tissues. This review examines these transport limitations, first from an experimental point of view and then from a modeling point of view. Various types of experimental tumor models which have been used in animals to represent human tumors are discussed. Then, mathematical models of extravascular transport are discussed from the prespective of two approaches: compartmental and distributed. Compartmental models lump one or more sections of a tissue or body into a "compartment" to describe the time course of disposition of a substance. These models contain "effective" parameters which represent the entire compartment. Distributed models consider the structural and morphological aspects of the tissue to determine the transport properties of that tissue. These distributed models describe both the temporal and spatial distribution of a substance in tissues. Each of these modeling techniques is described in detail with applications for cancer detection and treatment in mind.

Congenital brainstem disconnection associated with a syrinx of the brainstem.

PubMed

Barth, P G; de Vries, L S; Nikkels, P G J; Troost, D

2008-02-01

We report a case of congenital brainstem disconnection including the second detailed autopsy. A full-term newborn presented with irreversible apnoea and died on the fifth day. MRI revealed disconnection of the brainstem. The autopsy included a series of transverse sections of the mesencephalon, medulla oblongata and bridging tissue fragments. A fragile tube walled by mature brainstem tissue could be reconstructed. It enveloped a cylinder of fluid within the ventral pons extending to the mesencephalon and the lower brainstem. The aqueduct was patent and outside the lesion. The basilar artery was represented by a tiny median vessel. The ventral and lateral parts of the posterior brainstem were surrounded by heterotopic glial tissue. The olivary nucleus was absent and the cerebellar dentate nucleus was dysplastic. Considering the maturity of the remaining parts of the pons, the onset of structural decline is likely to be close to the time of birth. Probable causes are progressively insufficient perfusion through an hypoplastic basilar artery, and obstructed venous drainage through an abnormal glial barrier surrounding the posterior brainstem. The morphological findings can be characterized as a syrinx, known from disorders in which brainstem or spinal cord are damaged by a combination of mechanical and circulatory factors.

3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture.

PubMed

Ghezzi, Chiara E; Marelli, Benedetto; Omenetto, Fiorenzo G; Funderburgh, James L; Kaplan, David L

2017-01-01

The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear silk protein films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous silk films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single silk films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.

Lectins stain cells differentially in the coral, Montipora capitata

USGS Publications Warehouse

Work, Thierry M.; Farah, Yael

2014-01-01

A limitation in our understanding of coral disease pathology and cellular pathogenesis is a lack of reagents to characterize coral cells. We evaluated the utility of plant lectins to stain tissues of a dominant coral, Montipora capitata, from Hawaii. Of 22 lectins evaluated, nine of these stained structures in the upper or basal body wall of corals. Specific structures revealed by lectins that were not considered distinct or evident on routine hematoxylin and eosin sections of coral tissues included apical and basal granules in gastrodermis and epidermis, cnidoglandular tract and actinopharynx cell surface membranes, capsules of mature holotrichous isorhizas, and perivitelline and periseminal cells. Plant lectins could prove useful to further our understanding of coral physiology, anatomy, cell biology, and disease pathogenesis.

Laser tissue welding mediated with a protein solder

NASA Astrophysics Data System (ADS)

Small, Ward, IV; Heredia, Nicholas J.; Celliers, Peter M.; Da Silva, Luiz B.; Eder, David C.; Glinsky, Michael E.; London, Richard A.; Maitland, Duncan J.; Matthews, Dennis L.; Soltz, Barbara A.

1996-05-01

A study of laser tissue welding mediated with an indocyanine green dye-enhanced protein solder was performed. Freshly obtained sections of porcine artery were used for the experiments. Sample arterial wall thickness ranged from two to three millimeters. Incisions approximately four millimeters in length were treated using an 805 nanometer continuous- wave diode laser coupled to a one millimeter diameter fiber. Controlled parameters included the power delivered by the laser, the duration of the welding process, and the concentration of dye in the solder. A two-color infrared detection system was constructed to monitor the surface temperatures achieved at the weld site. Burst pressure measurements were made to quantify the strengths of the welds immediately following completion of the welding procedure.

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.

Separating spectral mixtures in hyperspectral image data using independent component analysis: validation with oral cancer tissue sections

NASA Astrophysics Data System (ADS)

Duann, Jeng-Ren; Jan, Chia-Ing; Ou-Yang, Mang; Lin, Chia-Yi; Mo, Jen-Feng; Lin, Yung-Jiun; Tsai, Ming-Hsui; Chiou, Jin-Chern

2013-12-01

Recently, hyperspectral imaging (HSI) systems, which can provide 100 or more wavelengths of emission autofluorescence measures, have been used to delineate more complete spectral patterns associated with certain molecules relevant to cancerization. Such a spectral fingerprint may reliably correspond to a certain type of molecule and thus can be treated as a biomarker for the presence of that molecule. However, the outcomes of HSI systems can be a complex mixture of characteristic spectra of a variety of molecules as well as optical interferences due to reflection, scattering, and refraction. As a result, the mixed nature of raw HSI data might obscure the extraction of consistent spectral fingerprints. Here we present the extraction of the characteristic spectra associated with keratinized tissues from the HSI data of tissue sections from 30 oral cancer patients (31 tissue samples in total), excited at two different wavelength ranges (330 to 385 and 470 to 490 nm), using independent and principal component analysis (ICA and PCA) methods. The results showed that for both excitation wavelength ranges, ICA was able to resolve much more reliable spectral fingerprints associated with the keratinized tissues for all the oral cancer tissue sections with significantly higher mean correlation coefficients as compared to PCA (p<0.001).

Raman Imaging of Plant Cell Walls in Sections of Cucumis sativus

PubMed Central

Zeise, Ingrid; Heiner, Zsuzsanna; Holz, Sabine; Joester, Maike; Büttner, Carmen

2018-01-01

Raman microspectra combine information on chemical composition of plant tissues with spatial information. The contributions from the building blocks of the cell walls in the Raman spectra of plant tissues can vary in the microscopic sub-structures of the tissue. Here, we discuss the analysis of 55 Raman maps of root, stem, and leaf tissues of Cucumis sativus, using different spectral contributions from cellulose and lignin in both univariate and multivariate imaging methods. Imaging based on hierarchical cluster analysis (HCA) and principal component analysis (PCA) indicates different substructures in the xylem cell walls of the different tissues. Using specific signals from the cell wall spectra, analysis of the whole set of different tissue sections based on the Raman images reveals differences in xylem tissue morphology. Due to the specifics of excitation of the Raman spectra in the visible wavelength range (532 nm), which is, e.g., in resonance with carotenoid species, effects of photobleaching and the possibility of exploiting depletion difference spectra for molecular characterization in Raman imaging of plants are discussed. The reported results provide both, specific information on the molecular composition of cucumber tissue Raman spectra, and general directions for future imaging studies in plant tissues. PMID:29370089

Raman Imaging of Plant Cell Walls in Sections of Cucumis sativus.

PubMed

Zeise, Ingrid; Heiner, Zsuzsanna; Holz, Sabine; Joester, Maike; Büttner, Carmen; Kneipp, Janina

2018-01-25

Raman microspectra combine information on chemical composition of plant tissues with spatial information. The contributions from the building blocks of the cell walls in the Raman spectra of plant tissues can vary in the microscopic sub-structures of the tissue. Here, we discuss the analysis of 55 Raman maps of root, stem, and leaf tissues of Cucumis sativus , using different spectral contributions from cellulose and lignin in both univariate and multivariate imaging methods. Imaging based on hierarchical cluster analysis (HCA) and principal component analysis (PCA) indicates different substructures in the xylem cell walls of the different tissues. Using specific signals from the cell wall spectra, analysis of the whole set of different tissue sections based on the Raman images reveals differences in xylem tissue morphology. Due to the specifics of excitation of the Raman spectra in the visible wavelength range (532 nm), which is, e.g., in resonance with carotenoid species, effects of photobleaching and the possibility of exploiting depletion difference spectra for molecular characterization in Raman imaging of plants are discussed. The reported results provide both, specific information on the molecular composition of cucumber tissue Raman spectra, and general directions for future imaging studies in plant tissues.

FROZEN THIN SECTIONS OF FRESH TISSUE FOR ELECTRON MICROSCOPY, WITH A DESCRIPTION OF PANCREAS AND LIVER

PubMed Central

Christensen, A. Kent

1971-01-01

A simple method has been developed that allows frozen thin sections of fresh-frozen tissue to be cut on a virtually unmodified ultramicrotome kept at room temperature. A bowl-shaped Dewar flask with a knifeholder in its depths replaces the stage of the microtome; a bar extends down into the bowl from the microtome's cutting arm and bears the frozen tissue near its lower end. When the microtome is operated, the tissue passes a glass or diamond knife in the depths of the bowl as in normal cutting. The cutting temperature is maintained by flushing the bowl with cold nitrogen gas, and can be set anywhere from about -160°C up to about -30°C. The microtome is set for a cutting thickness of 540–1000 A. Sections are picked up from the dry knife edge, and are placed on membrane-coated grids, flattened with the polished end of a copper rod, and either dried in nitrogen gas or freeze-dried. Throughout the entire process the tissue is kept cold and does not come in contact with any solvent. The morphology seen in frozen thin sections of rat pancreas and liver generally resembles that in conventional preparations, although freezing damage and low contrast limit the detail that can be discerned. Among unusual findings is a frequent abundance of mitochondrial granules in material prepared by this method. PMID:4942776

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

Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue Specimens for Enhanced Clinical Guidance

DTIC Science & Technology

2015-07-01

describe: 1) major activities ; 2) specific objectives; 3) significant results or key outcomes, including major findings, developments, or conclusions...project progresses to completion, the emphasis in reporting in this section should shift from reporting activities to reporting accomplishments. B...high-resolution axial (depth) imaging as well. Of course, both techniques are under active development, and their properties may change sub

Strategies for laboratory cost containment and for pathologist shortage: centralised pathology laboratories with microwave-stimulated histoprocessing and telepathology.

PubMed

Leong, Anthony S Y; Leong, F Joel W M

2005-02-01

The imposition of laboratory cost containment, often from external forces, dictates the necessity to develop strategies to meet laboratory cost savings. In addition, the national and worldwide shortage of anatomical pathologists makes it imperative to examine our current practice and laboratory set-ups. Some of the strategies employed in other areas of pathology and laboratory medicine include improvements in staff productivity and the adoption of technological developments that reduce manual intervention. However, such opportunities in anatomical pathology are few and far between. Centralisation has been an effective approach in bringing economies of scale, the adoption of 'best practices' and the consolidation of pathologists, but this has not been possible in anatomical pathology because conventional histoprocessing takes a minimum of 14 hours and clinical turnaround time requirements necessitate that the laboratory and pathologist be in proximity and on site. While centralisation of laboratories for clinical chemistry, haematology and even microbiology has been successful in Australia and other countries, the essential requirements for anatomical pathology laboratories are different. In addition to efficient synchronised courier networks, a method of ultra-rapid tissue processing and some expedient system of returning the prepared tissue sections to the remote laboratory are essential to maintain the turnaround times mandatory for optimal clinical management. The advent of microwave-stimulated tissue processing that can be completed in 30-60 minutes and the immediate availability of compressed digital images of entire tissue sections via telepathology completes the final components of the equation necessary for making centralised anatomical pathology laboratories a reality.

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.

The development of computer-aided system for tissue scaffolds (CASTS) system for functionally graded tissue-engineering scaffolds.

PubMed

Sudarmadji, Novella; Chua, Chee Kai; Leong, Kah Fai

2012-01-01

Computer-aided system for tissue scaffolds (CASTS) is an in-house parametric library of polyhedral units that can be assembled into customized tissue scaffolds. Thirteen polyhedral configurations are available to select, depending on the biological and mechanical requirements of the target tissue/organ. Input parameters include the individual polyhedral units and overall scaffold block as well as the scaffold strut diameter. Taking advantage of its repeatability and reproducibility, the scaffold file is then converted into .STL file and fabricated using selective laser sintering, a rapid prototyping system. CASTS seeks to fulfill anatomical, biological, and mechanical requirements of the target tissue/organ. Customized anatomical scaffold shape is achieved through a Boolean operation between the scaffold block and the tissue defect image. Biological requirements, such as scaffold pore size and porosity, are unique for different type of cells. Matching mechanical properties, such as stiffness and strength, between the scaffold and target organ is very important, particularly in the regeneration of load-bearing organ, i.e., bone. This includes mimicking the compressive stiffness variation across the bone to prevent stress shielding and ensuring that the scaffold can withstand the load normally borne by the bone. The stiffness variation is tailored by adjusting the scaffold porosity based on the porosity-stiffness relationship of the CASTS scaffolds. Two types of functional gradients based on the gradient direction include radial and axial/linear gradient. Radial gradient is useful in the case of regenerating a section of long bones while the gradient in linear direction can be used in short or irregular bones. Stiffness gradient in the radial direction is achieved by using cylindrical unit cells arranged in a concentric manner, in which the porosity decreases from the center of the structure toward the outside radius, making the scaffold stiffer at the outer radius and more porous at the center of the scaffold. On the other hand, the linear gradient is accomplished by varying the strut diameter along the gradient direction. The parameters to vary in both gradient types are the strut diameter, the unit cell dimension, and the boundaries between two scaffold regions with different stiffness.

Measuring the resolution of uncompressed plastic sections cut using an oscillating knife ultramicrotome.

PubMed

Sader, Kasim; Reedy, Michael; Popp, David; Lucaveche, Carmen; Trinick, John

2007-07-01

Thin sections of biological tissue embedded in plastic and cut with an ultramicrotome do not generally display useful details smaller than approximately 50 A in the electron microscope. However, there is evidence that before sectioning the embedded tissue can be substantially better preserved, which suggests that cutting is when major damage and loss of resolution occurs. We show here a striking example of such damage in embedded insect flight muscle fibres. X-ray diffraction of the embedded muscle gave patterns extending to 13A, whereas sections cut from the same block showed only approximately 50 A resolution. A possible source of this damage is the substantial compression that was imposed on sections during cutting. An oscillating knife ultramicrotome eliminates the compression and it seemed possible that sections cut with such a knife would show substantially improved preservation. We used the oscillating knife to cut sections from the embedded muscle and from embedded catalase crystals. Preservation with and without oscillation was assessed in Fourier transforms of micrographs. Sections cut with the knife oscillating did not show improved preservation over those cut without. Thus compression during cutting does not appear to be the major source of damage in plastic sections, and leaves unexplained the 50 A versus 13A discrepancy between block and section preservation. The results nevertheless suggest that improvements in ultramicrotomy will be important for bringing thin-sectioning and tomography of plastic-embedded cells and tissues to the point where macromolecule shapes can be resolved.

p53 and Bcl-2expression in response to boldenone induced liver cells injury.

PubMed

Tousson, Ehab; Alm-Eldeen, Abeer; El-Moghazy, Mostafa

2011-09-01

Boldenone is an anabolic steroid developed for veterinary use. Recently, it is used by bodybuilders in both off-season and pre-contest, where it is well known for increasing vascularity while preparing for a bodybuilding contest. So, the present study was designed to investigate the possible effect of using growth promoter boldenone undecylenate on the rabbit liver tissue. Thirty-two adult New Zealand rabbits were divided into four groups (8 animals each). Control group includes animals that injected intramuscularly with olive oil and dissected after 3 weeks. The experimental groups include animals that receive one, two and three intramuscular injections of 5 mg/kg body weight boldenone, respectively. The animals were dissected after 3, 6 and 9 weeks respectively, where the interval of each dose of boldenon was 3 weeks. Small pieces of the liver tissues were sent for the histopathological examination. Apoptotic p53 and antiapoptotic Bc1-2 proteins were localized immunohistochemically. Histological observations of the liver tissue showed that the sinusoidal congestion was the most prominent feature that extended from the centrilobular to the periportal regions. Hepatocellular vacuolation in the centrilobular region was also detected. Liver immunohistochemical observation showed a significant increase of the apoptotic protein p53 and a significant decrease in the antiapoptotic Bc1-2 proteins. The highest frequency of p53 positive cells was observed in the liver sections of three dose of boldenone injections, while the lowest in control group, also the highest frequency of Bcl-2 positive cells was observed in the liver sections of control group while the lowest in three dose of boldenone injections. The present results investigate that people should be careful if they want to use such steroids to enhance their strength and endurance.

Validation of a TFE3 break-apart FISH assay for Xp11.2 translocation renal cell carcinomas.

PubMed

Mosquera, Juan-Miguel; Dal Cin, Paola; Mertz, Kirsten D; Perner, Sven; Davis, Ian J; Fisher, David E; Rubin, Mark A; Hirsch, Michelle S

2011-09-01

Renal cell carcinomas (RCCs) with an Xp11.2 translocation predominantly affect young patients, and can present at an advanced stage. However, more cases in older patients and incidentally detected cancers at earlier stages are also being identified. As the histology of Xp11.2 RCCs overlaps with clear cell and papillary RCCs, it is not infrequent that Xp11.2 RCCs are overlooked and misdiagnosed. The objective of this study was to validate the use of fluorescence in-situ hybridization (FISH) for identifying Xp11.2 RCCs. One hundred fifty-eight consecutive, unselected renal tumors were evaluated in tissue microarrays, including 109 clear cell RCCs, 20 papillary RCCs, 3 RCCs with mixed papillary and clear cell features, 1 Xp11.2 translocation RCC, 8 chromophobe RCCs, 10 oncocytomas, and 7 angiomyolipomas. FISH evaluation was performed blinded to karyotype data, available in about two-thirds of cases. Furthermore, conventional sections of 4 Xp11.2 RCCs, 4 RCCs with mixed papillary and clear cell features, and 4 cases of alveolar soft part sarcoma (the latter for control purposes) were also assessed by FISH. Break-apart signals were homogeneously identified throughout tumor cells in 2 cases from the tissue microarrays including 1 known Xp11.2 RCC and 1 misdiagnosed Xp11.2 RCC. All conventional sections from the Xp11.2 RCC and alveolar soft part sarcoma cases were positive for the TFE3 rearrangement by FISH. All remaining cases were negative. Our study shows the clinical application of FISH in formalin-fixed, paraffin-embedded tissue for detection of Xp11.2 translocation RCCs and other tumors with this genetic aberration.

Immunohistochemical detection of a hypoxia marker in spontaneous canine tumours.

PubMed Central

Cline, J. M.; Thrall, D. E.; Page, R. L.; Franko, A. J.; Raleigh, J. A.

1990-01-01

An immunoperoxidase technique has been used to detect the in vivo binding of a 2-nitroimidazole hypoxia marker in histochemical sections of a variety of excised canine tumours. The binding occurred 10-12 cell diameters away from tumour blood vessels, consistent with the expected location of hypoxic cells in tissues in which oxygen concentration gradients are established by diffusion. Hypoxic fractions ranging from 4 to 13% have been estimated on the basis of morphometric analysis of multiple tumour sections. The binding of the marker was restricted to the cytoplasm of the cells. The marker appeared in regions adjacent to necrosis but also in regions free of necrosis. As in earlier autoradiography studies, binding was occasionally observed in cells adjacent to tumour blood vessels. Generally, binding to normal tissues was not observed. However, binding to smooth muscle cells surrounding arterioles in some sections of normal tissue and tumour tissue was observed. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1701659

Measurement of the 33S(n,α) cross-section at n_TOF(CERN): Applications to BNCT

PubMed Central

Sabaté-Gilarte, Marta; Praena, Javier; Porras, Ignacio; Quesada, José Manuel; Mastinu, Pierfrancesco

2016-01-01

Aim The main purpose of this work is to present a new (n,α) cross-section measurement for a stable isotope of sulfur, 33S, in order to solve existing discrepancies. Background 33S has been studied as a cooperating target for Boron Neutron Capture Therapy (BNCT) because of its large (n,α) cross-section in the epithermal neutron energy range, the most suitable one for BNCT. Although the most important evaluated databases, such as ENDF, do not show any resonances in the cross-section, experimental measurements which provided data from 10 keV to 1 MeV showed that the lowest-lying and strongest resonance of 33S(n,α) cross-section occurs at 13.5 keV. Nevertheless, the set of resonance parameters that describe such resonance shows important discrepancies (more than a factor of 2) between them. Materials and methods A new measurement of the 33S(n,α)30Si reaction cross-section was proposed to the ISOLDE and Neutron Time-of-Flight Experiments Committee of CERN. It was performed at n_TOF(CERN) in 2012 using MicroMegas detectors. Results In this work, we will present a brief overview of the experiment as well as preliminary results of the data analysis in the neutron energy range from thermal to 100 keV. These results will be taken into account to calculate the kerma-fluence factors corresponding to 33S in addition to 10B and those of a standard four-component ICRU tissue. Conclusions MCNP simulations of the deposited dose, including our experimental data, shows an important kerma rate enhancement at the surface of the tissue, mainly due to the presence of 33S. PMID:26933393

Measurement of the (33)S(n,α) cross-section at n_TOF(CERN): Applications to BNCT.

PubMed

Sabaté-Gilarte, Marta; Praena, Javier; Porras, Ignacio; Quesada, José Manuel; Mastinu, Pierfrancesco

2016-01-01

The main purpose of this work is to present a new (n,α) cross-section measurement for a stable isotope of sulfur, (33)S, in order to solve existing discrepancies. (33)S has been studied as a cooperating target for Boron Neutron Capture Therapy (BNCT) because of its large (n,α) cross-section in the epithermal neutron energy range, the most suitable one for BNCT. Although the most important evaluated databases, such as ENDF, do not show any resonances in the cross-section, experimental measurements which provided data from 10 keV to 1 MeV showed that the lowest-lying and strongest resonance of (33)S(n,α) cross-section occurs at 13.5 keV. Nevertheless, the set of resonance parameters that describe such resonance shows important discrepancies (more than a factor of 2) between them. A new measurement of the (33)S(n,α)(30)Si reaction cross-section was proposed to the ISOLDE and Neutron Time-of-Flight Experiments Committee of CERN. It was performed at n_TOF(CERN) in 2012 using MicroMegas detectors. In this work, we will present a brief overview of the experiment as well as preliminary results of the data analysis in the neutron energy range from thermal to 100 keV. These results will be taken into account to calculate the kerma-fluence factors corresponding to (33)S in addition to (10)B and those of a standard four-component ICRU tissue. MCNP simulations of the deposited dose, including our experimental data, shows an important kerma rate enhancement at the surface of the tissue, mainly due to the presence of (33)S.

Qualitative and Quantitative Analysis of Histone Deacetylases in Kidney Tissue Sections.

PubMed

Ververis, Katherine; Marzully, Selly; Samuel, Chrishan S; Hewitson, Tim D; Karagiannis, Tom C

2016-01-01

Fluorescent microscope imaging technologies are increasing in their applications and are being used on a wide scale. However methods used to quantify the level of fluorescence intensity are often not utilized-perhaps given the result may be immediately seen, quantification of the data may not seem necessary. However there are a number of reasons given to quantify fluorescent images including the importance of removing potential bias in the data upon observation as well as quantification of large numbers of images gives statistical power to detect subtle changes in experiments. In addition discreet localization of a protein could be detected without selection bias that may not be detectable by eye. Such data will be deemed useful when detecting the levels of HDAC enzymes within cells in order to develop more effective HDAC inhibitor compounds for use against multiple diseased states. Hence, we discuss a methodology devised to analyze fluorescent images using Image J to detect the mean fluorescence intensity of the 11 metal-dependent HDAC enzymes using murine kidney tissue sections as an example.

Hyperspectral microscopy and cluster analysis for oral cancer diagnosis

NASA Astrophysics Data System (ADS)

Jarman, Anneliese; Manickavasagam, Arunthathi; Hosny, Neveen; Festy, Frederic

2017-02-01

Oral cancer incidences have been increasing in recent years and late detection often leads to poor prognosis. Raman spectroscopy has been identified has a valuable diagnostic tool for cancer but its time consuming nature has prevented its clinical use. For Raman to become a realistic aid to histopathology, a rapid pre-screening technique is required to find small regions of interest on tissue sections [1]. The aim of this work is to investigate the feasibility of hyperspectral imaging in the visible spectral range as a fast imaging technique before Raman is performed. We have built a hyperspectral microscope which captures 300 focused and intensity corrected images with wavelength ranging from 450- 750 nm in around 30 minutes with sub-micron spatial resolution and around 10 nm spectral resolution. Hyperstacks of known absorbing samples, including fluorescent dyes and dried blood droplets, show excellent results with spectrally accurate transmission spectra and concentration-dependent intensity variations. We successfully showed the presence of different components from a non-absorbent saliva droplet sample. Data analysis is the greatest hurdle to the interpretation of more complex data such as unstained tissue sections.

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation.

PubMed

Inman, Kimberly E; Downs, Karen M

2006-10-01

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.

Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras

PubMed Central

Karagenç, Levent; Sandikci, Mustafa

2010-01-01

The objective of the current study was to determine the tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. Quail-chick chimeras were constructed by transferring dissociated cells from the area opaca of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in embryonic as well as extra-embryonic tissues of the recipient embryo were examined using the QCPN monoclonal antibody after 6 days of incubation in serial sections taken at 100-μm intervals. Data gathered in the present study demonstrated that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently the mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in tissues derived from the neural ectoderm and the surface ectoderm, including the optic cup, diencephalon and lens. Although the possibility of random incorporation of transplanted cells into these embryonic structures cannot be excluded, these results would suggest that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types in the recipient chick embryo, including those derived from the surface ectoderm and neural ectoderm. PMID:19900180

Transplantation of Tissue-Engineered Cartilage in an Animal Model (Xenograft and Autograft): Construct Validation.

PubMed

Nemoto, Hitoshi; Watson, Deborah; Masuda, Koichi

2015-01-01

Tissue engineering holds great promise for cartilage repair with minimal donor-site morbidity. The in vivo maturation of a tissue-engineered construct can be tested in the subcutaneous tissues of the same species for autografts or of immunocompromised animals for allografts or xenografts. This section describes detailed protocols for the surgical transplantation of a tissue-engineered construct into an animal model to assess construct validity.

Methods and means of 3D diffuse Mueller-matrix tomography of depolarizing optically anisotropic biological layers

NASA Astrophysics Data System (ADS)

Dubolazov, O. V.; Ushenko, V. O.; Trifoniuk, L.; Ushenko, Yu. O.; Zhytaryuk, V. G.; Prydiy, O. G.; Grytsyuk, M.; Kushnerik, L.; Meglinskiy, I.

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 birefringence of prostate histological sections are found. The interconnections between such distributions and parameters of linear and circular birefringence of prostate tissue histological sections are defined. The comparative investigations of coordinate distributions of phase anisotropy parameters formed by fibrillar networks of prostate tissues of different pathological states (adenoma and carcinoma) 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 birefringence are defined. The objective criteria of cause of Benign and malignant conditions differentiation are determined.

Evaporation process in histological tissue sections for neutron autoradiography.

PubMed

Espector, Natalia M; Portu, Agustina; Santa Cruz, Gustavo A; Saint Martin, Gisela

2018-05-01

The analysis of the distribution and density of nuclear tracks forming an autoradiography in a nuclear track detector (NTD) allows the determination of 10 B atoms concentration and location in tissue samples from Boron Neutron Capture Therapy (BNCT) protocols. This knowledge is of great importance for BNCT dosimetry and treatment planning. Tissue sections studied with this technique are obtained by cryosectioning frozen tissue specimens. After the slicing procedure, the tissue section is put on the NTD and the sample starts drying. The thickness varies from its original value allowing more particles to reach the detector and, as the mass of the sample decreases, the boron concentration in the sample increases. So in order to determine the concentration present in the hydrated tissue, the application of corrective coefficients is required. Evaporation mechanisms as well as various factors that could affect the process of mass variation are outlined in this work. Mass evolution for tissue samples coming from BDIX rats was registered with a semimicro analytical scale and measurements were analyzed with software developed to that end. Ambient conditions were simultaneously recorded, obtaining reproducible evaporation curves. Mathematical models found in the literature were applied for the first time to this type of samples and the best fit of the experimental data was determined. The correlation coefficients and the variability of the parameters were evaluated, pointing to Page's model as the one that best represented the evaporation curves. These studies will contribute to a more precise assessment of boron concentration in tissue samples by the Neutron Autoradiography technique.

Determination of elemental tissue composition following proton treatment using positron emission tomography

NASA Astrophysics Data System (ADS)

Cho, Jongmin; Ibbott, Geoffrey; Gillin, Michael; Gonzalez-Lepera, Carlos; Min, Chul Hee; Zhu, Xuping; El Fakhri, Georges; Paganetti, Harald; Mawlawi, Osama

2013-06-01

Positron emission tomography (PET) has been suggested as an imaging technique for in vivo proton dose and range verification after proton induced-tissue activation. During proton treatment, irradiated tissue is activated and decays while emitting positrons. In this paper, we assessed the feasibility of using PET imaging after proton treatment to determine tissue elemental composition by evaluating the resultant composite decay curve of activated tissue. A phantom consisting of sections composed of different combinations of 1H, 12C, 14N, and 16O was irradiated using a pristine Bragg peak and a 6 cm spread-out Bragg-peak (SOBP) proton beam. The beam ranges defined at 90% distal dose were 10 cm the delivered dose was 1.6 Gy for the near monoenergetic beam and 2 Gy for the SOBP beam. After irradiation, activated phantom decay was measured using an in-room PET scanner for 30 min in list mode. Decay curves from the activated 12C and 16O sections were first decomposed into multiple simple exponential decay curves, each curve corresponding to a constituent radioisotope, using a least-squares method. The relative radioisotope fractions from each section were determined. These fractions were used to guide the decay curve decomposition from the section consisting mainly of 12C + 16O and calculate the relative elemental composition of 12C and 16O. A Monte Carlo simulation was also used to determine the elemental composition of the 12C + 16O section. The calculated compositions of the 12C + 16O section using both approaches (PET and Monte Carlo) were compared with the true known phantom composition. Finally, two patients were imaged using an in-room PET scanner after proton therapy of the head. Their PET data and the technique described above were used to construct elemental composition (12C and 16O) maps that corresponded to the proton-activated regions. We compared the 12C and 16O compositions of seven ROIs that corresponded to the vitreous humor, adipose/face mask, adipose tissue, and brain tissue with ICRU 46 elemental composition data. The 12C and 16O compositions of the 12C + 16O phantom section were estimated to within a maximum difference of 3.6% for the near monoenergetic and SOBP beams over an 8 cm depth range. On the other hand, the Monte Carlo simulation estimated the corresponding 12C and 16O compositions in the 12C + 16O section to within a maximum difference of 3.4%. For the patients, the 12C and 16O compositions in the seven ROIs agreed with the ICRU elemental composition data, with a mean (maximum) difference of 9.4% (15.2%). The 12C and 16O compositions of the phantom and patients were estimated with relatively small differences. PET imaging may be useful for determining the tissue elemental composition and thereby improving proton treatment planning and verification.

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

PubMed Central

1986-01-01

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick- translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling. PMID:3084498

TransOmic analysis of forebrain sections in Sp2 conditional knockout embryonic mice using IR-MALDESI imaging of lipids and LC-MS/MS label-free proteomics

PubMed Central

Loziuk, Philip; Meier, Florian; Johnson, Caroline

2016-01-01

Quantitative methods for detection of biological molecules are needed more than ever before in the emerging age of “omics” and “big data.” Here, we provide an integrated approach for systematic analysis of the “lipidome” in tissue. To test our approach in a biological context, we utilized brain tissue selectively deficient for the transcription factor Specificity Protein 2 (Sp2). Conditional deletion of Sp2 in the mouse cerebral cortex results in developmental deficiencies including disruption of lipid metabolism. Silver (Ag) cationization was implemented for infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids, as these have been linked to regulation by Sp2. Combining Ag-doped and conventional IR-MALDESI imaging, this approach was extended to IR-MALDESI imaging of embryonic mouse brains. Further, our imaging technique was combined with bottom-up shotgun proteomic LC-MS/MS analysis and western blot for comparing Sp2 conditional knockout (Sp2-cKO) and wild-type (WT) cortices of tissue sections. This provided an integrated omics dataset which revealed many specific changes to fundamental cellular processes and biosynthetic pathways. In particular, step-specific altered abundances of nucleotides, lipids, and associated proteins were observed in the cerebral cortices of Sp2-cKO embryos. PMID:26942738

Re-engineering the Pancreas Tumor Microenvironment: A "Regenerative Program" Hacked.

PubMed

Evan, Gerard I; Hah, Nasun; Littlewood, Trevor D; Sodir, Nicole M; Campos, Tania; Downes, Michael; Evans, Ronald M

2017-04-01

The "hallmarks" of pancreatic ductal adenocarcinoma (PDAC) include proliferative, invasive, and metastatic tumor cells and an associated dense desmoplasia comprised of fibroblasts, pancreatic stellate cells, extracellular matrix, and immune cells. The oncogenically activated pancreatic epithelium and its associated stroma are obligatorily interdependent, with the resulting inflammatory and immunosuppressive microenvironment contributing greatly to the evolution and maintenance of PDAC. The peculiar pancreas-specific tumor phenotype is a consequence of oncogenes hacking the resident pancreas regenerative program, a tissue-specific repair mechanism regulated by discrete super enhancer networks. Defined as genomic regions containing clusters of multiple enhancers, super enhancers play pivotal roles in cell/tissue specification, identity, and maintenance. Hence, interfering with such super enhancer-driven repair networks should exert a disproportionately disruptive effect on tumor versus normal pancreatic tissue. Novel drugs that directly or indirectly inhibit processes regulating epigenetic status and integrity, including those driven by histone deacetylases, histone methyltransferase and hydroxylases, DNA methyltransferases, various metabolic enzymes, and bromodomain and extraterminal motif proteins, have shown the feasibility of disrupting super enhancer-dependent transcription in treating multiple tumor types, including PDAC. The idea that pancreatic adenocarcinomas rely on embedded super enhancer transcriptional mechanisms suggests a vulnerability that can be potentially targeted as novel therapies for this intractable disease. Clin Cancer Res; 23(7); 1647-55. ©2017 AACR See all articles in this CCR Focus section, "Pancreatic Cancer: Challenge and Inspiration." ©2017 American Association for Cancer Research.

Linear attenuation coefficients of tissues from 1 keV to 150 keV

NASA Astrophysics Data System (ADS)

Böke, Aysun

2014-09-01

The linear attenuation coefficients and three interaction processes have been computed for liver, kidney, muscle, fat and for a range of x-ray energies from 1 keV to 150 keV. Molecular photoelectric absorption cross sections were calculated from atomic cross section data. Total coherent (Rayleigh) and incoherent (Compton) scattering cross sections were obtained by numerical integration over combinations of F2m(x) with the Thomson formula and Sm(x) with the Klein-Nishina formula, respectively. For the coherent (Rayleigh) scattering cross section calculations, molecular form factors were obtained from recent experimental data in the literature for values of x<1 Å-1 and from the relativistic modified atomic form factors for values of x≥1 Å-1. With the inclusion of molecular interference effects in the coherent (Rayleigh) scattering, more accurate knowledge of the scatter from these tissues will be provided. The number of elements involved in tissue composition is 5 for liver, 47 for kidney, 44 for muscle and 3 for fat. The results are compared with previously published experimental and theoretical linear attenuation coefficients. In general, good agreement is obtained. The molecular form factors and scattering functions and cross sections are incorporated into a Monte Carlo program. The energy distributions of x-ray photons scattered from tissues have been simulated and the results are presented.

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.

Depth-Dependent Anisotropies of Amides and Sugar in Perpendicular and Parallel Sections of Articular Cartilage by Fourier Transform Infrared Imaging (FTIRI)

PubMed Central

Xia, Yang; Mittelstaedt, Daniel; Ramakrishnan, Nagarajan; Szarko, Matthew; Bidthanapally, Aruna

2010-01-01

Full thickness blocks of canine humeral cartilage were microtomed into both perpendicular sections and a series of 100 parallel sections, each 6 μm thick. Fourier Transform Infrared Imaging (FTIRI) was used to image each tissue section eleven times under different infrared polarizations (from 0° to 180° polarization states in 20° increments and with an additional 90° polarization), at a spatial resolution of 6.25 μm and a wavenumber step of 8 cm−1. With increasing depth from the articular surface, amide anisotropies increased in the perpendicular sections and decreased in the parallel sections. Both types of tissue sectioning identified a 90° difference between amide I and amide II in the superficial zone of cartilage. The fibrillar distribution in the parallel sections from the superficial zone was shown to not be random. Sugar had the greatest anisotropy in the upper part of the radial zone in the perpendicular sections. The depth-dependent anisotropic data were fitted with a theoretical equation that contained three signature parameters, which illustrate the arcade structure of collagens with the aid of a fibril model. Infrared imaging of both perpendicular and parallel sections provides the possibility of determining the three-dimensional macromolecular structures in articular cartilage. Being sensitive to the orientation of the macromolecular structure in healthy articular cartilage aids the prospect of detecting the early onset of the tissue degradation that may lead to pathological conditions such as osteoarthritis. PMID:21274999

Coregistration of Preoperative MRI with Ex Vivo Mesorectal Pathology Specimens to Spatially Map Post-treatment Changes in Rectal Cancer Onto In Vivo Imaging: Preliminary Findings.

PubMed

Antunes, Jacob; Viswanath, Satish; Brady, Justin T; Crawshaw, Benjamin; Ros, Pablo; Steele, Scott; Delaney, Conor P; Paspulati, Raj; Willis, Joseph; Madabhushi, Anant

2018-07-01

The objective of this study was to develop and quantitatively evaluate a radiology-pathology fusion method for spatially mapping tissue regions corresponding to different chemoradiation therapy-related effects from surgically excised whole-mount rectal cancer histopathology onto preoperative magnetic resonance imaging (MRI). This study included six subjects with rectal cancer treated with chemoradiation therapy who were then imaged with a 3-T T2-weighted MRI sequence, before undergoing mesorectal excision surgery. Excised rectal specimens were sectioned, stained, and digitized as two-dimensional (2D) whole-mount slides. Annotations of residual disease, ulceration, fibrosis, muscularis propria, mucosa, fat, inflammation, and pools of mucin were made by an expert pathologist on digitized slide images. An expert radiologist and pathologist jointly established corresponding 2D sections between MRI and pathology images, as well as identified a total of 10 corresponding landmarks per case (based on visually similar structures) on both modalities (five for driving registration and five for evaluating alignment). We spatially fused the in vivo MRI and ex vivo pathology images using landmark-based registration. This allowed us to spatially map detailed annotations from 2D pathology slides onto corresponding 2D MRI sections. Quantitative assessment of coregistered pathology and MRI sections revealed excellent structural alignment, with an overall deviation of 1.50 ± 0.63 mm across five expert-selected anatomic landmarks (in-plane misalignment of two to three pixels at 0.67- to 1.00-mm spatial resolution). Moreover, the T2-weighted intensity distributions were distinctly different when comparing fibrotic tissue to perirectal fat (as expected), but showed a marked overlap when comparing fibrotic tissue and residual rectal cancer. Our fusion methodology enabled successful and accurate localization of post-treatment effects on in vivo MRI. Copyright © 2018 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.

Electron transport in biomolecular gaseous and liquid systems: theory, experiment and self-consistent cross-sections

NASA Astrophysics Data System (ADS)

White, R. D.; Cocks, D.; Boyle, G.; Casey, M.; Garland, N.; Konovalov, D.; Philippa, B.; Stokes, P.; de Urquijo, J.; González-Magaña, O.; McEachran, R. P.; Buckman, S. J.; Brunger, M. J.; Garcia, G.; Dujko, S.; Petrovic, Z. Lj

2018-05-01

Accurate modelling of electron transport in plasmas, plasma-liquid and plasma-tissue interactions requires (i) the existence of accurate and complete sets of cross-sections, and (ii) an accurate treatment of electron transport in these gaseous and soft-condensed phases. In this study we present progress towards the provision of self-consistent electron-biomolecule cross-section sets representative of tissue, including water and THF, by comparison of calculated transport coefficients with those measured using a pulsed-Townsend swarm experiment. Water–argon mixtures are used to assess the self-consistency of the electron-water vapour cross-section set proposed in de Urquijo et al (2014 J. Chem. Phys. 141 014308). Modelling of electron transport in liquids and soft-condensed matter is considered through appropriate generalisations of Boltzmann’s equation to account for spatial-temporal correlations and screening of the electron potential. The ab initio formalism is applied to electron transport in atomic liquids and compared with available experimental swarm data for these noble liquids. Issues on the applicability of the ab initio formalism for krypton are discussed and addressed through consideration of the background energy of the electron in liquid krypton. The presence of self-trapping (into bubble/cluster states/solvation) in some liquids requires a reformulation of the governing Boltzmann equation to account for the combined localised–delocalised nature of the resulting electron transport. A generalised Boltzmann equation is presented which is highlighted to produce dispersive transport observed in some liquid systems.

Paraffin tissue microarrays constructed with a cutting board and cutting board arrayer.

PubMed

Vogel, Ulrich Felix

2010-05-01

Paraffin tissue microarrays (PTMAs) are blocks of paraffin containing up to 1300 paraffin tissue core biopsies (PTCBs). Normally, these PTCBs are punched from routine paraffin tissue blocks, which contain tissues of differing thicknesses. Therefore, the PTCBs are of different lengths. In consequence, the sections of the deeper portions of the PTMA do not contain all of the desired PTCBs. To overcome this drawback, cutting boards were constructed from panels of plastic with a thickness of 4 mm. Holes were drilled into the plastic and filled completely with at least one PTCB per hole. After being trimmed to a uniform length of 4 mm, these PTCBs were pushed from the cutting board into corresponding holes in a recipient block by means of a plate with steel pins. Up to 1000 sections per PTMA were cut without any significant loss of PTCBs, thereby increasing the efficacy of the PTMA technique.

Autofluorescence of bovine ligamentum nuchae, cartilage, heart valve and lung measured by microscopy and fibre optics.

PubMed

Swatland, H J

1988-09-01

The fluorescence of bovine tissues was measured post mortem by microscopy of frozen sections and by using optical fibres to excite fluorescence and to measure fluorescence emission spectra. Mechanical disruption of the tissue (by comminution or sectioning) did not appreciably change tissue fluorescence spectra. Ligamentum nuchae had the strongest fluorescence and lung tissue had the weakest. In samples measured with a minimum prior exposure to ultraviolet light, the peak fluorescence emission was at 410 or 420 nm (with excitation at 365 nm). Exposure to ultraviolet light for about 1 minute shifted the fluorescence peak to 450 to 470 nm. Further exposure (about 30 minutes) caused a loss of the 450 to 470 nm fluorescence peak, while emissions above 530 nm were maintained or strengthened. Microscopy showed that the fluorescence that was measured by fibre optics from intact connective tissues originated mostly from collagen and elastin fibres.

Technical Note: Method to correlate whole-specimen histopathology of radical prostatectomy with diagnostic MR imaging

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

McGrath, Deirdre M., E-mail: d.mcgrath@sheffield.ac.uk; Lee, Jenny; Foltz, Warren D.

Purpose: Validation of MRI-guided tumor boundary delineation for targeted prostate cancer therapy is achieved via correlation with gold-standard histopathology of radical prostatectomy specimens. Challenges to accurate correlation include matching the pathology sectioning plane with the in vivo imaging slice plane and correction for the deformation that occurs between in vivo imaging and histology. A methodology is presented for matching of the histological sectioning angle and position to the in vivo imaging slices. Methods: Patients (n = 4) with biochemical failure following external beam radiotherapy underwent diagnostic MRI to confirm localized recurrence of prostate cancer, followed by salvage radical prostatectomy. High-resolutionmore » 3-D MRI of the ex vivo specimens was acquired to determine the pathology sectioning angle that best matched the in vivo imaging slice plane, using matching anatomical features and implanted fiducials. A novel sectioning device was developed to guide sectioning at the correct angle, and to assist the insertion of reference dye marks to aid in histopathology reconstruction. Results: The percentage difference in the positioning of the urethra in the ex vivo pathology sections compared to the positioning in in vivo images was reduced from 34% to 7% through slicing at the best match angle. Reference dye marks were generated, which were visible in ex vivo imaging, in the tissue sections before and after processing, and in histology sections. Conclusions: The method achieved an almost fivefold reduction in the slice-matching error and is readily implementable in combination with standard MRI technology. The technique will be employed to generate datasets for correlation of whole-specimen prostate histopathology with in vivo diagnostic MRI using 3-D deformable registration, allowing assessment of the sensitivity and specificity of MRI parameters for prostate cancer. Although developed specifically for prostate, the method is readily adaptable to other types of whole tissue specimen, such as mastectomy or liver resection.« less

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.

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.

Automated classification of immunostaining patterns in breast tissue from the human protein atlas.

PubMed

Swamidoss, Issac Niwas; Kårsnäs, Andreas; Uhlmann, Virginie; Ponnusamy, Palanisamy; Kampf, Caroline; Simonsson, Martin; Wählby, Carolina; Strand, Robin

2013-01-01

The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many applications, ranging from antibody quality control to tumor grading.

PIXE analysis of mineral matter in thin sections of human lung

NASA Astrophysics Data System (ADS)

Annegarn, H. J.; Pillay, A. E.; Da Vies, J. C. A.; Faure, D.; Sellschop, J. P. F.

1988-12-01

It is postulated that insoluble mineral residues in the lungs of deceased miners may provide a quantitative measure of the integrated lifetime dust exposure. For epidemiological surveys rapid instrumental techniques are required to analyse representative samples of lung tissue. Particle-induced X-ray emission (PIXE) has been evaluated for analysis of microtomed slices of wax-embedded lung and lymph node (Hilar gland) tissue from deceased miners. The 50 μm slices, mounted on Mylar backings and placed in a He atmosphere, were irradiated using 3.2 MeV protons. PIXE analysis provided adequate sensitivity for key mineral elements including Si, Cr and Ti. The porous, nonuniform nature of lung tissue made it impossible to measure the tissue mass in the irradiated area, preventing the calculation of mass concentrations. Instead, biological sulphur was used as an internal standard, assuming that the fraction of S in soft, fat-free tissue is constant. Results are presented for lung and lymph node tissue from gold, chrome, copper, platinum and asbestos miners. Si mineral residues in lymph node tissue were found to be concentrated by a factor 50 relative to lung. Cr residues were clearly observed in the chrome miner's lung, but no excess of Cu was present in the copper miner's lung. There is evidence of preferential Si removal relative to Ti. Results warrant further development of PIXE for scanning of large numbers of lung samples prepared in the above manner.

The future of medical diagnostics: review paper

PubMed Central

2011-01-01

While histopathology of excised tissue remains the gold standard for diagnosis, several new, non-invasive diagnostic techniques are being developed. They rely on physical and biochemical changes that precede and mirror malignant change within tissue. The basic principle involves simple optical techniques of tissue interrogation. Their accuracy, expressed as sensitivity and specificity, are reported in a number of studies suggests that they have a potential for cost effective, real-time, in situ diagnosis. We review the Third Scientific Meeting of the Head and Neck Optical Diagnostics Society held in Congress Innsbruck, Innsbruck, Austria on the 11th May 2011. For the first time the HNODS Annual Scientific Meeting was held in association with the International Photodynamic Association (IPA) and the European Platform for Photodynamic Medicine (EPPM). The aim was to enhance the interdisciplinary aspects of optical diagnostics and other photodynamic applications. The meeting included 2 sections: oral communication sessions running in parallel to the IPA programme and poster presentation sessions combined with the IPA and EPPM posters sessions. PMID:21861912

Bilateral respiratory epithelial adenomatoid hamartoma of the olfactory cleft penetrating into the endocranium.

PubMed

Mladina, Ranko; Skitarelić, Neven; Poje, Gorazd; Vuković, Katarina

2011-09-01

Respiratory epithelial adenomatoid hamartomas (REAHs) of the nose and paranasal sinuses are relatively rare. These tumors usually do not extend over the boundaries of the nose and sinuses. The authors presented a 65-year-old man experiencing progressive hyposmia, followed by intermittent stubborn headache. The symptoms lasted for almost 2 years and were getting worse very slowly. Fiberendoscopy showed relatively discrete polypoid tissue occupying the olfactory cleft bilaterally. The computed tomography and magnetic resonance imaging suggested the possible lack of the cribriform plate and the unity and uniformity of the tissues located both in the endocranium and high in the nasal cavity. The clinical picture resembled very much a esthesineuroblastoma.The patient underwent endoscopic sinus surgery under the general hypotensive anesthesia. Frozen sections during the surgery showed REAH. The entire tumor was removed in a piece meal way, including both olfactory bulbs because they were involved within the pathologic tissue as well.This case showed that REAH could also be a locally aggressive process, penetrating even into the endocranium.

Immunocytochemical methods to study the distribution of Orientia tsutsugamushi in Leptotrombidium (Acari: Trombiculidae) chiggers.

PubMed

Myint, K S; Linthicum, K J; Tanskul, P; Lerdthusnee, K; Vaughn, D W; Manomuth, C; Mongkolsirichaikul, D; Hansukjariya, P; Hastriter, M W

1998-07-01

Immunocytochemical methods were developed and tested for their ability to detect the distribution of Orientia tsutsugamushi in paraffin sections of adult chiggers (Leptotrombidium imphalum Vercammen-Grandjean & Langston). Rickettsial antigen was detected by application of a simple direct or amplified immunocytochemistry procedure and an indirect immunofluorescent procedure. In the direct procedure alkaline phosphatase conjugation to the mouse polyclonal antibody to the Karp strain was followed by the HistoMark Red test system to detect rickettsial antigen. The amplification procedure used a similar method but used an unlabeled primary antibody followed by secondary biotinylated antimouse IgG, streptavidin-alkaline phosphatase, and the HistoMark Red test system. The immunofluorescent procedure included a biotinylated secondary antibody followed by addition of a streptavidin-FITC conjugate. Specific tissue tropisms in infected chiggers were observed in the salivary glands, nervous tissue, and ovaries of adult female mites in all procedures; however, nonspecific fluorescence of the chigger limited definitive identification of tissue tropisms with the indirect immunofluorescent procedure.

Validity for the simplified water displacement instrument to measure arm lymphedema as a result of breast cancer surgery.

PubMed

Sagen, Ase; Kåresen, Rolf; Skaane, Per; Risberg, May Arna

2009-05-01

To evaluate concurrent and construct validity for the Simplified Water Displacement Instrument (SWDI), an instrument for measuring arm volumes and arm lymphedema as a result of breast cancer surgery. Validity design. Hospital setting. Women (N=23; mean age, 64+/-11y) were examined 6 years after breast cancer surgery with axillary node dissection. Not applicable. The SWDI was included for measuring arm volumes to estimate arm lymphedema as a result of breast cancer surgery. A computed tomography (CT) scan was included to examine the cross-sectional areas (CSAs) in square millimeters for the subcutaneous tissue, for the muscle tissue, and for measuring tissue density in Hounsfield units. Magnetic resonance imaging (MRI) with T2-weighted sequences was included to show increased signal intensity in subcutaneous and muscle tissue areas. The affected arm volume measured by the SWDI was significantly correlated to the total CSA of the affected upper limb (R=.904) and also to the CSA of the subcutaneous tissue and muscles tissue (R=.867 and R=.725), respectively (P<.001). The CSA of the subcutaneous tissue for the upper limb was significantly larger compared with the control limb (11%). Tissue density measured in Hounsfield units did not correlate significantly with arm volume (P>.05). The affected arm volume was significantly larger (5%) than the control arm volume (P<.05). Five (22%) women had arm lymphedema defined as a 10% increase in the affected arm volume compared with the control arm volume, and an increased signal intensity was identified in all 5 women on MRI (T2-weighted, kappa=.777, P<.001). The SWDI showed high concurrent and construct validity as shown with significant correlations between the CSA (CT) of the subcutaneous and muscle areas of the affected limb and the affected arm volume (P>.001). There was a high agreement between those subjects who were diagnosed with arm lymphedema by using the SWDI and the increased signal intensity on MRI, with a kappa value of .777 (P<.001). High construct validity for the SWDI was confirmed for arm lymphedema as a volume increase, but it was not confirmed for lymphedema without an increase in arm volume (swelling). The SWDI is a simple and valid tool for estimating arm volume and arm lymphedema after breast cancer surgery.

An MRI-based leg model used to simulate biomechanical phenomena during cuff algometry: a finite element study.

PubMed

Manafi-Khanian, Bahram; Arendt-Nielsen, Lars; Graven-Nielsen, Thomas

2016-03-01

Cuff pressure stimulation is applicable for assessing deep-tissue pain sensitivity by exciting a variety of deep-tissue nociceptors. In this study, the relative transfer of biomechanical stresses and strains from the cuff via the skin to the muscle and the somatic tissue layers around bones were investigated. Cuff pressure was applied on the lower leg at three different stimulation intensities (mild pressure to pain). Three-dimensional finite element models including bones and three different layers of deep tissues were developed based on magnetic resonance images (MRI). The skin indentation maps at mild pressure, pain threshold, and intense painful stimulations were extracted from MRI and applied to the model. The mean stress under the cuff position around tibia was 4.6, 4.9 and around fibula 14.8, 16.4 times greater than mean stress of muscle surface in the same section at pain threshold and intense painful stimulations, respectively. At the same stimulation intensities, the mean strains around tibia were 36.4, 42.3 % and around fibula 32.9, 35.0 %, respectively, of mean strain on the muscle surface. Assuming strain as the ideal stimulus for nociceptors the results suggest that cuff algometry is less capable to challenge the nociceptors of tissues around bones as compared to more superficially located muscles.

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

Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

PubMed Central

Leigh, Steven Y.; Chen, Ye; Liu, Jonathan T.C.

2014-01-01

A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively ‘labels’ in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues. PMID:24940534

Multiphoton microscopic imaging of histological sections without hematoxylin and eosin staining differentiates carcinoma in situ lesion from normal oesophagus

NASA Astrophysics Data System (ADS)

Chen, Jianxin; Xu, Jian; Kang, Deyong; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Jiang, Xingshan

2013-10-01

Multiphoton microscopy (MPM) has become a powerful, important tool for tissues imaging at the molecular level. In this paper, this technique was extended to histological investigations, differentiating carcinoma in situ (CIS) lesion from normal oesophagus by imaging histological sections without hematoxylin and eosin (H&E) staining. The results show that the histology procedures of dehydration, paraffin embedding, and de-paraffinizing highlighted two photon excited fluorescence of cytoplasm and nucleolus of epithelial cell and collagen in stroma. MPM has the ability to identify the characteristics of CIS lesion including changes of squamous cells and full epithelium, identification of basement membrane, especially prominent nucleolus. The studies described here show that MPM has the potential for future retrospective studies of tumor staging by employing on histological section specimens without H&E staining.

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.

Optical Brain Imaging: A Powerful Tool for Neuroscience.

PubMed

Zhu, Xinpei; Xia, Yanfang; Wang, Xuecen; Si, Ke; Gong, Wei

2017-02-01

As the control center of organisms, the brain remains little understood due to its complexity. Taking advantage of imaging methods, scientists have found an accessible approach to unraveling the mystery of neuroscience. Among these methods, optical imaging techniques are widely used due to their high molecular specificity and single-molecule sensitivity. Here, we overview several optical imaging techniques in neuroscience of recent years, including brain clearing, the micro-optical sectioning tomography system, and deep tissue imaging.

Modeling Analysis of Biomechanical Changes of Middle Ear and Cochlea in Otitis Media

NASA Astrophysics Data System (ADS)

Gan, Rong Z.; Zhang, Xiangming; Guan, Xiying

2011-11-01

A comprehensive finite element (FE) model of the human ear including the ear canal, middle ear, and spiral cochlea was developed using histological sections of human temporal bone. The cochlea was modeled with three chambers separated by the basilar membrane and Reissner's membrane and filled with perilymphatic fluid. The viscoelastic material behavior was applied to middle ear soft tissues based on dynamic measurements of tissues in our lab. The model was validated using the experimental data obtained in human temporal bones and then used to simulate various stages of otitis media (OM) including the changes of morphology, mechanical properties, pressure, and fluid level in the middle ear. Function alterations of the middle ear and cochlea in OM were derived from the model and compared with the measurements from temporal bones. This study indicates that OM can be simulated in the FE model to predict the hearing loss induced by biomechanical changes of the middle ear and cochlea.

Seasonal gametogenesis of host sea anemone ( Entacmaea quadricolor) inhabiting Hong Kong waters

NASA Astrophysics Data System (ADS)

Bi, Ying; Zhang, Bin; Zhang, Zhifeng; Qiu, Jianwen

2015-02-01

Studying gonadal development of annual cycle can reveal the process of gametogenesis and reproductive period, and evaluate fertility and source utilization of a species. Host sea anemones are conspicuous members of tropical and subtropical reef ecosystems, but little is known about its biology including reproductive seasonality. Here we reported a one-year study on the gametogenesis and reproduction of host sea anemone ( Entacmaea quadricolor) inhabiting Hong Kong waters. E. quadricolor tissues were sampled in 12 occasions from 5 m and 15 m depths of water, respectively. Histological sectioning of the tissues showed that E. quadricolor was dioecious, and populational ratio of female to male was 1:1.6. The gonadal development was asynchronous within an annual cycle, which included proliferating, growing, maturing, spawning, and resting stages. The spawning occurred between August and October when surface seawater temperature reached the annual maximum (28°C), suggesting that temperature is an important factor modulating the gonadal development and mature of E. quadricolor.

Realistic 3D computer model of the gerbil middle ear, featuring accurate morphology of bone and soft tissue structures.

PubMed

Buytaert, Jan A N; Salih, Wasil H M; Dierick, Manual; Jacobs, Patric; Dirckx, Joris J J

2011-12-01

In order to improve realism in middle ear (ME) finite-element modeling (FEM), comprehensive and precise morphological data are needed. To date, micro-scale X-ray computed tomography (μCT) recordings have been used as geometric input data for FEM models of the ME ossicles. Previously, attempts were made to obtain these data on ME soft tissue structures as well. However, due to low X-ray absorption of soft tissue, quality of these images is limited. Another popular approach is using histological sections as data for 3D models, delivering high in-plane resolution for the sections, but the technique is destructive in nature and registration of the sections is difficult. We combine data from high-resolution μCT recordings with data from high-resolution orthogonal-plane fluorescence optical-sectioning microscopy (OPFOS), both obtained on the same gerbil specimen. State-of-the-art μCT delivers high-resolution data on the 3D shape of ossicles and other ME bony structures, while the OPFOS setup generates data of unprecedented quality both on bone and soft tissue ME structures. Each of these techniques is tomographic and non-destructive and delivers sets of automatically aligned virtual sections. The datasets coming from different techniques need to be registered with respect to each other. By combining both datasets, we obtain a complete high-resolution morphological model of all functional components in the gerbil ME. The resulting 3D model can be readily imported in FEM software and is made freely available to the research community. In this paper, we discuss the methods used, present the resulting merged model, and discuss the morphological properties of the soft tissue structures, such as muscles and ligaments.

Risk factors for eye bank preparation failure of Descemet membrane endothelial keratoplasty tissue.

PubMed

Vianna, Lucas M M; Stoeger, Christopher G; Galloway, Joshua D; Terry, Mark; Cope, Leslie; Belfort, Rubens; Jun, Albert S

2015-05-01

To assess the results of a single eye bank preparing a high volume of Descemet membrane endothelial keratoplasty (DMEK) tissues using multiple technicians to provide an overview of the experience and to identify possible risk factors for DMEK preparation failure. Cross-sectional study. setting: Lions VisionGift and Wilmer Eye Institute at Johns Hopkins Hospital. All 563 corneal tissues processed by technicians at Lions VisionGift for DMEK between October 2011 and May 2014 inclusive. Tissues were divided into 2 groups: DMEK preparation success and DMEK preparation failure. We compared donor characteristics, including past medical history. The overall tissue preparation failure rate was 5.2%. Univariate analysis showed diabetes mellitus (P = .000028) and its duration (P = .023), hypertension (P = .021), and hyperlipidemia or obesity (P = .0004) were more common in the failure group. Multivariate analysis showed diabetes mellitus (P = .0001) and hyperlipidemia or obesity (P = .0142) were more common in the failure group. Elimination of tissues from donors either with diabetes or with hyperlipidemia or obesity reduced the failure rate from 5.2% to 2.2%. Trends toward lower failure rates occurring with increased technician experience also were found. Our work showed that tissues from donors with diabetes mellitus (especially with longer disease duration) and hyperlipidemia or obesity were associated with higher failure rates in DMEK preparation. Elimination of tissues from donors either with diabetes mellitus or with hyperlipidemia or obesity reduced the failure rate. In addition, our data may provide useful initial guidelines and benchmark values for eye banks seeking to establish and maintain DMEK programs. Copyright © 2015 Elsevier Inc. All rights reserved.

Method Optimization for Extracting High-Quality RNA From the Human Pancreas Tissue.

PubMed

Jun, Eunsung; Oh, Juyun; Lee, Song; Jun, Hye-Ryeong; Seo, Eun Hye; Jang, Jin-Young; Kim, Song Cheol

2018-06-01

Nucleic acid sequencing is frequently used to determine the molecular basis of diseases. Therefore, proper storage of biological specimens is essential to inhibit nucleic acid degradation. RNA isolated from the human pancreas is generally of poor quality because of its high concentration of endogenous RNase. In this study, we optimized the method for extracting high quality RNA from paired tumor and normal pancreatic tissues obtained from eight pancreatic cancer patients post-surgery. RNA integrity number (RIN) was checked to evaluate the integrity of RNA, we tried to extract the RNA with an RIN value of 8 or higher that allows for the latest genetic analysis. The effect of several parameters, including the method used for tissue lysis, RNAlater treatment, tissue weight at storage, and the time to storage after surgical resection, on the quantity and quality of RNA extracted was examined. Data showed that the highest quantity of RNA was isolated using a combination of manual and mechanical methods of tissue lysis. Additionally, sectioning the tissues into small pieces (<100 mg) and treating them with RNAlater solution prior to storage increased RNA stability. Following these guidelines, high quality RNA was obtained from 100% (8/8) of tumor tissues and 75% (6/8) of normal tissues. High-quality RNA was still stable under repeated freezing and thawing. The application of these results during sample handling and storage in clinical settings will facilitate the genetic diagnosis of diseases and their subsequent treatment. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Online, absolute quantitation of propranolol from spatially distinct 20-μm and 40-μm dissections of brain, liver, and kidney thin tissue sections by laser microdissection – liquid vortex capture – mass spectrometry

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

Kertesz, Vilmos; Vavrek, Marissa; Freddo, Carol

Here, spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser cut and drop sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm x 20more » μm or 40 μm x 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d 7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolold-d 7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser cut and drop sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings.« less

Online, absolute quantitation of propranolol from spatially distinct 20-μm and 40-μm dissections of brain, liver, and kidney thin tissue sections by laser microdissection – liquid vortex capture – mass spectrometry

DOE PAGES

Kertesz, Vilmos; Vavrek, Marissa; Freddo, Carol; ...

2016-05-23

Here, spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser cut and drop sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm x 20more » μm or 40 μm x 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d 7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolold-d 7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser cut and drop sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings.« less

Predominant mucosal expression of 5-HT4(+h) receptor splice variants in pig stomach and colon

PubMed Central

Priem, Evelien KV; De Maeyer, Joris H; Vandewoestyne, Mado; Deforce, Dieter; Lefebvre, Romain A

2013-01-01

AIM: To investigate cellular 5-HT4(-h/+h) receptor distribution, particularly in the epithelial layer, by laser microdissection and polymerase chain reaction (PCR) in porcine gastrointestinal (GI) tissues. METHODS: A stepwise approach was used to evaluate RNA quality and to study cell-specific 5-HT4 receptor mRNA expression in the porcine gastric fundus and colon descendens. After freezing, staining and laser microdissection and pressure catapulting (LMPC), RNA quality was evaluated by the Experion automated electrophoresis system. 5-HT4 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were examined by endpoint reverse transcription (RT)-PCR in mucosal and muscle-myenteric plexus (MMP) tissue fractions, in mucosal and MMP parts of hematoxylin and eosin (HE) stained tissue sections and in microdissected patches of the epithelial and circular smooth muscle cell layer in these sections. Pig gastric fundus tissue sections were also stained immunohistochemically (IHC) for enterochromaffin cells (EC cells; MAB352); these cells were isolated by LMPC and examined by endpoint RT-PCR. RESULTS: After HE staining, the epithelial and circular smooth muscle cell layer of pig colon descendens and the epithelial cell layer of gastric fundus were identified morphologically and isolated by LMPC. EC cells of pig gastric fundus were successfully stained by IHC and isolated by LMPC. Freezing, HE and IHC staining, and LMPC had no influence on RNA quality. 5-HT4 receptor and GAPDH mRNA expressions were detected in mucosa and MMP tissue fractions, and in mucosal and MMP parts of HE stained tissue sections of pig colon descendens and gastric fundus. In the mucosa tissue fractions of both GI regions, the expression of h-exon containing receptor [5-HT4(+h) receptor] mRNA was significantly higher (P < 0.01) compared to 5-HT4(-h) receptor expression, and a similar trend was obtained in the mucosal part of HE stained tissue sections. Large microdissected patches of the epithelial and circular smooth muscle cell layer of pig colon descendens and of the epithelial cell layer of pig gastric fundus, also showed 5-HT4 receptor and GAPDH mRNA expression. No 5-HT4 receptor mRNA expression was detected in gastric LMPC-isolated EC cells from IHC stained tissues, which cells were positive for GAPDH. CONCLUSION: Porcine GI mucosa predominantly expresses 5-HT4(+h) receptor splice variants, suggesting their contribution to the 5-HT4 receptor-mediated mucosal effects of 5-HT. PMID:23840113

Functional and Anatomical Outcomes of Facial Nerve Injury With Application of Polyethylene Glycol in a Rat Model.

PubMed

Brown, Brandon L; Asante, Tony; Welch, Haley R; Sandelski, Morgan M; Drejet, Sarah M; Shah, Kishan; Runge, Elizabeth M; Shipchandler, Taha Z; Jones, Kathryn J; Walker, Chandler L

2018-05-17

Functional and anatomical outcomes after surgical repair of facial nerve injury may be improved with the addition of polyethylene glycol (PEG) to direct suture neurorrhaphy. The application of PEG has shown promise in treating spinal nerve injuries, but its efficacy has not been evaluated in treatment of cranial nerve injuries. To determine whether PEG in addition to neurorrhaphy can improve functional outcomes and synkinesis after facial nerve injury. In this animal experiment, 36 rats underwent right facial nerve transection and neurorrhaphy with addition of PEG. Weekly behavioral scoring was done for 10 rats for 6 weeks and 14 rats for 16 weeks after the operations. In the 16-week study, the buccal branches were labeled and tissue analysis was performed. In the 6-week study, the mandibular and buccal branches were labeled and tissue analysis was performed. Histologic analysis was performed for 10 rats in a 1-week study to assess the association of PEG with axonal continuity and Wallerian degeneration. Six rats served as the uninjured control group. Data were collected from February 8, 2016, through July 10, 2017. Polyethylene glycol applied to the facial nerve after neurorrhaphy. Functional recovery was assessed weekly for the 16- and 6-week studies, as well as motoneuron survival, amount of regrowth, specificity of regrowth, and aberrant branching. Short-term effects of PEG were assessed in the 1-week study. Among the 40 male rats included in the study, PEG addition to neurorrhaphy showed no functional benefit in eye blink reflex (mean [SEM], 3.57 [0.88] weeks; 95% CI, -2.8 to 1.9 weeks; P = .70) or whisking function (mean [SEM], 4.00 [0.72] weeks; 95% CI, -3.6 to 2.4 weeks; P = .69) compared with suturing alone at 16 weeks. Motoneuron survival was not changed by PEG in the 16-week (mean, 132.1 motoneurons per tissue section; 95% CI, -21.0 to 8.4; P = .13) or 6-week (mean, 131.1 motoneurons per tissue section; 95% CI, -11.0 to 10.0; P = .06) studies. Compared with controls, neither surgical group showed differences in buccal branch regrowth at 16 (36.9 motoneurons per tissue section; 95% CI, -14.5 to 22.0; P = .28) or 6 (36.7 motoneurons per tissue section; 95% CI, -7.8 to 18.5; P = .48) weeks or in the mandibular branch at 6 weeks (25.2 motoneurons per tissue section; 95% CI, -14.5 to 15.5; P = .99). Addition of PEG had no advantage in regrowth specificity compared with suturing alone at 16 weeks (15.3% buccal branch motoneurons with misguided projections; 95% CI, -7.2% to 11.0%; P = .84). After 6 weeks, the number of motoneurons with misguided projections to the mandibular branch showed no advantage of PEG treatment compared with suturing alone (12.1% buccal branch motoneurons with misguided projections; 95% CI, -8.2% to 9.2%; P = .98). In the 1-week study, improved axonal continuity and muscular innervation were not observed in PEG-treated rats. Although PEG has shown efficacy in treating other nervous system injuries, PEG in addition to neurorraphy was not beneficial in a rat model of facial nerve injury. The addition of PEG to suturing may not be warranted in the surgical repair of facial nerve injury. NA.

Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.

PubMed

Fumoto, Shintaro; Nishimura, Koyo; Nishida, Koyo; Kawakami, Shigeru

2016-01-01

Evaluation methods for determining the distribution of transgene expression in the body and the in vivo fate of viral and non-viral vectors are necessary for successful development of in vivo gene delivery systems. Here, we evaluated the spatial distribution of transgene expression using tissue clearing methods. After hydrodynamic injection of plasmid DNA into mice, whole tissues were subjected to tissue clearing. Tissue clearing followed by confocal laser scanning microscopy enabled evaluation of the three-dimensional distribution of transgene expression without preparation of tissue sections. Among the tested clearing methods (ClearT2, SeeDB, and CUBIC), CUBIC was the most suitable method for determining the spatial distribution of transgene expression in not only the liver but also other tissues such as the kidney and lung. In terms of the type of fluorescent protein, the observable depth for green fluorescent protein ZsGreen1 was slightly greater than that for red fluorescent protein tdTomato. We observed a depth of ~1.5 mm for the liver and 500 μm for other tissues without preparation of tissue sections. Furthermore, we succeeded in multicolor deep imaging of the intracellular fate of plasmid DNA in the murine liver. Thus, tissue clearing would be a powerful approach for determining the spatial distribution of plasmid DNA and transgene expression in various murine tissues.

Morphological, ultrastructural and functional imaging of frozen/thawed and vitrified/warmed human ovarian tissue retrieved from oncological patients.

PubMed

Fabbri, R; Vicenti, R; Macciocca, M; Martino, N A; Dell'Aquila, M E; Pasquinelli, G; Morselli-Labate, A M; Seracchioli, R; Paradisi, R

2016-08-01

Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Polysaccharide‐Based Controlled Release Systems for Therapeutics Delivery and Tissue Engineering: From Bench to Bedside

PubMed Central

Miao, Tianxin; Wang, Junqing; Zeng, Yun; Chen, Xiaoyuan

2018-01-01

Abstract Polysaccharides or polymeric carbohydrate molecules are long chains of monosaccharides that are linked by glycosidic bonds. The naturally based structural materials are widely applied in biomedical applications. This article covers four different types of polysaccharides (i.e., alginate, chitosan, hyaluronic acid, and dextran) and emphasizes their chemical modification, preparation approaches, preclinical studies, and clinical translations. Different cargo fabrication techniques are also presented in the third section. Recent progresses in preclinical applications are then discussed, including tissue engineering and treatment of diseases in both therapeutic and monitoring aspects. Finally, clinical translational studies with ongoing clinical trials are summarized and reviewed. The promise of new development in nanotechnology and polysaccharide chemistry helps clinical translation of polysaccharide‐based drug delivery systems. PMID:29721408

Evaluation of different tissue de-paraffinization procedures for infrared spectral imaging.

PubMed

Nallala, Jayakrupakar; Lloyd, Gavin Rhys; Stone, Nicholas

2015-04-07

In infrared spectral histopathology, paraffin embedded tissues are often de-paraffinized using chemical agents such as xylene and hexane. These chemicals are known to be toxic and the routine de-waxing procedure is time consuming. A comparative study was carried out to identify alternate de-paraffinization methods by using paraffin oil and electronic de-paraffinization (using a mathematical computer algorithm) and their effectiveness was compared to xylene and hexane. Sixteen adjacent tissue sections obtained from a single block of a normal colon tissue were de-paraffinized using xylene, hexane and paraffin oil (+ hexane wash) at five different time points each for comparison. One section was reserved unprocessed for electronic de-paraffinization based on a modified extended multiplicative signal correction (EMSC). IR imaging was carried out on these tissue sections. Coefficients based on the fit of a pure paraffin model to the IR images were then calculated to estimate the amount of paraffin remaining after processing. Results indicate that on average xylene removes more paraffin in comparison to hexane and paraffin oil although the differences were small. This makes paraffin oil, followed by a hexane wash, an interesting and less toxic alternative method of de-paraffinization. However, none of the chemical methods removed paraffin completely from the tissues at any given time point. Moreover, paraffin was removed more easily from the glandular regions than the connective tissue regions indicating a form of differential paraffin retention based on the histology. In such cases, the use of electronic de-paraffinization to neutralize such variances across different tissue regions might be considered. Moreover it is faster, reduces scatter artefacts by index matching and enables samples to be easily stored for further analysis if required.

Wavelet analysis in two-dimensional tomography

NASA Astrophysics Data System (ADS)

Burkovets, Dimitry N.

2002-02-01

The diagnostic possibilities of wavelet-analysis of coherent images of connective tissue in its pathological changes diagnostics. The effectiveness of polarization selection in obtaining wavelet-coefficients' images is also shown. The wavelet structures, characterizing the process of skin psoriasis, bone-tissue osteoporosis have been analyzed. The histological sections of physiological normal and pathologically changed samples of connective tissue of human skin and spongy bone tissue have been analyzed.

Optimization of a Histopathological Biomarker for Sphingomyelin Accumulation in Acid Sphingomyelinase Deficiency

PubMed Central

Johnson, Jennifer; Maloney, Colleen L.; Yandl, Emily; Griffiths, Denise; Thurberg, Beth L.; Ryan, Susan

2012-01-01

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy. PMID:22614361

Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

PubMed

Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

2018-01-01

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

Cell-level temperature distributions in skeletal muscle post spinal cord injury as related to deep tissue injury.

PubMed

Ruschkewitz, Yael; Gefen, Amit

2010-02-01

Deep tissue injury (DTI) is a severe pressure ulcer, which initiates in skeletal muscle tissue under intact skin. Patients with spinal cord injury (SCI) are especially vulnerable to DTI, due to their impaired motosensory capacities. The underlying mechanisms that lead to DTI are, however, still poorly understood. This study focuses on cell-level temperature distributions in muscles of patients with SCI, which typically contain thinner muscle fibers and fewer capillaries. It has been shown previously by our group that ischemic muscles of rat models of DTI cool down mildly and locally, which is very likely to slow the diffusivity of metabolites in the ischemic regions. However, it is unclear how these temperature decreases affect diffusivity at the scale of individual muscle cells in the microanatomy of SCI patients. We hypothesize that a 2 degrees C drop in the temperature of inflowing capillary blood, as shown in our animal studies, has a substantial effect on lowering the diffusivity of metabolites in skeletal muscle, but the pathological microanatomy in the chronic phase of SCI is less dominant in affecting the local temperatures in and around muscle cells. In order to test this hypothesis, two-dimensional finite element (FE) models of cross sections through the microanatomy of muscle tissue were developed using COMSOL Multiphysics software for normal and SCI muscles. The models included muscle cells, extracellular matrix (ECM), and capillaries, each with its own geometrical, thermal, and heat production properties. The SCI model configuration specifically included reduced cross section of myofibrils in favor of more ECM, less capillaries, and decreased blood inflow rate. After a 20-s heat transfer simulation, it was found that temperatures around the cells of the SCI muscle were approximately 2 degrees C lower than that in the normal muscle, that is, heat production from the muscle cell metabolism did not compensate for the lower inflowing blood temperature in the SCI model. We conclude that the temperature and rate of inflowing capillary blood are the dominant factors determining the localized temperatures in the microarchitecture of an ischemic SCI muscle tissue. The altered SCI microanatomy was shown to be less influential. Taken together with the Stokes-Einstein theory, our results indicate that diffusivity of metabolites would be approximately 50% less around the cells of SCI muscle due to local cooling, which is yet another factor compromising tissue viability in the patients with SCI.

Body frame size in school children is related to the amount of adipose tissue in different depots but not to adipose distribution.

PubMed

Guzmán-de la Garza, Francisco J; González Ayala, Alejandra E; Gómez Nava, Marisol; Martínez Monsiváis, Leislie I; Salinas Martínez, Ana M; Ramírez López, Erik; Mathiew Quirós, Alvaro; Garcia Quintanilla, Francisco

2017-09-10

The main aim of this study was to test the hypothesis that body frame size is related to the amount of fat in different adipose tissue depots and to fat distribution in schoolchildren. Children aged between 5 and 10 years were included in this cross-sectional study (n = 565). Body frame size, adiposity markers (anthropometric, skinfolds thickness, and ultrasound measures), and fat distribution indices were analyzed. Correlation coefficients adjusted by reliability were estimated and analyzed by sex; the significance of the difference between two correlation coefficients was assessed using the Fisher z-transformation. The sample included primarily urban children; 58.6% were normal weight, 16.1% overweight, 19.6% obese, and the rest were underweight. Markers of subcutaneous adiposity, fat mass and fat-free mass, and preperitoneal adiposity showed higher and significant correlations with the sum of the biacromial + bitrochanteric diameter than with the elbow diameter, regardless of sex. The fat distribution conicity index presented significant but weak correlations; and visceral adipose tissue, hepatic steatosis, and the waist-for-hip ratio were not significantly correlated with body frame size measures. Body frame size in school children was related to the amount of adipose tissue in different depots, but not adipose distribution. More studies are needed to confirm this relationship and its importance to predict changes in visceral fat deposition during growth. © 2017 Wiley Periodicals, Inc.

Immunohistochemical characterization of neoplastic cells of breast origin.

PubMed

Noriega, Mariadelasmercedes; Paesani, Fernando; Perazzo, Florencia; Lago, Néstor; Krupitzki, Hugo; Nieto, Silvana; Garcia, Alejandro; Avagnina, Alejandra; Elsner, Boris; Denninghoff, Valeria Cecilia

2012-06-22

After skin cancer, breast cancer is the most common malignancy in women. Tumors of unknown origin account for 5-15% of malignant neoplasms, with 1.5% being breast cancer. An immunohistochemical panel with conventional and newer markers, such as mammaglobin, was selected for the detection of neoplastic cells of breast origin. The specific objectives are: 1) to determine the sensitivity and specificity of the panel, with a special emphasis on the inclusion of the mammaglobin marker, and 2) to compare immunohistochemistry performed on whole tissue sections and on tissue micro-array. Twenty-nine metastatic breast tumors were included and assumed as tumors of unknown origin. Other 48 biopsies of diverse tissues were selected and assumed as negative controls. Tissue Micro-Array was performed. Immunohistochemistry for mammaglobin, gross cystic disease fluid protein-15, estrogen receptor, progesterone receptor and cytokeratin 7 was done. Mammaglobin positive staining was observed in 10/29 cases, in 13/29 cases for gross cystic disease fluid protein-15, in 20/29 cases for estrogen receptor, in 9/29 cases for progesterone receptor, and in 25/29 cases for cytokeratin 7. Among the negative controls, mammaglobin was positive in 2/48, and gross cystic disease fluid protein-15 in 4/48. The inclusion of MAG antibody in the immunohistochemical panel for the detection of tumors of unknown origin contributed to the detection of metastasis of breast cancer. The diagnostic strategy with the highest positive predictive value (88%) included hormone receptors and mammaglobin in serial manner.

Obesity, regional body fat distribution, and the metabolic syndrome in older men and women.

PubMed

Goodpaster, Bret H; Krishnaswami, Shanthi; Harris, Tamara B; Katsiaras, Andreas; Kritchevsky, Steven B; Simonsick, Eleanor M; Nevitt, Michael; Holvoet, Paul; Newman, Anne B

2005-04-11

The metabolic syndrome is a disorder that includes dyslipidemia, insulin resistance, and hypertension and is associated with an increased risk of diabetes and cardiovascular disease. We determined whether patterns of regional fat deposition are associated with metabolic syndrome in older adults. A cross-sectional study was performed that included a random, population-based, volunteer sample of Medicare-eligible adults within the general communities of Pittsburgh, Pa, and Memphis, Tenn. The subjects consisted of 3035 men and women aged 70 to 79 years, of whom 41.7% were black. Metabolic syndrome was defined by Adult Treatment Panel III criteria, including serum triglyceride level, high-density lipoprotein cholesterol level, glucose level, blood pressure, and waist circumference. Visceral, subcutaneous abdominal, intermuscular, and subcutaneous thigh adipose tissue was measured by computed tomography. Visceral adipose tissue was associated with the metabolic syndrome in men who were of normal weight (odds ratio, 95% confidence interval: 2.1, 1.6-2.9), overweight (1.8, 1.5-2.1), and obese (1.2, 1.0-1.5), and in women who were of normal weight (3.3, 2.4-4.6), overweight (2.4, 2.0-3.0), and obese (1.7, 1.4-2.1), adjusting for race. Subcutaneous abdominal adipose tissue was associated with the metabolic syndrome only in normal-weight men (1.3, 1.1-1.7). Intermuscular adipose tissue was associated with the metabolic syndrome in normal-weight (2.3, 1.6-3.5) and overweight (1.2, 1.1-1.4) men. In contrast, subcutaneous thigh adipose tissue was inversely associated with the metabolic syndrome in obese men (0.9, 0.8-1.0) and women (0.9, 0.9-1.0). In addition to general obesity, the distribution of body fat is independently associated with the metabolic syndrome in older men and women, particularly among those of normal body weight.

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.

Automation of 3D reconstruction of neural tissue from large volume of conventional serial section transmission electron micrographs.

PubMed

Mishchenko, Yuriy

2009-01-30

We describe an approach for automation of the process of reconstruction of neural tissue from serial section transmission electron micrographs. Such reconstructions require 3D segmentation of individual neuronal processes (axons and dendrites) performed in densely packed neuropil. We first detect neuronal cell profiles in each image in a stack of serial micrographs with multi-scale ridge detector. Short breaks in detected boundaries are interpolated using anisotropic contour completion formulated in fuzzy-logic framework. Detected profiles from adjacent sections are linked together based on cues such as shape similarity and image texture. Thus obtained 3D segmentation is validated by human operators in computer-guided proofreading process. Our approach makes possible reconstructions of neural tissue at final rate of about 5 microm3/manh, as determined primarily by the speed of proofreading. To date we have applied this approach to reconstruct few blocks of neural tissue from different regions of rat brain totaling over 1000microm3, and used these to evaluate reconstruction speed, quality, error rates, and presence of ambiguous locations in neuropil ssTEM imaging data.

Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections.

PubMed

Wegner, Kyle A; Keikhosravi, Adib; Eliceiri, Kevin W; Vezina, Chad M

2017-08-01

The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

Quantitation of repaglinide and metabolites in mouse whole-body thin tissue sections using droplet-based liquid microjunction surface sampling-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

PubMed

Chen, Weiqi; Wang, Lifei; Van Berkel, Gary J; Kertesz, Vilmos; Gan, Jinping

2016-03-25

Herein, quantitation aspects of a fully automated autosampler/HPLC-MS/MS system applied for unattended droplet-based surface sampling of repaglinide dosed thin tissue sections with subsequent HPLC separation and mass spectrometric analysis of parent drug and various drug metabolites were studied. Major organs (brain, lung, liver, kidney and muscle) from whole-body thin tissue sections and corresponding organ homogenates prepared from repaglinide dosed mice were sampled by surface sampling and by bulk extraction, respectively, and analyzed by HPLC-MS/MS. A semi-quantitative agreement between data obtained by surface sampling and that by employing organ homogenate extraction was observed. Drug concentrations obtained by the two methods followed the same patterns for post-dose time points (0.25, 0.5, 1 and 2 h). Drug amounts determined in the specific tissues was typically higher when analyzing extracts from the organ homogenates. In addition, relative comparison of the levels of individual metabolites between the two analytical methods also revealed good semi-quantitative agreement. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitation of repaglinide and metabolites in mouse whole-body thin tissue sections using droplet-based liquid microjunction surface sampling-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

DOE PAGES

Chen, Weiqi; Wang, Lifei; Van Berkel, Gary J.; ...

2015-11-03

Herein, quantitation aspects of a fully automated autosampler/HPLC-MS/MS system applied for unattended droplet-based surface sampling of repaglinide dosed thin tissue sections with subsequent HPLC separation and mass spectrometric analysis of parent drug and various drug metabolites was studied. Major organs (brain, lung, liver, kidney, muscle) from whole-body thin tissue sections and corresponding organ homogenates prepared from repaglinide dosed mice were sampled by surface sampling and by bulk extraction, respectively, and analyzed by HPLC-MS/MS. A semi-quantitative agreement between data obtained by surface sampling and that by employing organ homogenate extraction was observed. Drug concentrations obtained by the two methods followed themore » same patterns for post-dose time points (0.25, 0.5, 1 and 2 h). Drug amounts determined in the specific tissues was typically higher when analyzing extracts from the organ homogenates. Furthermore, relative comparison of the levels of individual metabolites between the two analytical methods also revealed good semi-quantitative agreement.« less

Quantitation of repaglinide and metabolites in mouse whole-body thin tissue sections using droplet-based liquid microjunction surface sampling-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

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

Chen, Weiqi; Wang, Lifei; Van Berkel, Gary J.

Herein, quantitation aspects of a fully automated autosampler/HPLC-MS/MS system applied for unattended droplet-based surface sampling of repaglinide dosed thin tissue sections with subsequent HPLC separation and mass spectrometric analysis of parent drug and various drug metabolites was studied. Major organs (brain, lung, liver, kidney, muscle) from whole-body thin tissue sections and corresponding organ homogenates prepared from repaglinide dosed mice were sampled by surface sampling and by bulk extraction, respectively, and analyzed by HPLC-MS/MS. A semi-quantitative agreement between data obtained by surface sampling and that by employing organ homogenate extraction was observed. Drug concentrations obtained by the two methods followed themore » same patterns for post-dose time points (0.25, 0.5, 1 and 2 h). Drug amounts determined in the specific tissues was typically higher when analyzing extracts from the organ homogenates. Furthermore, relative comparison of the levels of individual metabolites between the two analytical methods also revealed good semi-quantitative agreement.« less

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.

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.

Novel Immunohistochemical Techniques Using Discrete Signal Amplification Systems for Human Cutaneous Peripheral Nerve Fiber Imaging

PubMed Central

Wang, Ningshan; Gibbons, Christopher H.; Freeman, Roy

2011-01-01

Confocal imaging uses immunohistochemical binding of specific antibodies to visualize tissues, but technical obstacles limit more widespread use of this technique in the imaging of peripheral nerve tissue. These obstacles include same-species antibody cross-reactivity and weak fluorescent signals of individual and co-localized antigens. The aims of this study were to develop new immunohistochemical techniques for imaging of peripheral nerve fibers. Three-millimeter punch skin biopsies of healthy individuals were fixed, frozen, and cut into 50-µm sections. Tissues were stained with a variety of antibody combinations with two signal amplification systems, streptavidin-biotin-fluorochrome (sABC) and tyramide-horseradish peroxidase-fluorochrome (TSA), used simultaneously to augment immunohistochemical signals. The combination of the TSA and sABC amplification systems provided the first successful co-localization of sympathetic adrenergic and sympathetic cholinergic nerve fibers in cutaneous human sweat glands and vasomotor and pilomotor systems. Primary antibodies from the same species were amplified individually without cross-reactivity or elevated background interference. The confocal fluorescent signal-to-noise ratio increased, and image clarity improved. These modifications to signal amplification systems have the potential for widespread use in the study of human neural tissues. PMID:21411809

[Registration and 3D rendering of serial tissue section images].

PubMed

Liu, Zhexing; Jiang, Guiping; Dong, Wu; Zhang, Yu; Xie, Xiaomian; Hao, Liwei; Wang, Zhiyuan; Li, Shuxiang

2002-12-01

It is an important morphological research method to reconstruct the 3D imaging from serial section tissue images. Registration of serial images is a key step to 3D reconstruction. Firstly, an introduction to the segmentation-counting registration algorithm is presented, which is based on the joint histogram. After thresholding of the two images to be registered, the criterion function is defined as counting in a specific region of the joint histogram, which greatly speeds up the alignment process. Then, the method is used to conduct the serial tissue image matching task, and lies a solid foundation for 3D rendering. Finally, preliminary surface rendering results are presented.

Next-gen tissue: preservation of molecular and morphological fidelity in prostate tissue.

PubMed

Gillard, Marc; Tom, Westin R; Antic, Tatjana; Paner, Gladell P; Lingen, Mark W; VanderWeele, David J

2015-01-01

Personalization of cancer therapy requires molecular evaluation of tumor tissue. Traditional tissue preservation involves formalin fixation, which degrades the quality of nucleic acids. Strategies to bank frozen prostate tissue can interfere with diagnostic studies. PAXgene is an alternative fixative that preserves protein and nucleic acid quality. Portions of prostates obtained from autopsy specimens were fixed in either 10% buffered formalin or PAXgene, and processed and embedded in paraffin. Additional sections were immediately embedded in OCT and frozen. DNA and RNA were extracted from the formalin-fixed, PAXgene-fixed, or frozen tissue. Quantitative PCR was used to compare the quality of DNA and RNA obtained from all three tissue types. In addition, 5 μm sections were cut from specimens devoid of cancer and from prostate cancer specimens obtained at prostatectomy and fixed in PAXgene. They were either stained with hematoxylin and eosin or interrogated with antibodies for p63, PSA and p504. Comparable tissue morphology was observed in both the formalin and PAXgene-fixed specimens. Similarly, immunohistochemical expression of the P63, PSA and P504 proteins was comparable between formalin and PAXgene fixation techniques. DNA from the PAXgene-fixed tissue was of similar quality to that from frozen tissue. RNA was also amplified with up to 8-fold greater efficiency in the PAXgene fixed tissue compared to the formalin-fixed tissue. Prostate specimens fixed with PAXgene have preserved histologic morphology, stain appropriately, and have preserved quality of nucleic acids. PAXgene fixation facilitates the use of prostatectomy tissue for molecular biology techniques such as next-generation sequencing.

Periodontal repair in dogs: examiner reproducibility in the supraalveolar periodontal defect model.

PubMed

Koo, Ki-Tae; Polimeni, Giuseppe; Albandar, Jasim M; Wikesjö, Ulf M E

2004-06-01

Histometric assessments are routinely used to evaluate biologic events ascertained in histologic sections acquired from animal and human studies. The objective of this study was to evaluate the intra- and inter-examiner reproducibility of histometric assessments in the supraalveolar periodontal defect model. Histometric analysis using incandescent and polarized light microscopy, an attached digital camera system, and a PC-based image analysis system including a custom program for the supraalveolar periodontal defect model was performed on histologic sections acquired from one jaw quadrant in each of 12 dogs. The animals had received an experimental protocol including implantation of a coral biomaterial and guided tissue regeneration (GTR) barrier devices, and were evaluated following a 4-week healing interval. Histometric parameters were recorded and repeated within a 3-month interval by two examiners following brief training. Intra- and inter-examiner reproducibility was assessed using the intra-class correlation coefficient (ICC). Most parameters showed high intra-examiner ICCs. Parameters including defect height, connective tissue repair, bone regeneration (height/area), formation of a junctional epithelium, positioning of the GTR device, ankylosis, root resorption, and defect area yielded an ICC> or 0..9. The ICCs for bone density and biomaterial density were somewhat lower (0.8 and 0.7, respectively). The inter-examiner reproducibility was somewhat lower compared to the intra-examiner reproducibility. Nevertheless, the ICCs were generally high (ICC range: 0.6-0.9). Histometric evaluations in the supraalveolar periodontal defect model yield highly reproducible results, in particular when a single examiner performs the histometric measurements, even when the examiner was exposed to limited training.

Histological Assessment of PAXgene Tissue Fixation and Stabilization Reagents

PubMed Central

Kap, Marcel; Smedts, Frank; Oosterhuis, Wolter; Winther, Rosa; Christensen, Nanna; Reischauer, Bilge; Viertler, Christian; Groelz, Daniel; Becker, Karl-Friedrich; Zatloukal, Kurt; Langer, Rupert; Slotta-Huspenina, Julia; Bodo, Koppany; de Jong, Bas; Oelmuller, Uwe; Riegman, Peter

2011-01-01

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities. PMID:22110732

Correlation of endoscopic optical coherence tomography with histology

NASA Astrophysics Data System (ADS)

Westphal, Volker; Rollins, Andrew M.; Willis, Joseph; Sivak, Michael J., Jr.; Izatt, Joseph A.

2000-04-01

Optical Coherence Tomography (OCT) is a noninvasive optical imaging technique that allows high-resolution cross- sectional imaging of tissue microstructure. We have recently developed a system for endoscopic OCT (EOCT) to examine the gastrointestinal tract of humans in vivo. Compared to endoscopic ultrasonic devices it offers a higher resolution and does not require coupling gels or fluids. EOCT may lead to a versatile tool for biopsy site selection or optical biopsy itself. The EOCT unit is comprised of an interferometer unit with a high speed scanning reference arm and an endoscopically compatible radially scanning probe as the sample arm. Fast data acquisition allows real-time display. Temporal averaging for speckle reduction and a transformation to correct nonlinear scanning were included in the EOCT control software, both in real-time. During in vivo clinical trials, we have observe the structure of the mucosa and submucosa in several gastrointestinal organs as well as glands, blood vessels, pits, villi and crypts. The purpose of this study was to correlate images acquired in vitro with EOCT to corresponding histological sections. EOCT images were obtained on fresh specimens, which were then fixed in formalin and submitted for standard histology. Tissues examined were normal specimens, which were then fixed in formalin and submitted for standard histology. Tissues examined were normal specimens of stomach, ileum, colon and rectum. It was shown that he thickness of the mucosa correlates well with the first bright layer in EOCT. The R2-value was determined to be 0.69. The submucosa and the muscularis propria could be identified. Furthermore, we were able to show the effect of pressure on the tissue on the visible details in the EOCT images.

Imaging cellular and subcellular structure of human brain tissue using micro computed tomography

NASA Astrophysics Data System (ADS)

Khimchenko, Anna; Bikis, Christos; Schweighauser, Gabriel; Hench, Jürgen; Joita-Pacureanu, Alexandra-Teodora; Thalmann, Peter; Deyhle, Hans; Osmani, Bekim; Chicherova, Natalia; Hieber, Simone E.; Cloetens, Peter; Müller-Gerbl, Magdalena; Schulz, Georg; Müller, Bert

2017-09-01

Brain tissues have been an attractive subject for investigations in neuropathology, neuroscience, and neurobiol- ogy. Nevertheless, existing imaging methodologies have intrinsic limitations in three-dimensional (3D) label-free visualisation of extended tissue samples down to (sub)cellular level. For a long time, these morphological features were visualised by electron or light microscopies. In addition to being time-consuming, microscopic investigation includes specimen fixation, embedding, sectioning, staining, and imaging with the associated artefacts. More- over, optical microscopy remains hampered by a fundamental limit in the spatial resolution that is imposed by the diffraction of visible light wavefront. In contrast, various tomography approaches do not require a complex specimen preparation and can now reach a true (sub)cellular resolution. Even laboratory-based micro computed tomography in the absorption-contrast mode of formalin-fixed paraffin-embedded (FFPE) human cerebellum yields an image contrast comparable to conventional histological sections. Data of a superior image quality was obtained by means of synchrotron radiation-based single-distance X-ray phase-contrast tomography enabling the visualisation of non-stained Purkinje cells down to the subcellular level and automated cell counting. The question arises, whether the data quality of the hard X-ray tomography can be superior to optical microscopy. Herein, we discuss the label-free investigation of the human brain ultramorphology be means of synchrotron radiation-based hard X-ray magnified phase-contrast in-line tomography at the nano-imaging beamline ID16A (ESRF, Grenoble, France). As an example, we present images of FFPE human cerebellum block. Hard X-ray tomography can provide detailed information on human tissues in health and disease with a spatial resolution below the optical limit, improving understanding of the neuro-degenerative diseases.

Imaging denatured collagen strands in vivo and ex vivo via photo-triggered hybridization of caged collagen mimetic peptides.

PubMed

Li, Yang; Foss, Catherine A; Pomper, Martin G; Yu, S Michael

2014-01-31

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Automated MALDI matrix deposition method with inkjet printing for imaging mass spectrometry.

PubMed

Baluya, Dodge L; Garrett, Timothy J; Yost, Richard A

2007-09-01

Careful matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is critical for producing reproducible analyte ion signals. Traditional methods for matrix deposition are often considered an art rather than a science, with significant sample-to-sample variability. Here we report an automated method for matrix deposition, employing a desktop inkjet printer (<$200) with 5760 x 1440 dpi resolution and a six-channel piezoelectric head that delivers 3 pL/drop. The inkjet printer tray, designed to hold CDs and DVDs, was modified to hold microscope slides. Empty ink cartridges were filled with MALDI matrix solutions, including DHB in methanol/water (70:30) at concentrations up to 40 mg/mL. Various samples (including rat brain tissue sections and standards of small drug molecules) were prepared using three deposition methods (electrospray, airbrush, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed that matrix crystals were formed evenly across the sample. There was minimal background signal after storing the matrix in the cartridges over a 6-month period. Overall, the mass spectral images gathered from inkjet-printed tissue specimens were of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods.

A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.

PubMed

Kino-Oka, Masahiro; Maeda, Yoshikatsu; Yamamoto, Takeyuki; Sugawara, Katsura; Taya, Masahito

2005-03-01

For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).

Acoustic property reconstruction of a pygmy sperm whale (Kogia breviceps) forehead based on computed tomography imaging.

PubMed

Song, Zhongchang; Xu, Xiao; Dong, Jianchen; Xing, Luru; Zhang, Meng; Liu, Xuecheng; Zhang, Yu; Li, Songhai; Berggren, Per

2015-11-01

Computed tomography (CT) imaging and sound experimental measurements were used to reconstruct the acoustic properties (density, velocity, and impedance) of the forehead tissues of a deceased pygmy sperm whale (Kogia breviceps). The forehead was segmented along the body axis and sectioned into cross section slices, which were further cut into sample pieces for measurements. Hounsfield units (HUs) of the corresponding measured pieces were obtained from CT scans, and regression analyses were conducted to investigate the linear relationships between the tissues' HUs and velocity, and HUs and density. The distributions of the acoustic properties of the head at axial, coronal, and sagittal cross sections were reconstructed, revealing that the nasal passage system was asymmetric and the cornucopia-shaped spermaceti organ was in the right nasal passage, surrounded by tissues and airsacs. A distinct dense theca was discovered in the posterior-dorsal area of the melon, which was characterized by low velocity in the inner core and high velocity in the outer region. Statistical analyses revealed significant differences in density, velocity, and acoustic impedance between all four structures, melon, spermaceti organ, muscle, and connective tissue (p < 0.001). The obtained acoustic properties of the forehead tissues provide important information for understanding the species' bioacoustic characteristics.

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.

Filter paper-assisted cell transfer (FaCT) technique: A novel cell-sampling technique for intraoperative diagnosis of central nervous system tumors.

PubMed

Kawamura, Jumpei; Kamoshida, Shingo; Shimakata, Takaaki; Hayashi, Yurie; Sakamaki, Kuniko; Denda, Tamami; Kawai, Kenji; Kuwao, Sadahito

2017-04-01

Intraoperative diagnosis of central nervous system (CNS) tumors provides critical guidance to surgeons in the determination of surgical resection margins and treatment. The techniques and preparations used for the intraoperative diagnosis of CNS tumors include frozen sectioning and cytologic methods (squash smear and touch imprint). Cytologic specimens, which do not have freezing artifacts, are important as an adjuvant tool to frozen sections. However, if the amount of submitted tissue samples is limited, then it is difficult to prepare both frozen sections and squash smears or touch imprint specimens from a single sample at the same time. Therefore, the objective of this study was to derive cells directly from filter paper on which tumor samples are placed. The authors established the filter paper-assisted cell transfer (FaCT) smear technique, in which tumor cells are transferred onto a glass slide directly from the filter paper sample spot after the biopsy is removed. Cell yields and diagnostic accuracy of the FaCT smears were assessed in 40 CNS tumors. FaCT smears had ample cell numbers and well preserved cell morphology sufficient for cytologic diagnosis, even if the submitted tissues were minimal. The overall diagnostic concordance rates between frozen sections and FaCT smears were 90% and 87.5%, respectively (no significant differences). When combining FaCT smears with frozen sections, the diagnostic concordance rate rose to 92.5%. The current results suggest that the FaCT smear technique is a simple and effective processing method that has significant value for intraoperative diagnosis of CNS tumors. Cancer Cytopathol 2017;125:277-282. © 2016 American Cancer Society. © 2017 American Cancer Society.

Skin cancer texture analysis of OCT images based on Haralick, fractal dimension and the complex directional field features

NASA Astrophysics Data System (ADS)

Raupov, Dmitry S.; Myakinin, Oleg O.; Bratchenko, Ivan A.; Kornilin, Dmitry V.; Zakharov, Valery P.; Khramov, Alexander G.

2016-04-01

Optical coherence tomography (OCT) is usually employed for the measurement of tumor topology, which reflects structural changes of a tissue. We investigated the possibility of OCT in detecting changes using a computer texture analysis method based on Haralick texture features, fractal dimension and the complex directional field method from different tissues. These features were used to identify special spatial characteristics, which differ healthy tissue from various skin cancers in cross-section OCT images (B-scans). Speckle reduction is an important pre-processing stage for OCT image processing. In this paper, an interval type-II fuzzy anisotropic diffusion algorithm for speckle noise reduction in OCT images was used. The Haralick texture feature set includes contrast, correlation, energy, and homogeneity evaluated in different directions. A box-counting method is applied to compute fractal dimension of investigated tissues. Additionally, we used the complex directional field calculated by the local gradient methodology to increase of the assessment quality of the diagnosis method. The complex directional field (as well as the "classical" directional field) can help describe an image as set of directions. Considering to a fact that malignant tissue grows anisotropically, some principal grooves may be observed on dermoscopic images, which mean possible existence of principal directions on OCT images. Our results suggest that described texture features may provide useful information to differentiate pathological from healthy patients. The problem of recognition melanoma from nevi is decided in this work due to the big quantity of experimental data (143 OCT-images include tumors as Basal Cell Carcinoma (BCC), Malignant Melanoma (MM) and Nevi). We have sensitivity about 90% and specificity about 85%. Further research is warranted to determine how this approach may be used to select the regions of interest automatically.

The CD117 immunohistochemistry tissue microarray survey for quality assurance and interlaboratory comparison: a College of American Pathologists Cell Markers Committee Study.

PubMed

Dorfman, David M; Bui, Marilyn M; Tubbs, Raymond R; Hsi, Eric D; Fitzgibbons, Patrick L; Linden, Michael D; Rickert, Robert R; Roche, Patrick C

2006-06-01

We have developed tissue microarray-based surveys to allow laboratories to compare their performance in staining predictive immunohistochemical markers, including proto-oncogene CD117 (c-kit), which is characteristically expressed in gastrointestinal stromal tumors (GISTs). GISTs exhibit activating mutations in the c-kit proto-oncogene, which render them amenable to treatment with imatinib mesylate. Consequently, correct identification of c-Kit expression is important for the diagnosis and treatment of GISTs. To analyze CD117 immunohistochemical staining performance by a large number of clinical laboratories. A mechanical device was used to construct tissue microarrays consisting of 3 x 1-mm cores of 10 tumor samples, which can be used to generate hundreds of tissue sections from the arrayed cases, suitable for large-scale interlaboratory comparison of immunohistochemical staining. An initial survey of 63 laboratories and a second survey of 90 laboratories, performed in 2004 and 2005, exhibited >81% concordance for 7 of 10 cores, including all 4 GIST cases, which were immunoreactive for CD117 with >95% staining concordance. Three of the cores achieved less than 81% concordance of results, possibly due to the presence of foci of necrosis in one core and CD117-positive mast cells in 2 cores of CD117-negative neoplasms. There was good performance among a large number of laboratories performing CD117 immunohistochemical staining, with consistently higher concordance of results for CD117-positive GIST cases than for nonimmunoreactive cases. Tissue microarrays for CD117 and other predictive markers should be useful for interlaboratory comparisons, quality assurance, and education of participants regarding staining nuances such as the expression of CKIT by nonneoplastic mast cells.

Use of optical coherence tomography in delineating airways microstructure: comparison of OCT images to histopathological sections

NASA Astrophysics Data System (ADS)

Yang, Ying; Whiteman, Suzanne; Gey van Pittius, Daniel; He, Yonghong; Wang, Ruikang K.; Spiteri, Monica A.

2004-04-01

An ideal diagnostic system for the human airways should be able to detect and define early development of premalignant pathological lesions, to facilitate optimal curative treatment and prevent irreversible and/or invasive lung disease. There is great need for exploration of safe, repeatable imaging techniques which can run at real-time and with high spatial resolution. In this study, optical coherence tomography (OCT) was utilized to acquire cross-sectional images of upper and lower airways using fresh pig lung resections as a model system. Obtained OCT images were compared with parallel tissue characterization by conventional histological analysis. Our objective was to determine whether OCT differentiates the composite structural layers and inherent anatomical variations along different airway locations. The data show that OCT can clearly display the multilayered structure of the airways. The subtle architectural differences in three separate anatomical locations including trachea, main bronchus and tertiary bronchus were clearly delineated. Images of the appropriate anatomical profiles, with depth of up to 2 mm and 10 µm spatial resolution were obtained by our current OCT system, which was sufficient for recognition of the epithelium, subepithelial tissues and cartilage. In addition, the relative thickness of individual structural components was accurately reflected and comparable to histological sections. These data support OCT as a highly feasible, optical biopsy tool, which merits further exploration for early diagnosis of human airway epithelial pathology.

Tissue banking in Asia Pacific region: past, present and future.

PubMed

Nather, Aziz; Mandy, Foong Shi Yun; Ning, Tan; Kaiying, Wang

2018-04-25

Tissue banking in the Asia Pacific regions is driven by two main forces-firstly the International Atomic Energy Agency (IAEA) via Regional Co-operative Agreement projects and secondly by the Asia Pacific Association of Surgical Tissue Banking (APASTB). This overview is written in three sections: (1) History of tissue banking in individual country in the region. (2) History of APASTB. (3) History of IAEA programme in Asia Pacific region. The current status and future of the tissue banking programme in the region will be discussed.

9 CFR 310.15 - Disposition of thyroid glands and laryngeal muscle tissue.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Disposition of thyroid glands and laryngeal muscle tissue. 310.15 Section 310.15 Animals and Animal Products FOOD SAFETY AND INSPECTION... of thyroid glands and laryngeal muscle tissue. (a) Livestock thyroid glands and laryngeal muscle...

9 CFR 310.15 - Disposition of thyroid glands and laryngeal muscle tissue.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Disposition of thyroid glands and laryngeal muscle tissue. 310.15 Section 310.15 Animals and Animal Products FOOD SAFETY AND INSPECTION... of thyroid glands and laryngeal muscle tissue. (a) Livestock thyroid glands and laryngeal muscle...

9 CFR 310.15 - Disposition of thyroid glands and laryngeal muscle tissue.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Disposition of thyroid glands and laryngeal muscle tissue. 310.15 Section 310.15 Animals and Animal Products FOOD SAFETY AND INSPECTION... of thyroid glands and laryngeal muscle tissue. (a) Livestock thyroid glands and laryngeal muscle...

9 CFR 310.15 - Disposition of thyroid glands and laryngeal muscle tissue.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Disposition of thyroid glands and laryngeal muscle tissue. 310.15 Section 310.15 Animals and Animal Products FOOD SAFETY AND INSPECTION... of thyroid glands and laryngeal muscle tissue. (a) Livestock thyroid glands and laryngeal muscle...

9 CFR 310.15 - Disposition of thyroid glands and laryngeal muscle tissue.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Disposition of thyroid glands and laryngeal muscle tissue. 310.15 Section 310.15 Animals and Animal Products FOOD SAFETY AND INSPECTION... of thyroid glands and laryngeal muscle tissue. (a) Livestock thyroid glands and laryngeal muscle...

21 CFR 1270.42 - Human tissue offered for import.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Human tissue offered for import. 1270.42 Section 1270.42 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN TISSUE INTENDED...

21 CFR 1270.42 - Human tissue offered for import.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Human tissue offered for import. 1270.42 Section 1270.42 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN TISSUE INTENDED...

21 CFR 1270.42 - Human tissue offered for import.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Human tissue offered for import. 1270.42 Section 1270.42 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN TISSUE INTENDED...

21 CFR 1270.42 - Human tissue offered for import.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Human tissue offered for import. 1270.42 Section 1270.42 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN TISSUE INTENDED...

21 CFR 1270.42 - Human tissue offered for import.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Human tissue offered for import. 1270.42 Section 1270.42 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION HUMAN TISSUE INTENDED...

A Method to Prevent Protein Delocalization in Imaging Mass Spectrometry of Non-Adherent Tissues: Application to Small Vertebrate Lens Imaging

PubMed Central

Anderson, David M. G.; Floyd, Kyle A.; Barnes, Stephen; Clark, Judy M.; Clark, John I.; Mchaourab, Hassane; Schey, Kevin L.

2015-01-01

MALDI imaging requires careful sample preparation to obtain reliable, high quality images of small molecules, peptides, lipids, and proteins across tissue sections. Poor crystal formation, delocalization of analytes, and inadequate tissue adherence can affect the quality, reliability, and spatial resolution of MALDI images. We report a comparison of tissue mounting and washing methods that resulted in an optimized method using conductive carbon substrates that avoids thaw mounting or washing steps, minimizes protein delocalization, and prevents tissue detachment from the target surface. Application of this method to image ocular lens proteins of small vertebrate eyes demonstrates the improved methodology for imaging abundant crystallin protein products. This method was demonstrated for tissue sections from rat, mouse, and zebrafish lenses resulting in good quality MALDI images with little to no delocalization. The images indicate, for the first time in mouse and zebrafish, discrete localization of crystallin protein degradation products resulting in concentric rings of distinct protein contents that may be responsible for the refractive index gradient of vertebrate lenses. PMID:25665708

Shedding quantitative fluorescence light on novel regulatory mechanisms in skeletal biomedicine and biodentistry.

PubMed

Lee, Ji-Won; Iimura, Tadahiro

2017-02-01

Digitalized fluorescence images contain numerical information such as color (wavelength), fluorescence intensity and spatial position. However, quantitative analyses of acquired data and their validation remained to be established. Our research group has applied quantitative fluorescence imaging on tissue sections and uncovered novel findings in skeletal biomedicine and biodentistry. This review paper includes a brief background of quantitative fluorescence imaging and discusses practical applications by introducing our previous research. Finally, the future perspectives of quantitative fluorescence imaging are discussed.

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

PubMed Central

Loibner, Martina; Oberauner-Wappis, Lisa; Viertler, Christian; Groelz, Daniel; Zatloukal, Kurt

2017-01-01

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections. PMID:29364207

Mechanisms of Intraductal Tumor Spread

DTIC Science & Technology

2004-08-01

59 (2): 119-127, 2002. "• "A geometric model for image analysis in cytology" Ortiz de Solorzano C., R . Malladi , Lockett S. In: Geometric methods in...gland tissue sections". Fernandez-Gonzalez R ., Deschamps T., Idica A.K., Malladi R ., Ortiz de Solorzano C. Journal of Biomedical Optics 9(3):445-453...normal and neoplastic mammary gland tissue sections". Fernandez-Gonzalez R ., Deschamps T., Idica A.K., Malladi R ., Ortiz de Solorzano C., Proceedings

Histopathological changes in Wistar albino rats exposed to aqueous extract of unripe Carica papaya.

PubMed

Oduola, Taofeeq; Bello, Ibrahim; Idowu, Thomas; Avwioro, Godwin; Adeosun, Ganiyu; Olatubosun, Luqman

2010-05-01

Exposure of animals to xenobiotics may or may not trigger adverse response at cellular levels. Aqueous extract of unripe Carica papaya is consumed by sickle cell patients as antisickling agent in Western Nigeria. This study was undertaken to investigate the effects of Carica papaya on certain organs in Wister albino rats exposed to aqueous extract of unripe Carica papaya. Different doses of aqueous extract of unripe Carica papaya were administered orally daily for 42 days to six groups of rats. At the end of exposure, the animals were sacrificed and tissue sections were prepared from livers, kidneys, hearts and small intestines using standard techniques. Histopathological results showed that no pathological changes were observed in tissue sections of experimental animals when compared with tissue sections of the same organs in control animals. No pathological changes were elicited in the organs of rats exposed to aqueous extract of unripe Carica papaya.

Comparative Studies of Effect of Auxin and Ethylene on Permeability and Synthesis of RNA and Protein 1

PubMed Central

Sacher, Joseph A.; Salminen, Seppo O.

1969-01-01

The effects of ethylene on permeability and RNA and protein synthesis were assayed over a 6 to 26 hr period in tissue sections from avocado (Persea gratissima Gaertn. F., var. Fuerte), both pulp and peel of banana (Musa sapientum L., var. Gros Michel), bean endocarp (Phaseolus vulgaris L., var. Kentucky Wonder Pole beans) and leaves of Rhoeo discolor. Ethylene had no effect on permeability in 4 of the 5 tissues, but sometimes enhanced solute uptake in banana peel; it had either no effect or an inhibitory effect on synthesis of RNA and protein in sections from fruits of avocado and banana. Auxin (α-naphthalene acetic acid) stimulated synthesis of RNA and protein in bean endocarp and Rhoeo leaf sections, whereas ethylene inhibited both basal and auxin-induced synthesis. It is concluded that in these tissues the auxin effect is not an ethylene effect. PMID:16657212

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase.

PubMed

Van Noorden, C J

1984-01-01

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Advances in Surgical Reconstructive Techniques in the Management of Penile, Urethral, and Scrotal Cancer.

PubMed

Bickell, Michael; Beilan, Jonathan; Wallen, Jared; Wiegand, Lucas; Carrion, Rafael

2016-11-01

This article reviews the most up-to-date surgical treatment options for the reconstructive management of patients with penile, urethral, and scrotal cancer. Each organ system is examined individually. Techniques and discussion for penile cancer reconstruction include Mohs surgery, glans resurfacing, partial and total glansectomy, and phalloplasty. Included in the penile cancer reconstruction section is the use of penile prosthesis in phalloplasty patients after penectomy, tissue engineering in phallic regeneration, and penile transplantation. Reconstruction following treatment of primary urethral carcinoma and current techniques for scrotal cancer reconstruction using split-thickness skin grafts and flaps are described. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for polyomavirus DNA and antigen.

PubMed

Kidney, B A; Haines, D M; Ellis, J A; Burnham, M; Jackson, M L

2001-06-01

To determine whether vaccine site-associated sarcomas (VSS) from cats contain polyomavirus antigen or DNA. 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. Sections from each tissue block were evaluated for polyomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-murine polyomavirus polyclonal antiserum as the primary antibody. The DNA was extracted from sections of each tissue block, and a polymerase chain reaction assay was performed, using primers designed to amplify regions of the bovine polyomavirus genome and consensus polyomavirus primers designed to detect unknown polyomaviruses. Polyomavirus antigen and DNA were not detected in any of the VSS. Results suggest that polyomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

Mueller-matrix mapping of optically anisotropic fluorophores of biological tissues in the diagnosis of cancer

NASA Astrophysics Data System (ADS)

Ushenko, Yu A.; Sidor, M. I.; Bodnar, G. B.; Koval', G. D.

2014-08-01

We report the results of studying the polarisation manifestations of laser autofluorescence of optically anisotropic structures in biological tissues. A Mueller-matrix model is proposed to describe their complex anisotropy (linear and circular birefringence, linear and circular dichroism). The relationship is established between the mechanisms of optical anisotropy and polarisation manifestations of laser autofluorescence of histological sections of rectal tissue biopsy in different spectral regions. The ranges of changes in the statistical moments of the 1st-to-4th orders, which describe the distribution of the azimuth-invariant elements of Mueller matrices of rectal tissue autofluorescence, are found. Effectiveness of laser autofluorescence polarimetry is determined and the histological sections of biopsy of benign (polyp) and malignant (adenocarcinoma) tumours of the rectal wall are differentiated for the first time.

Dentine sialoprotein and collagen I expression after experimental pulp capping in humans using emdogain gel.

PubMed

Fransson, H; Petersson, K; Davies, J R

2011-03-01

To characterize the hard tissue formed in human teeth experimentally pulp capped either with calcium hydroxide or with Emdogain Gel (Biora AB, Malmö, Sweden) - a derivative of enamel matrix (EMD), using two markers for dentine; dentine sialoprotein (DSP) and type 1 collagen (Col I). Affinity-purified rabbit anti-Col I and anti-DSP polyclonal antibodies were used to stain histological sections from nine pairs of contra-lateral premolars that had been experimentally pulp amputated and randomly capped with EMDgel or calcium hydroxide. Twelve weeks after the teeth had been pulp capped, they were extracted, fixed, demineralized and serially sectioned prior to immunohistochemical staining. In the calcium hydroxide treated teeth DSP was seen in the new hard tissue which formed a bridge. DSP was also seen in the newly formed hard tissue in the EMDgel-treated teeth. Proliferated pulp tissue partly filled the space initially occupied by EMDgel and DSP-stained hard tissue was observed alongside exposed dentine surfaces as well as in isolated masses within the proliferated pulp tissue, although the new hard tissue did not cover the pulp exposure. DSP staining was also seen in the cells lining the hard tissue in both groups. Col I staining was seen in the newly formed hard tissue in both groups. The new hard tissue formed after pulp capping with EMDgel or calcium hydroxide contained DSP and Col I, considered to be markers for dentine. Thus, the newly formed hard tissue can be characterized as dentine rather than unspecific hard tissue. © 2010 International Endodontic Journal.

Histologic effects of a high-repetition pulsed Nd:YAG laser on intraoral soft tissue

NASA Astrophysics Data System (ADS)

White, Joel M.; Goodis, Harold E.; Yessik, Michael J.; Myers, Terry D.

1995-05-01

High-repetition rate, fiberoptic-delivered Nd:YAG lasers have increased oral soft tissue laser applications. This study focused on three parameters: the temperature rise occurring in deeper tissue during excision, the histology of thermal coagulation during excision of oral tissue, and effects of accidental exposure to adjacent hard tissue. Thermocouples were placed 5.0 +/- 0.5 mm in bone below fresh bovine gingiva and at the same depth in tongue; temperatures in the underlying tissue were measured during laser excision. An Nd:YAG laser with 100 microsecond(s) pulse duration was used to excise the tissue using a 200 or 300 micrometers diameter fiber in contact with the tissue. The soft tissue was excised using constant force and rate with laser powers of 1.5, 3, 5, and 10 W, and a variety of pulse rates. The tissue was bioprepared, sectioned and stained with hematoxylin and eosin. The width and depth of the tissue removed as well as lateral and deep thermal coagulation were measured in histologic sections with a measuring microscope (10x). Multifactor randomized ANOVA showed that probe diameter and repetition rates were not significant variables (p

Histochemical detection of lead and zinc in plant tissues

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

Tung, G.; Temple, P.J.

1975-01-01

Histochemical studies on uptake and localization of lead and zinc in plant tissues were carried out. A histochemical stain technique was developed to differentiate zinc from lead. Lead was detected in plant tissues by soaking fresh plant materials in freshly prepared sodium rhodizonate stain (0.2% Na rhodizonate acidified to pH3 with glacial acetic acid). Samples were evacuated 5 min and soaked for 30 min before embedding in the congealed stain, then sectioned with a cryostat and examined under a light microscope. Lead particles in plant tissues were stained scarlet-red. Gelatinous, proteinaceous or saccharic embedding materials normally used to prepare plantmore » sampled for sectioning in the cryostat interfered with the color reaction. Sectioning plant samples without staining whole tissues resulted in a weakened response to the stain. Color of stained sample materials were retained for several months if stored in a frozen condition. This technique was used to detect lead both inside and on the surface of plant samples collected in the vicinity of highway and industrial lead sources and to trace the pathways of lead uptake from the air or from contaminated soils. A sodium rhodizonate technique was also developed to be specific for zinc in plant tissues. Plant samples were soaked in a neutral Na-rhodizonate in phosphate buffer at pH 7.5 for observation. The color of zinc developed to produce a purplish or reddish-brown color.« less

Outer membrane proteins of pathogenic spirochetes

PubMed Central

Cullen, Paul A.; Haake, David A.; Adler, Ben

2009-01-01

Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

High resolution macroscopy (HRMac) of the eye using nonlinear optical imaging

NASA Astrophysics Data System (ADS)

Winkler, Moritz; Jester, Bryan E.; Nien-Shy, Chyong; Chai, Dongyul; Brown, Donald J.; Jester, James V.

2010-02-01

Non-linear optical (NLO) imaging using femtosecond lasers provides a non-invasive means of imaging the structural organization of the eye through the generation of second harmonic signals (SHG). While NLO imaging is able to detect collagen, the small field of view (FoV) limits the ability to study how collagen is structurally organized throughout the larger tissue. To address this issue we have used computed tomography on optical and mechanical sectioned tissue to greatly expand the FoV and provide high resolution macroscopic (HRMac) images that cover the entire tissue (cornea and optic nerve head). Whole, fixed cornea (13 mm diameter) or optic nerve (3 mm diameter) were excised and either 1) embedded in agar and sectioned using a vibratome (200-300 um), or 2) embedded in LR White plastic resin and serially sectioned (2 um). Vibratome and plastic sections were then imaged using a Zeiss LSM 510 Meta and Chameleon femtosecond laser to generate NLO signals and assemble large macroscopic 3-dimensional tomographs with high resolution that varied in size from 9 to 90 Meg pixels per plane having a resolution of 0.88 um lateral and 2.0 um axial. 3-D reconstructions allowed for regional measurements within the cornea and optic nerve to quantify collagen content, orientation and organization over the entire tissue. We conclude that NLO based tomography to generate HRMac images provides a powerful new tool to assess collagen structural organization. Biomechanical testing combined with NLO tomography may provide new insights into the relationship between the extracellular matrix and tissue mechanics.

A Tissue-Specific Approach to the Analysis of Metabolic Changes in Caenorhabditis elegans

PubMed Central

Pujol, Claire; Ipsen, Sabine; Brodesser, Susanne; Mourier, Arnaud; Tolnay, Markus; Frank, Stephan; Trifunović, Aleksandra

2011-01-01

The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens. PMID:22162770

Improvised double-embedding technique of minute biopsies: a mega boon to histopathology laboratory.

PubMed

Yadav, Lokendra; Thomas, Sarega; Kini, Usha

2015-01-01

Optimal orientation of minute mucosal biopsies is essential for a definite diagnosis in gastrointestinal pathology or to visualize neural plexuses in Hirschsprung disease. The problem of minute size of the biopsy and its orientation gets compounded when they are from neonates and mandates exhaustive strip cuts, thus delaying reporting. A modified agar-paraffin technique is aimed to make tissue embedding efficient and user-friendly by inking mapping biopsies (one or more) either fresh or fixed with surgical coloring inks followed by embedding first in agar after orientation and followed thereafter by processing, re-embedding in paraffin wax, sectioning and staining. The tissues in agar paraffin block were found to be well processed, firm, held secure and well preserved. The blocks were easy to cut, with serial sections of thickness 2-3 μ and easy to spread. The colored inks remained permanently on the tissues both in the block as well as on the sections which helped in easy identification of tissues. Agar did not interfere with any stain such as Hematoxylin and Eosin or with histochemical stains, enzyme histochemistry or immunohistochemistry. Inking biopsies and pooling them in a block when obtained from the same patient reduced the number of tissue blocks. The modified agar-paraffin embedding technique is a simple reliable user friendly method that can greatly improve the quality of diagnostic information from minute biopsies by optimal orientation, better quality of sections, faster turnaround time and cost-effectiveness by economizing on the number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure.

Periapical tissue response after use of intermediate restorative material, gutta-percha, reinforced zinc oxide cement, and mineral trioxide aggregate as retrograde root-end filling materials: a histologic study in dogs.

PubMed

Wälivaara, Dan-Åke; Abrahamsson, Peter; Isaksson, Sten; Salata, Luiz Antonio; Sennerby, Lars; Dahlin, Christer

2012-09-01

To investigate the periapical tissue response of 4 different retrograde root-filling materials, ie, intermediate restorative material, thermoplasticized gutta-percha, reinforced zinc oxide cement (Super-EBA), and mineral trioxide aggregate (MTA), in conjunction with an ultrasonic root-end preparation technique in an animal model. Vital roots of the third and fourth right mandibular premolars in 6 healthy mongrel dogs were apicectomized and sealed with 1 of the materials using a standardized surgical procedure. After 120 days, the animals were sacrificed and the specimens were analyzed radiologically, histologically, and scanning electron microscopically. The Fisher exact test was performed on the 2 outcome values. Twenty-three sections were analyzed histologically. Evaluation showed better re-establishment of the periapical tissues and generally lower inflammatory infiltration in the sections from teeth treated with the intermediate restorative material and the MTA. New root cement on the resected dentin surfaces was seen on all sections regardless of the used material. New hard tissue formation, directly on the surface of the material, was seen only in the MTA sections. There was no statistical difference in outcome among the tested materials. The results from this dog model favor the intermediate restorative material and MTA as retrograde fillings when evaluating the bone defect regeneration. MTA has the most favorable periapical tissue response when comparing the biocompatibility of the materials tested. Copyright © 2012 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Identification of tissular origin of particles based on autofluorescence multispectral image analysis at the macroscopic scale

NASA Astrophysics Data System (ADS)

Corcel, Mathias; Devaux, Marie-Françoise; Guillon, Fabienne; Barron, Cécile

2017-06-01

Powders produced from plant materials are heterogeneous in relation to native plant heterogeneity, and during grinding, dissociation often occurred at the tissue scale. The tissue composition of powdery samples could be modified through dry fractionation diagrams and impact their end-uses properties. If tissue identification is often made on native plant structure, this characterization is not straightforward in destructured samples such powders. Taking advantage of the autofluorescence properties of cell wall components, multispectral image acquisition is envisioned to identify the tissular origin of particles. Images were acquired on maize stem sections and ground tissues isolated from the same stem by hand dissection. The variability in fluorescence intensity profiles was analysed using principal component analysis. The correspondence between fluorescence profiles and the different tissues observed in maize sections was assessed based on histology or known compositional heterogeneity. Similar variability was encountered in fluorescence profiles extracted from powder leading to the potential ability to predict tissular origin based on this autofluorescence multispectral signal.

Histomolecular interpretation of pleomorphic adenomas of the salivary gland by matrix-assisted laser desorption ionization imaging and spatial segmentation.

PubMed

Ernst, Günther; Guntinas-Lichius, Orlando; Hauberg-Lotte, Lena; Trede, Dennis; Becker, Michael; Alexandrov, Theodore; von Eggeling, Ferdinand

2015-07-01

Despite efforts in localization of key proteins using immunohistochemistry, the complex proteomic composition of pleomorphic adenomas has not yet been characterized. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging) allows label-free and spatially resolved detection of hundreds of proteins directly from tissue sections and of histomorphological regions by finding colocalized molecular signals. Spatial segmentation of MALDI imaging data is an algorithmic method for finding regions of similar proteomic composition as functionally similar regions. We investigated 2 pleomorphic adenomas by applying spatial segmentation to the MALDI imaging data of tissue sections. The spatial segmentation subdivided the tissue in a good accordance with the tissue histology. Numerous molecular signals colocalized with histologically defined tissue regions were found. Our study highlights the cellular transdifferentiation within the pleomorphic adenoma. It could be shown that spatial segmentation of MALDI imaging data is a promising approach in the emerging field of digital histological analysis and characterization of tumors. © 2014 Wiley Periodicals, Inc.

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.

Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system.

PubMed

Laurinaviciene, Aida; Plancoulaine, Benoit; Baltrusaityte, Indra; Meskauskas, Raimundas; Besusparis, Justinas; Lesciute-Krilaviciene, Daiva; Raudeliunas, Darius; Iqbal, Yasir; Herlin, Paulette; Laurinavicius, Arvydas

2014-01-01

Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools. Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue. Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores. Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools.

Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system

PubMed Central

2014-01-01

Background Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools. Methods Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue. Results Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores. Conclusions Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools. PMID:25565007

Basic mechanism of button battery ingestion injuries and novel mitigation strategies after diagnosis and removal.

PubMed

Jatana, Kris R; Rhoades, Keith; Milkovich, Scott; Jacobs, Ian N

2017-06-01

Button battery (BB) injuries continue to be a significant source of morbidity and mortality, and there is a need to confirm the mechanism of injury for development of additional mitigation strategies. Cadaveric piglet esophageal model. Lithium, silver oxide, alkaline, and zinc-air BBs were placed in thawed sections of cadaveric piglet esophagus, bathed in normal saline. Severity of gross visual burn, pH, and temperature were recorded every 30 minutes for 6 hours. In other esophageal tissue specimens, the lithium BB was removed after 24, 36, and 48 hours and the site was irrigated with either 0.25% or 3% acetic acid. Separately, ReaLemon® juice, orange juice, Coke®, Dasani® water, Pepsi®, and saline were infused over a vertically suspended esophagus with a CR2032 lithium battery every 5 minutes for 2 hours while tissue temperature and pH were measured. A gradual rise in tissue pH and minimal change in temperature was noted for all BBs. ReaLemon® and orange juice applied every 5 minutes were most effective at neutralization of tissue pH with minimal change in tissue temperature. After BB removal (24, 36, 48 hours), irrigation of esophageal tissue specimens with 50-150 mL 0.25% acetic acid neutralized the highly alkaline tissue pH. BB appear to cause an isothermic hydrolysis reaction resulting in an alkaline caustic injury. Potential new mitigation strategies include application of neutralizing weakly acidic solutions that may reduce esophageal injury progression. NA Laryngoscope, 127:1276-1282, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

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

Computer Three-Dimensional Reconstruction of the Atrioventricular Node

PubMed Central

Li, Jue; Greener, Ian D.; Inada, Shin; Nikolski, Vladimir P.; Yamamoto, Mitsuru; Hancox, Jules C.; Zhang, Henggui; Billeter, Rudi; Efimov, Igor R.; Dobrzynski, Halina; Boyett, Mark R.

2009-01-01

Because of its complexity, the atrioventricular node (AVN), remains 1 of the least understood regions of the heart. The aim of the study was to construct a detailed anatomic model of the AVN and relate it to AVN function. The electric activity of a rabbit AVN preparation was imaged using voltage-dependent dye. The preparation was then fixed and sectioned. Sixty-five sections at 60- to 340-μm intervals were stained for histology and immunolabeled for neurofilament (marker of nodal tissue) and connexin43 (gap junction protein). This revealed multiple structures within and around the AVN, including transitional tissue, inferior nodal extension, penetrating bundle, His bundle, atrial and ventricular muscle, central fibrous body, tendon of Todaro, and valves. A 3D anatomically detailed mathematical model (≈13 million element array) of the AVN and surrounding atrium and ventricle, incorporating all cell types, was constructed. Comparison of the model with electric activity recorded in experiments suggests that the inferior nodal extension forms the slow pathway, whereas the transitional tissue forms the fast pathway into the AVN. In addition, it suggests the pacemaker activity of the atrioventricular junction originates in the inferior nodal extension. Computer simulation of the propagation of the action potential through the anatomic model shows how, because of the complex structure of the AVN, reentry (slow-fast and fast-slow) can occur. In summary, a mathematical model of the anatomy of the AVN has been generated that allows AVN conduction to be explored. PMID:18309098

Large area 3-D optical coherence tomography imaging of lumpectomy specimens for radiation treatment planning

NASA Astrophysics Data System (ADS)

Wang, Cuihuan; Kim, Leonard; Barnard, Nicola; Khan, Atif; Pierce, Mark C.

2016-02-01

Our long term goal is to develop a high-resolution imaging method for comprehensive assessment of tissue removed during lumpectomy procedures. By identifying regions of high-grade disease within the excised specimen, we aim to develop patient-specific post-operative radiation treatment regimens. We have assembled a benchtop spectral-domain optical coherence tomography (SD-OCT) system with 1320 nm center wavelength. Automated beam scanning enables "sub-volumes" spanning 5 mm x 5 mm x 2 mm (500 A-lines x 500 B-scans x 2 mm in depth) to be collected in under 15 seconds. A motorized sample positioning stage enables multiple sub-volumes to be acquired across an entire tissue specimen. Sub-volumes are rendered from individual B-scans in 3D Slicer software and en face (XY) images are extracted at specific depths. These images are then tiled together using MosaicJ software to produce a large area en face view (up to 40 mm x 25 mm). After OCT imaging, specimens were sectioned and stained with HE, allowing comparison between OCT image features and disease markers on histopathology. This manuscript describes the technical aspects of image acquisition and reconstruction, and reports initial qualitative comparison between large area en face OCT images and HE stained tissue sections. Future goals include developing image reconstruction algorithms for mapping an entire sample, and registering OCT image volumes with clinical CT and MRI images for post-operative treatment planning.

Immunohistochemical evalulation of activated Ras and Rac1 as potential downstream effectors of aquaporin-5 in breast cancer in vivo.

PubMed

Jensen, Helene H; Login, Frédéric H; Park, Ji-Young; Kwon, Tae-Hwan; Nejsum, Lene N

2017-11-25

Aberrant levels of aquaporin-5 (AQP5) expression have been observed in several types of cancer, including breast cancer, where AQP5 overexpression is associated with metastasis and poor prognosis. In cultured cancer cells, AQP5 facilitates cell migration and activates Ras signaling. Both increased cell migration and Ras activation are associated with cancer metastasis, but so far it is unknown if AQP5 also affects these processes in vivo. Therefore, we investigated if high AQP5 expression in breast cancer tissue correlated with increased activation of Ras and of Rac1, which is a GTPase also involved in cell migration. This was accomplished by immunohistochemical analysis of invasive ductal carcinoma of breast tissue sections from human patients, followed by qualitative and quantitative correlation analysis between AQP5 and activated Ras and Rac1. Immunohistochemistry revealed that activation of Ras and Rac1 was positively correlated. There was, however, no correlation between high AQP5 expression and activation of Ras, whereas a nonsignificant, but positive, tendency between the levels of AQP5 and activated Rac1 levels was observed. In summary, this is the first report that correlates AQP5 expression levels to downstream signaling partners in breast cancer tissue sections. The results suggest Rac1 as a potential downstream signaling partner of AQP5 in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Hydroxylapatite nanoparticles: fabrication methods and medical applications

NASA Astrophysics Data System (ADS)

Okada, Masahiro; Furuzono, Tsutomu

2012-12-01

Hydroxylapatite (or hydroxyapatite, HAp) exhibits excellent biocompatibility with various kinds of cells and tissues, making it an ideal candidate for tissue engineering, orthopedic and dental applications. Nanosized materials offer improved performances compared with conventional materials due to their large surface-to-volume ratios. This review summarizes existing knowledge and recent progress in fabrication methods of nanosized (or nanostructured) HAp particles, as well as their recent applications in medical and dental fields. In section 1, we provide a brief overview of HAp and nanoparticles. In section 2, fabrication methods of HAp nanoparticles are described based on the particle formation mechanisms. Recent applications of HAp nanoparticles are summarized in section 3. The future perspectives in this active research area are given in section 4.

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

Enhanced Sensitivity for High Spatial Resolution Lipid Analysis by Negative Ion Mode MALDI Imaging Mass Spectrometry

PubMed Central

Angel, Peggi M.; Spraggins, Jeffrey M.; Baldwin, H. Scott; Caprioli, Richard

2012-01-01

We have achieved enhanced lipid imaging to a ~10 μm spatial resolution using negative ion mode matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry, sublimation of 2,5-dihydroxybenzoic acid as the MALDI matrix and a sample preparation protocol that uses aqueous washes. We report on the effect of treating tissue sections by washing with volatile buffers at different pHs prior to negative ion mode lipid imaging. The results show that washing with ammonium formate, pH 6.4, or ammonium acetate, pH 6.7, significantly increases signal intensity and number of analytes recorded from adult mouse brain tissue sections. Major lipid species measured were glycerophosphoinositols, glycerophosphates, glycerolphosphoglycerols, glycerophosphoethanolamines, glycerophospho-serines, sulfatides, and gangliosides. Ion images from adult mouse brain sections that compare washed and unwashed sections are presented and show up to fivefold increases in ion intensity for washed tissue. The sample preparation protocol has been found to be applicable across numerous organ types and significantly expands the number of lipid species detectable by imaging mass spectrometry at high spatial resolution. PMID:22243218

Cloning of Helicobacter suis cholesterol α-glucosyltransferase and production of an antibody capable of detecting it in formalin-fixed, paraffin-embedded gastric tissue sections.

PubMed

Kawakubo, Masatomo; Horiuchi, Kazuki; Komura, Hitomi; Sato, Yoshiko; Kato, Masayoshi; Ikeyama, Meguru; Fukushima, Mana; Yamada, Shigenori; Ishizone, Satoshi; Matsumoto, Takehisa; Ota, Hiroyoshi; Sagara, Junji; Nakayama, Jun

2017-10-01

Helicobacter suis (H. suis), formerly called Helicobacter heilmannii type 1 (H. heilmannii), is a gram-negative bacterium of the Helicobacter species. This pathogen infects the stomach of humans and animals such as dogs, cats, pigs, and rodents, the latter giving rise to zoonotic infection. Here, we generated a H. suis-specific antibody useful for immunohistochemistry with formalin-fixed, paraffin-embedded tissue sections. To do so, we began by cloning the gene encoding H. suis cholesterol α-glucosyltransferase (αCgT). αCgT is the key enzyme responsible for biosynthesis of cholesteryl α-D-glucopyranoside (CGL), a major cell wall component of Helicobacter species including H. suis. The deduced amino acid sequence of H. suis αCgT had 56% identity with the corresponding Helicobacter pylori (H. pylori). We then developed a polyclonal antibody (anti-Hh-I205R) by immunizing rabbits with a 205 amino acid H. suis αCgT fragment. Immunohistochemistry with the anti-Hh-I205R antibody could differentiate H. suis from H. pylori in gastric mucosa sections derived from mice infected with either pathogen. We then probed formalin-fixed, paraffin-embedded sections of human gastric mucosa positive for H. suis infection with the anti-Hh-I205R antibody and detected positive staining. These results indicate that anti-Hh-I205R antibody is specific for H. suis αCgT and useful to detect H. suis in gastric specimens routinely analyzed in pathological examinations.

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.

[Expression of OPG/RANK/RANKL in the rat dental pulp tissue of periodontitis combined with vascular calcification and its clinical significance].

PubMed

Pan, Ke-Qing; Zhang, Peng-Mei; Deng, Jing; Lou, Xiu-Xiu; Meng, Yun; Liu, Gui-Rong

2016-08-01

To study the expression and possible role of OPG/RANK/RANKLin the rat dental pulp of periodontitis combined with vascular calcification. Thirty-six male Wister rats were randomly divided into 4 groups: control group(group C), periodontitis group(group CP), vascular calcification group(group VDN) and compound group(group CP+VDN). Each group underwent corresponding management to establish animal model. When the model was successful, the maxillae including molars were sectioned, pulp tissue was examined by H-E staining; Immunohistochemical staining method was used to evaluate the expression and ratio of OPG and RANKL in pulp tissues. Statistical analysis was carried out using SPSS 19.0 software package. The pulp tissue of group CP, VDN, CP+VDN showed varied degrees of damage, neutrophil infiltration, pulp vascular congestion, odontoblasts vacuolar changes, pulp necrosis by H-E staining, and the changes in CP+VDN group was the most significant, followed by CP group, VDN group. Immunohistochemistry showed OPG in pulp tissues in group CP, VDN, CP+VDN were significantly lower than that in normal group (P<0.05), and the expression in group CP+VDN was the least;Expression of RANKL in pulp tissues in group CP, VDN, CP+VDN were significantly higher than that in normal group(P<0.05)，and the expression in group CP+VDN was the highest. The ratio of OPG/RANKL in normal group was the highest, and the ratio in CP+VDN group was the lowest. Periodontitis and vascular calcification can damage the pulp tissue, periodontitis compound with vascular calcification may aggravate the injury; OPG/RANKL/RANK system may play an important role in pulp tissue injury.

SOME PECULIARITIES IN INCLUSION AND DISTRIBUTION OF P$sup 32$ IN THE RABBIT EYE TISSUES (in Russian)

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

Medvedovskaya, Ts.P.; Likharev, I.A.

1963-11-01

Data on levels of inclusion and correlations between the activities of P/ sup 32/ in the eye tissue of rabbits at different periods during two months after a single introduction of the isotope are presented. Experiments were staged on 21 rabbits (42 eyes). P/sup 32/ was introduced subcutaneously in a concentration of 250 C/kg. Distribution of the isotope was studied with the radiometric method. The content of the activity in the eye tissues was calculated in mu C and in percentage relation to the total activity of the eyeball The initial quantity of the isotope in the eye tissues inmore » percentage relation to the originally introduced amount was also calculated, as well as the content of activity in the tissues per 1 g of humid weight in mu C. In single subcutaneous introduction of P/sup 32/ it penetrates into all tissues of the rabbit eye. The eyeball recieves a total of 0.01%, of the activity introduced. On the first day the greatest concentration of activity is seen in the eye tissues with a rich vascular network, where intensive metabolic processes take place. The share of the crystalline lens in the included P/sup 32/ is negligible. Later on the named tissues rapidly eliminated P/sup 32/, whereas in the crystalline lens it is only on the 6th day that the maximum activity sets on. In a month and later about 50% of the activity preserved in the eye is concentrated in the crystalline lens. The most radiosensitive sections of the crystalline lens mainly accumulate P/sup 32/. (auth)« less

Distinctive Glycerophospholipid Profiles of Human Seminoma and Adjacent Normal Tissues by Desorption Electrospray Ionization Imaging Mass Spectrometry

NASA Astrophysics Data System (ADS)

Masterson, Timothy A.; Dill, Allison L.; Eberlin, Livia S.; Mattarozzi, Monica; Cheng, Liang; Beck, Stephen D. W.; Bianchi, Federica; Cooks, R. Graham

2011-08-01

Desorption electrospray ionization mass spectrometry (DESI-MS) has been successfully used to discriminate between normal and cancerous human tissue from different anatomical sites. On the basis of this, DESI-MS imaging was used to characterize human seminoma and adjacent normal tissue. Seminoma and adjacent normal paired human tissue sections (40 tissues) from 15 patients undergoing radical orchiectomy were flash frozen in liquid nitrogen and sectioned to 15 μm thickness and thaw mounted to glass slides. The entire sample was two-dimensionally analyzed by the charged solvent spray to form a molecular image of the biological tissue. DESI-MS images were compared with formalin-fixed, hematoxylin and eosin (H&E) stained slides of the same material. Increased signal intensity was detected for two seminolipids [seminolipid (16:0/16:0) and seminolipid (30:0)] in the normal tubule testis tissue; these compounds were undetectable in seminoma tissue, as well as from the surrounding fat, muscle, and blood vessels. A glycerophosphoinositol [PI(18:0/20:4)] was also found at increased intensity in the normal testes tubule tissue when compared with seminoma tissue. Ascorbic acid (i.e., vitamin C) was found at increased amounts in seminoma tissue when compared with normal tissue. DESI-MS analysis was successfully used to visualize the location of several types of molecules across human seminoma and normal tissues. Discrimination between seminoma and adjacent normal testes tubules was achieved on the basis of the spatial distributions and varying intensities of particular lipid species as well as ascorbic acid. The increased presence of ascorbic acid within seminoma compared with normal seminiferous tubules was previously unknown.

Techniques of Celloidin Removal From Temporal Bone Sections

PubMed Central

O’Malley, Jennifer T.; Burgess, Barbara J.; Jones, Diane D.; Adams, Joe C.; Merchant, Saumil N.

2009-01-01

Objectives We sought to determine whether the technique of celloidin removal influences the results of immunostaining in celloidin-embedded cochleae. Methods We compared four protocols of celloidin removal, including those using clove oil, acetone, ether-alcohol, and methanol saturated with sodium hydroxide. By optimally fixing our tissue (perfused mice), and keeping constant the fixative type (formalin plus acetic acid), fixation time (25 hours), and decalcification time (ethylenediaminetetraacetic acid for 7 days), we determined whether the technique of celloidin removal influenced the immunostaining results. Six antibodies were used with each removal method: prostaglandin D synthase, sodium, potassium adenosine triphosphatase (Na+,K+-ATPase), aquaporin 1, connective tissue growth factor, tubulin, and 200 kd neurofilament. Results Clove oil, acetone, and ether-alcohol resulted in incomplete removal of the celloidin, thereby negatively affecting the results of immunostaining. The methanol–sodium hydroxide method was effective in completely removing the celloidin; it produced the cleanest and most reproducible immunostaining for all six antibodies. Conclusions Freshly prepared methanol saturated with sodium hydroxide and diluted 1:2 with methanol was the best solvent for removing celloidin from mouse temporal bone sections, resulting in consistent and reproducible immunostaining with the six antibodies tested. PMID:19663375

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.

Sodium hypochlorite with reduced surface tension does not improve in situ pulp tissue dissolution.

PubMed

De-Deus, Gustavo; de Berredo Pinho, Marco André; Reis, Claudia; Fidel, Sandra; Souza, Erick; Zehnder, Matthias

2013-08-01

Sodium hypochlorite (NaOCl) solutions with added wetting agents are advertised to dissolve necrotic tissue in root canals faster than their counterparts without a lowered surface tension. This was tested in the current study, and the null hypothesis formulated was that there was no difference between a commercially available NaOCl solution with a lowered surface tension (Chlor-XTRA; Vista Dental Products, Racine, WI) and a counterpart containing the same amount of available chlorine without added wetting agents regarding the soft tissue that remains in oval-shaped canals after mechanical preparation and irrigation. Formerly vital extracted teeth (N = 44, 22 pairs) with similar anatomy were radiographically paired and chemomechanically prepared. In 1 tooth from each pair, a 5.25% NaOCl solution with reduced surface tension was used; in the other, a pure, technical-grade NaOCl solution of 5.25% was used. The percentage of remaining pulp tissue (PRPT) was histologically assessed in root cross-sections. The non-Gaussian raw data were subjected to Kruskal-Wallis and Mann-Whitney U tests to verify the respective effect of the cross-section level and solution on the PRPT. The relationship between the cross-section level and the PRPT was estimated by the Spearman correlation test. The alpha-type error was set at 5%. The cross-section level significantly influenced the PRPT (P < .05), whereas the PRPT was not influenced by the solution used (P > .05). A significant inverse correlation was found between the cross-section level and the PRPT (P < .05, r = -0.330). The lower the distance to the apex, the higher the PRPT regardless of the solution used. Contrary to the advertised statement, the dental solution with a reduced surface tension did not dissolve vital pulp tissue in oval root canals any better than a conventional NaOCl solution of similar strength. Closer to the apex, pulp tissue dissolution is less efficient irrespective of the solution. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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.

Infrared Microtransmission And Microreflectance Of Biological Systems

NASA Astrophysics Data System (ADS)

Hill, Steve L.; Krishnan, K.; Powell, Jay R.

1989-12-01

The infrared microsampling technique has been successfully applied to a variety of biological systems. A microtomed tissue section may be prepared to permit both visual and infrared discrimination. Infrared structural information may be obtained for a single cell, and computer-enhanced images of tissue specimens may be calculated from spectral map data sets. An analysis of a tissue section anomaly may gg suest eitherprotein compositional differences or a localized concentration of foreign matterp. Opaque biological materials such as teeth, gallstones, and kidney stones may be analyzed by microreflectance spectroscop. Absorption anomalies due to specular dispersion are corrected with the Kraymers-Kronig transformation. Corrected microreflectance spectra may contribute to compositional analysis and correlate diseased-related spectral differences to visual specimen anomalies.