Microimaging FT-IR of oral cavity tumours. Part III: Cells, inoculated tissues and human tissues

NASA Astrophysics Data System (ADS)

Conti, C.; Ferraris, P.; Giorgini, E.; Pieramici, T.; Possati, L.; Rocchetti, R.; Rubini, C.; Sabbatini, S.; Tosi, G.; Mariggiò, M. A.; Lo Muzio, L.

2007-05-01

The biochemistry of healthy and tumour cell cultures, inoculated tissues and oral cavity tissues have been studied by FT-IR Microscopy with the aim to relate spectral patterns with microbiological and histopathological findings. 'Supervised' and 'unsupervised' procedures of data handling afforded a satisfactory degree of accordance between spectroscopic and the other two techniques. In particular, changes in frequency and intensity of proteins, connective and nucleic acids vibrational modes as well as the visualization of biochemical single wave number or band ratio images, allowed an evaluation of the pathological changes. The spectroscopic patterns of inoculated tissues resulted quite similar to human tissues; differences of both types of sections with cellular lines could be explained by the influence of the environment.

Multiview hyperspectral topography of tissue structural and functional characteristics

NASA Astrophysics Data System (ADS)

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

2016-01-01

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

Ultrastructural evaluation of multiple pass low energy versus single pass high energy radio-frequency treatment.

PubMed

Kist, David; Burns, A Jay; Sanner, Roth; Counters, Jeff; Zelickson, Brian

2006-02-01

The radio-frequency (RF) device is a system capable of volumetric heating of the mid to deep dermis and selective heating of the fibrous septa strands and fascia layer. Clinically, these effects promote dermal collagen production, and tightening of these deep subcutaneous structures. A new technique of using multiple low energy passes has been described which results in lower patient discomfort and fewer side effects. This technique has also been anecdotally described as giving more reproducible and reliable clinical results of tissue tightening and contouring. This study will compare ultrastructural changes in collagen between a single pass high energy versus up to five passes of a multiple pass lower energy treatment. Three subjects were consented and treated in the preauricular region with the RF device using single or multiple passes (three or five) in the same 1.5 cm(2) treatment area with a slight delay between passes to allow tissue cooling. Biopsies from each treatment region and a control biopsy were taken immediately, 24 hours or 6 months post treatment for electron microscopic examination of the 0-1 mm and 1-2 mm levels. Sections of tissue 1 mm x 1 mm x 80 nm were examined with an RCA EMU-4 Transmission Electron Microscope. Twenty sections from 6 blocks from each 1 mm depth were examined by 2 blinded observers. The morphology and degree of collagen change in relation to area examined was compared to the control tissue, and estimated using a quantitative scale. Ultrastructural examination of tissue showed that an increased amount of collagen fibril changes with increasing passes at energies of 97 J (three passes) and 122 J (five passes), respectively. The changes seen after five multiple passes were similar to those detected after much more painful single pass high-energy treatments. This ultrastructural study shows changes in collagen fibril morphology with an increased effect demonstrated at greater depths of the skin with multiple low-fluence passes and at lesser depths with single pass higher fluence settings. Findings suggest that similar collagen fibril alteration can occur with multiple pass low-energy treatments and single pulse high-energy treatments. The lower fluence multiple pass approach is associated with less patient discomfort, less side effects, and more consistent clinical results. Copyright 2005 Wiley-Liss, Inc.

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

PubMed

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

1998-10-01

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

Interpreting CARS images of tissue within the C-H-stretching region

NASA Astrophysics Data System (ADS)

Dietzek, Benjamin; Meyer, Tobias; Medyukhina, Anna; Bergner, Norbert; Krafft, Christoph; Romeike, Bernd F. M.; Reichart, Rupert; Kalff, Rolf; Schmitt, Michael; Popp, Jürgen

2014-03-01

Single band coherent anti-Stokes Raman scattering (CARS) microscopy within the CH-stretching region is applied to detect individual cells and nuclei of human brain tissue and brain tumors - an information which allows for histopathologic grading of the tissue. The CARS image contrast within the C-H-stretching region correlated to the tissue composition. Based on the specific application example of identifying nuclei within (coherent) Raman images of neurotissue sections, we shall derive general design parameters for lasers optimally suited to serve in a clinical environment and discuss the potential of recently developed methods to analyze spectrally resolved CARS images and image segmentation algorithms.

Multi-stained whole slide image alignment in digital pathology

NASA Astrophysics Data System (ADS)

Déniz, Oscar; Toomey, David; Conway, Catherine; Bueno, Gloria

2015-03-01

In Digital Pathology, one of the most simple and yet most useful feature is the ability to view serial sections of tissue simultaneously on a computer monitor. This enables the pathologist to evaluate the histology and expression of multiple markers for a patient in a single review. However, the rate limiting step in this process is the time taken for the pathologist to open each individual image, align the sections within the viewer, with a maximum of four slides at a time, and then manually move around the section. In addition, due to tissue processing and pre-analytical steps, sections with different stains have non-linear variations between the two acquisitions, that is, they will stretch and change shape from section to section. To date, no solution has come close to a workable solution to automatically align the serial sections into one composite image. This research work address this problem to obtain an automated serial section alignment tool enabling the pathologists to simply scroll through the various sections in a single viewer. To this aim a multi-resolution intensity-based registration method using mutual information as a similarity metric, an optimizer based on an evolutionary process and a bilinear transformation has been used. To characterize the performance of the algorithm 40 cases x 5 different serial sections stained with hematoxiline-eosine (HE), estrogen receptor (ER), progesterone receptor (PR), Ki67 and human epidermal growth factor receptor 2 (Her2), have been considered. The qualitative results obtained are promising, with average computation time of 26.4s for up to 14660x5799 images running interpreted code.

Applications of Mass Spectrometry Imaging to Cancer.

PubMed

Arentz, G; Mittal, P; Zhang, C; Ho, Y-Y; Briggs, M; Winderbaum, L; Hoffmann, M K; Hoffmann, P

2017-01-01

Pathologists play an essential role in the diagnosis and prognosis of benign and cancerous tumors. Clinicians provide tissue samples, for example, from a biopsy, which are then processed and thin sections are placed onto glass slides, followed by staining of the tissue with visible dyes. Upon processing and microscopic examination, a pathology report is provided, which relies on the pathologist's interpretation of the phenotypical presentation of the tissue. Targeted analysis of single proteins provide further insight and together with clinical data these results influence clinical decision making. Recent developments in mass spectrometry facilitate the collection of molecular information about such tissue specimens. These relatively new techniques generate label-free mass spectra across tissue sections providing nonbiased, nontargeted molecular information. At each pixel with spatial coordinates (x/y) a mass spectrum is acquired. The acquired mass spectrums can be visualized as intensity maps displaying the distribution of single m/z values of interest. Based on the sample preparation, proteins, peptides, lipids, small molecules, or glycans can be analyzed. The generated intensity maps/images allow new insights into tumor tissues. The technique has the ability to detect and characterize tumor cells and their environment in a spatial context and combined with histological staining, can be used to aid pathologists and clinicians in the diagnosis and management of cancer. Moreover, such data may help classify patients to aid therapy decisions and predict outcomes. The novel complementary mass spectrometry-based methods described in this chapter will contribute to the transformation of pathology services around the world. © 2017 Elsevier Inc. All rights reserved.

Brain Slice Staining and Preparation for Three-Dimensional Super-Resolution Microscopy

PubMed Central

German, Christopher L.; Gudheti, Manasa V.; Fleckenstein, Annette E.; Jorgensen, Erik M.

2018-01-01

Localization microscopy techniques – such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM) – provide the highest precision for single molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single molecule localizations of synaptic vesicle and active zone proteins in three-dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging. PMID:28924666

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.

Imaging of single cells and tissue using MeV ions

NASA Astrophysics Data System (ADS)

Watt, F.; Bettiol, A. A.; van Kan, J. A.; Ynsa, M. D.; Minqin, Ren; Rajendran, R.; Huifang, Cui; Fwu-Shen, Sheu; Jenner, A. M.

2009-06-01

With the attainment of sub-100 nm high energy (MeV) ion beams, comes the opportunity to image cells and tissue at nano-dimensions. The advantage of MeV ion imaging is that the ions will penetrate whole cells, or relatively thick tissue sections, without any significant loss of resolution. In this paper, we demonstrate that whole cells (cultured N2A neuroblastoma cells ATCC) and tissue sections (rabbit pancreas tissue) can be imaged at sub-100 nm resolutions using scanning transmission ion microscopy (STIM), and that sub-cellular structural details can be identified. In addition to STIM imaging we have also demonstrated for the first time, that sub-cellular proton induced fluorescence imaging (on cultured N2A neuroblastoma cells ATCC) can also be carried out at resolutions of 200 nm, compared with 300-400 nm resolutions achieved by conventional optical fluorescence imaging. The combination of both techniques offers a potentially powerful tool in the quest for elucidating cell function, particularly when it should be possible in the near future to image down to sub-50 nm.

Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot

NASA Astrophysics Data System (ADS)

Prentice, Boone M.; Chumbley, Chad W.; Caprioli, Richard M.

2017-01-01

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R2) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

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.

Quantum Dots for Molecular Pathology

PubMed Central

True, Lawrence D.; Gao, Xiaohu

2007-01-01

Assessing malignant tumors for expression of multiple biomarkers provides data that are critical for patient management. Quantum dot-conjugated probes to specific biomarkers are powerful tools that can be applied in a multiplex manner to single tissue sections of biopsies to measure expression levels of multiple biomarkers. PMID:17251330

Soft Tissue Tumours: Their Natural History and Treatment

PubMed Central

Cade, Stanford

1951-01-01

A series of 153 patients, the largest yet recorded from a single source, suffering from soft tissue sarcoma is discussed. References to the literature show the rarity of such tumours, the vagueness of the nomenclature and the disappointing results of treatment. Of the 153 patients only 7 have no histological confirmation of the diagnosis. In 146, sections and histological reports are available. 148 patients have been followed up either to death or to date. ImagesFig. 1Fig. 2Fig. 3Fig. 4 PMID:14808223

Multiclonal Invasion in Breast Tumors Identified by Topographic Single Cell Sequencing.

PubMed

Casasent, Anna K; Schalck, Aislyn; Gao, Ruli; Sei, Emi; Long, Annalyssa; Pangburn, William; Casasent, Tod; Meric-Bernstam, Funda; Edgerton, Mary E; Navin, Nicholas E

2018-01-11

Ductal carcinoma in situ (DCIS) is an early-stage breast cancer that infrequently progresses to invasive ductal carcinoma (IDC). Genomic evolution has been difficult to delineate during invasion due to intratumor heterogeneity and the low number of tumor cells in the ducts. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS) to measure genomic copy number profiles of single tumor cells while preserving their spatial context in tissue sections. We applied TSCS to 1,293 single cells from 10 synchronous patients with both DCIS and IDC regions in addition to exome sequencing. Our data reveal a direct genomic lineage between in situ and invasive tumor subpopulations and further show that most mutations and copy number aberrations evolved within the ducts prior to invasion. These results support a multiclonal invasion model, in which one or more clones escape the ducts and migrate into the adjacent tissues to establish the invasive carcinomas. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2018-04-30

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

Mid-infrared fiber-coupled supercontinuum spectroscopic imaging using a tapered chalcogenide photonic crystal fiber

NASA Astrophysics Data System (ADS)

Rosenberg Petersen, Christian; Prtljaga, Nikola; Farries, Mark; Ward, Jon; Napier, Bruce; Lloyd, Gavin Rhys; Nallala, Jayakrupakar; Stone, Nick; Bang, Ole

2018-02-01

We present the first demonstration of mid-infrared spectroscopic imaging of human tissue using a fiber-coupled supercontinuum source spanning from 2-7.5 μm. The supercontinuum was generated in a tapered large mode area chalcogenide photonic crystal fiber in order to obtain broad bandwidth, high average power, and single-mode output for good imaging properties. Tissue imaging was demonstrated in transmission by raster scanning over a sub-mm region of paraffinized colon tissue on CaF2 substrate, and the signal was measured using a fiber-coupled grating spectrometer. This demonstration has shown that we can distinguish between epithelial and surrounding connective tissues within a paraffinized section of colon tissue by imaging at discrete wavelengths related to distinct chemical absorption features.

Multiview hyperspectral topography of tissue structural and functional characteristics

NASA Astrophysics Data System (ADS)

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

2012-12-01

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

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

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.

A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF.

PubMed

Théron, Laëtitia; Centeno, Delphine; Coudy-Gandilhon, Cécile; Pujos-Guillot, Estelle; Astruc, Thierry; Rémond, Didier; Barthelemy, Jean-Claude; Roche, Frédéric; Feasson, Léonard; Hébraud, Michel; Béchet, Daniel; Chambon, Christophe

2016-10-26

Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m / z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.

A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF

PubMed Central

Théron, Laëtitia; Centeno, Delphine; Coudy-Gandilhon, Cécile; Pujos-Guillot, Estelle; Astruc, Thierry; Rémond, Didier; Barthelemy, Jean-Claude; Roche, Frédéric; Feasson, Léonard; Hébraud, Michel; Béchet, Daniel; Chambon, Christophe

2016-01-01

Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation. PMID:28248242

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

PubMed

Ginsberg, Stephen D; Che, Shaoli

2004-08-01

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

Thermomicrocapillaries as temperature biosensors in single cells

NASA Astrophysics Data System (ADS)

Herth, Simone; Giesguth, Miriam; Wedel, Waldemar; Reiss, Günther; Dietz, Karl-Josef

2013-03-01

Temperature is an important physical parameter in biology and its deviation from optimum can cause damage in biosystems. Thermocouples based on the Seebeck effect can be structured on glass microcapillaries to obtain thermomicrocapillaries (TMCs) usable in a micromanipulation setup. The suitability of the setup was proven by monitoring the temperature increase upon illumination of leaves and single cells following insertion of the TMC. The increase was 1.5 K in green tissue and 0.75 K in white leaf sections due to lower absorption. In single cells of trichomes, the increase was 0.5 K due to heat dissipation to the surrounding air.

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.

Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains

NASA Astrophysics Data System (ADS)

Chen, Youting; Chen, Jing; Chen, Hong; Hong, Zhipeng; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Yanling; Chen, Jianxin

2014-09-01

Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear-cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

Serial sectioning for examination of photoreceptor cell architecture by focused ion beam technology

PubMed Central

Mustafi, Debarshi; Avishai, Amir; Avishai, Nanthawan; Engel, Andreas; Heuer, Arthur; Palczewski, Krzysztof

2011-01-01

Structurally deciphering complex neural networks requires technology with sufficient resolution to allow visualization of single cells and their intimate surrounding connections. Scanning electron microscopy (SEM), coupled with serial ion ablation (SIA) technology, presents a new avenue to study these networks. SIA allows ion ablation to remove nanometer sections of tissue for SEM imaging, resulting in serial section data collection for three-dimensional reconstruction. Here we highlight a method for preparing retinal tissues for imaging of photoreceptors by SIA-SEM technology. We show that this technique can be used to visualize whole rod photoreceptors and the internal disc elements from wild-type (wt) mice. The distance parameters of the discs and photoreceptors are in good agreement with previous work with other methods. Moreover, we show that large planes of retinal tissue can be imaged at high resolution to display the packing of normal rods. Finally, SIA-SEM imaging of retinal tissue from a mouse model (Nrl−/−) with phenotypic changes akin to the human disease enhanced S-cone syndrome (ESCS) revealed a structural profile of overall photoreceptor ultrastructure and internal elements that accompany this disease. Overall, this work presents a new method to study photoreceptor cells at high structural resolution that has a broad applicability to the visual neuroscience field. PMID:21439323

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

PubMed Central

Lavenant, Gwendoline Thiery; Zavalin, Andrey I.; Caprioli, Richard M.

2013-01-01

Targeted multiplex Imaging Mass Spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This manuscript describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet. PMID:23397138

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

NASA Astrophysics Data System (ADS)

Thiery-Lavenant, Gwendoline; Zavalin, Andre I.; Caprioli, Richard M.

2013-04-01

Targeted multiplex imaging mass spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This article describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.

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

A Military-Relevant Model of Closed Concussive Head Injury: Longitudinal Studies Characterizing and Validating Single and Repetitive mTBI

DTIC Science & Technology

2013-10-01

processed at FD Neurotechnologies Inc. (Ellicott City, MD, USA). A series of coronal free floating brain sections (40 μm; 960 µm interval from +4.0...axonal damage. Another series of coronal sections was stained using FD NeuroSilverTM Kit II (FD Neurotechnologies Inc., Ellicott City, MD, USA) for... Neurotechnologies Inc for processing and staining. Tissue processing and staining protocols have been optimized for fluorescent staining of AQ4 co

Comparison of a new hydro-surgical technique to traditional methods for the preparation of full-thickness skin grafts from canine cadaveric skin and report of a single clinical case.

PubMed

Townsend, F I; Ralphs, S C; Coronado, G; Sweet, D C; Ward, J; Bloch, C P

2012-01-01

To compare the hydro-surgical technique to traditional techniques for removal of subcutaneous tissue in the preparation of full-thickness skin grafts. Ex vivo experimental study and a single clinical case report. Four canine cadavers and a single clinical case. Four sections of skin were harvested from the lateral flank of recently euthanatized dogs. Traditional preparation methods used included both a blade or scissors technique, each of which were compared to the hydro-surgical technique individually. Preparation methods were compared based on length of time for removal of the subcutaneous tissue from the graft, histologic grading, and measurable thickness as compared to an untreated sample. The hydro-surgical technique had the shortest skin graft preparation time as compared to traditional techniques (p = 0.002). There was no significant difference in the histological grading or measurable subcutaneous thickness between skin specimens. The hydro-surgical technique provides a rapid, effective debridement of subcutaneous tissue in the preparation of full-thickness skin grafts. There were not any significant changes in histological grade and subcutaneous tissue remaining among all treatment types. Additionally the hydro-surgical technique was successfully used to prepare a full-thickness meshed free skin graft in the reconstruction of a traumatic medial tarsal wound in a dog.

Identification of regions of normal grey matter and white matter from pathologic glioblastoma and necrosis in frozen sections using Raman imaging.

PubMed

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

2015-11-01

In neurosurgical applications, a tool capable of distinguishing grey matter, white matter, and areas of tumor and/or necrosis in near-real time could greatly aid in tumor resection decision making. Raman spectroscopy is a non-destructive spectroscopic technique which provides molecular information about the tissue under examination based on the vibrational properties of the constituent molecules. With careful measurement and data processing, a spatial step and repeat acquisition of Raman spectra can be used to create Raman images. Forty frozen brain tissue sections were imaged in their entirety using a 300-µm-square measurement grid, and two or more regions of interest within each tissue were also imaged using a 25 µm-square step size. Molecular correlates for histologic features of interest were identified within the Raman spectra, and novel imaging algorithms were developed to compare molecular features across multiple tissues. In previous work, the relative concentration of individual biomolecules was imaged. Here, the relative concentrations of 1004, 1300:1344, and 1660 cm(-1), which correspond primarily to protein and lipid content, were simultaneously imaged across all tissues. This provided simple interpretation of boundaries between grey matter, white matter, and diseased tissue, and corresponded with findings from adjacent hematoxylin and eosin-stained sections. This novel, yet simple, multi-channel imaging technique allows clinically-relevant resolution with straightforward molecular interpretation of Raman images not possible by imaging any single peak. This method can be applied to either surgical or laboratory tools for rapid, non-destructive imaging of grey and white matter.

Periodontal tissue regeneration by combined applications of recombinant human osteogenic protein-1 and bone morphogenetic protein-2. A pilot study in Chacma baboons (Papio ursinus).

PubMed

Ripamonti, U; Crooks, J; Petit, J C; Rueger, D C

2001-08-01

Native and recombinant human bone morphogenetic/osteogenic proteins (BMPs/ OPs) singly initiate bone induction in vivo. The finding of synchronous but spatially different BMPs/OPs expression during periodontal tissue morphogenesis suggests novel therapeutic approaches using morphogen combinations based on recapitulation of embryonic development. Twelve furcation defects prepared in the first and second mandibular molars of three adult baboons (Papio ursinus) were used to assess whether qualitative histological aspects of periodontal tissue regeneration could be enhanced and tissue morphogenesis modified by combined or single applications of recombinant hOP-1 and hBMP-2. Doses of BMPs/OPs were 100 microg of each protein per 1 g of insoluble collagenous bone matrix as carrier. Approximately 200 mg of carrier matrix was used per furcation defect. Undecalcified sections cut for histological analysis 60 d after healing of hOP-1-treated specimens showed substantial cementogenesis with scattered remnants of the collagenous carrier. hBMP-2 applied alone induced greater amounts of mineralized bone and osteoid when compared to hOP-1 alone or to combined morphogen applications. Combined applications of hOP-1 and hBMP-2 did not enhance alveolar bone regeneration or new attachment formation over and above the single applications of the morphogens. The results of this study, which is the first to attempt to address the structure-activity relationship amongst BMP/OP family members, indicate that tissue morphogenesis induced by hOP-1 and hBMP-2 is qualitatively different when the morphogens are applied singly, with hOP-1 inducing substantial cementogenesis. hBMP-2 treated defects, on the other hand, showed limited cementum formation but a temporal enhancement of alveolar bone regeneration and remodelling. The demonstration of therapeutic mosaicism in periodontal regeneration will require extensive testing of ratios and doses of recombinant morphogen combinations for optimal tissue engineering in clinical contexts.

A multiple-well method for immunohistochemical testing of many reagents on a single microscopic slide.

PubMed

McKeever, P E; Letica, L H; Shakui, P; Averill, D R

1988-09-01

Multiple wells (M-wells) have been made over tissue sections on single microscopic slides to simultaneously localize binding specificity of many antibodies. More than 20 individual 4-microliter wells over tissue have been applied/slide, representing more than a 5-fold improvement in wells/slide and a 25-fold reduction in reagent volume over previous methods. More than 30 wells/slide have been applied over cellular monolayers. To produce the improvement, previous strategies of placing specimens into wells were changed to instead create wells over the specimen. We took advantage of the hydrophobic properties of paint to surround the wells and to segregate the various different primary antibodies. Segregation was complete on wells alternating with and without primary monoclonal antibody. The procedure accommodates both frozen and paraffin sections, yielding slides which last more than a year. After monoclonal antibody detection, standard histologic stains can be applied as counterstains. M-wells are suitable for localizing binding of multiple reagents or sample unknowns (polyclonal or monoclonal antibodies, hybridoma supernatants, body fluids, lectins) to either tissues or cells. Their small sample volume and large number of sample wells/slide could be particularly useful for early screening of hybridoma supernatants and for titration curves in immunohistochemistry (McKeever PE, Shakui P, Letica LH, Averill DR: J Histochem Cytochem 36:931, 1988).

Semi-automatic central-chest lymph-node definition from 3D MDCT images

NASA Astrophysics Data System (ADS)

Lu, Kongkuo; Higgins, William E.

2010-03-01

Central-chest lymph nodes play a vital role in lung-cancer staging. The three-dimensional (3D) definition of lymph nodes from multidetector computed-tomography (MDCT) images, however, remains an open problem. This is because of the limitations in the MDCT imaging of soft-tissue structures and the complicated phenomena that influence the appearance of a lymph node in an MDCT image. In the past, we have made significant efforts toward developing (1) live-wire-based segmentation methods for defining 2D and 3D chest structures and (2) a computer-based system for automatic definition and interactive visualization of the Mountain central-chest lymph-node stations. Based on these works, we propose new single-click and single-section live-wire methods for segmenting central-chest lymph nodes. The single-click live wire only requires the user to select an object pixel on one 2D MDCT section and is designed for typical lymph nodes. The single-section live wire requires the user to process one selected 2D section using standard 2D live wire, but it is more robust. We applied these methods to the segmentation of 20 lymph nodes from two human MDCT chest scans (10 per scan) drawn from our ground-truth database. The single-click live wire segmented 75% of the selected nodes successfully and reproducibly, while the success rate for the single-section live wire was 85%. We are able to segment the remaining nodes, using our previously derived (but more interaction intense) 2D live-wire method incorporated in our lymph-node analysis system. Both proposed methods are reliable and applicable to a wide range of pulmonary lymph nodes.

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue

PubMed Central

Koob, A.O.; Bruns, L.; Prassler, C.; Masliah, E.; Klopstock, T.; Bender, A.

2016-01-01

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. PMID:22402104

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

PubMed

Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

2012-06-15

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

Top-down mass spectrometry imaging of intact proteins by laser ablation ESI FT-ICR MS.

PubMed

Kiss, András; Smith, Donald F; Reschke, Brent R; Powell, Matthew J; Heeren, Ron M A

2014-05-01

Laser ablation ESI (LAESI) is a recent development in MS imaging. It has been shown that lipids and small metabolites can be imaged in various samples such as plant material, tissue sections or bacterial colonies without any sample pretreatment. Further, LAESI has been shown to produce multiply charged protein ions from liquids or solid surfaces. This presents a means to address one of the biggest challenges in MS imaging; the identification of proteins directly from biological tissue surfaces. Such identification is hindered by the lack of multiply charged proteins in common MALDI ion sources and the difficulty of performing tandem MS on such large, singly charged ions. We present here top-down identification of intact proteins from tissue with a LAESI ion source combined with a hybrid ion-trap FT-ICR mass spectrometer. The performance of the system was first tested with a standard protein with electron capture dissociation and infrared multiphoton dissociation fragmentation to prove the viability of LAESI FT-ICR for top-down proteomics. Finally, the imaging of a tissue section was performed, where a number of intact proteins were measured and the hemoglobin α chain was identified directly from tissue using CID and infrared multiphoton dissociation fragmentation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single element ultrasonic imaging of limb geometry: an in-vivo study with comparison to MRI

NASA Astrophysics Data System (ADS)

Zhang, Xiang; Fincke, Jonathan R.; Anthony, Brian W.

2016-04-01

Despite advancements in medical imaging, current prosthetic fitting methods remain subjective, operator dependent, and non-repeatable. The standard plaster casting method relies on prosthetist experience and tactile feel of the limb to design the prosthetic socket. Often times, many fitting iterations are required to achieve an acceptable fit. Use of improper socket fittings can lead to painful pathologies including neuromas, inflammation, soft tissue calcification, and pressure sores, often forcing the wearer to into a wheelchair and reducing mobility and quality of life. Computer software along with MRI/CT imaging has already been explored to aid the socket design process. In this paper, we explore the use of ultrasound instead of MRI/CT to accurately obtain the underlying limb geometry to assist the prosthetic socket design process. Using a single element ultrasound system, multiple subjects' proximal limbs were imaged using 1, 2.25, and 5 MHz single element transducers. Each ultrasound transducer was calibrated to ensure acoustic exposure within the limits defined by the FDA. To validate image quality, each patient was also imaged in an MRI. Fiducial markers visible in both MRI and ultrasound were used to compare the same limb cross-sectional image for each patient. After applying a migration algorithm, B-mode ultrasound cross-sections showed sufficiently high image resolution to characterize the skin and bone boundaries along with the underlying tissue structures.

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.

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

NASA Astrophysics Data System (ADS)

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

2016-12-01

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

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.

Elastic light single-scattering spectroscopy for detection of dysplastic tissues

NASA Astrophysics Data System (ADS)

Canpolat, Murat; Denkçeken, Tuba; Akman, Ayşe.; Alpsoy, Erkan; Tuncer, Recai; Akyüz, Mahmut; Baykara, Mehmet; Yücel, Selçuk; Başsorgun, Ibrahim; ćiftçioǧlu, M. Akif; Gökhan, Güzide Ayşe.; Gürer, ElifInanç; Peştereli, Elif; Karaveli, Šeyda

2013-11-01

Elastic light single-scattering spectroscopy (ELSSS) system has been developed and tested in diagnosis of cancerous tissues of different organs. ELSSS system consists of a miniature visible light spectrometer, a single fiber optical probe, a halogen tungsten light source and a laptop. Measurements were performed on excised brain, skin, cervix and prostate tumor specimens and surrounding normal tissues. Single fiber optical probe with a core diameter of 100 μm was used to deliver white light to and from tissue. Single optical fiber probe mostly detects singly scattered light from tissue rather than diffused light. Therefore, measured spectra are sensitive to size of scatters in tissue such as cells, nuclei, mitochondria and other organelles of cells. Usually, nuclei of tumor cells are larger than nuclei of normal cells. Therefore, spectrum of singly scattered light of tumor tissue is different than normal tissue. The spectral slopes were shown to be positive for normal brain, skin and prostate and cervix tissues and negative for the tumors of the same tissues. Signs of the spectral slopes were used as a discrimination parameter to differentiate tumor from normal tissues for the three organ tissues. Sensitivity and specificity of the system in differentiation between tumors from normal tissues were 93% and %100 for brain, 87% and 85% for skin, 93.7% and 46.1% for cervix and 98% and 100% for prostate.

Scanning X-ray diffraction on cardiac tissue: automatized data analysis and processing.

PubMed

Nicolas, Jan David; Bernhardt, Marten; Markus, Andrea; Alves, Frauke; Burghammer, Manfred; Salditt, Tim

2017-11-01

A scanning X-ray diffraction study of cardiac tissue has been performed, covering the entire cross section of a mouse heart slice. To this end, moderate focusing by compound refractive lenses to micrometer spot size, continuous scanning, data acquisition by a fast single-photon-counting pixel detector, and fully automated analysis scripts have been combined. It was shown that a surprising amount of structural data can be harvested from such a scan, evaluating the local scattering intensity, interfilament spacing of the muscle tissue, the filament orientation, and the degree of anisotropy. The workflow of data analysis is described and a data analysis toolbox with example data for general use is provided. Since many cardiomyopathies rely on the structural integrity of the sarcomere, the contractile unit of cardiac muscle cells, the present study can be easily extended to characterize tissue from a diseased heart.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

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

PubMed Central

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

2013-01-01

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

Pathogenic role of the eight probably/possibly carcinogenic HPV types 26, 53, 66, 67, 68, 70, 73 and 82 in cervical cancer.

PubMed

Halec, Gordana; Alemany, Laia; Lloveras, Belen; Schmitt, Markus; Alejo, Maria; Bosch, Franz X; Tous, Sara; Klaustermeier, Jo Ellen; Guimerà, Nuria; Grabe, Niels; Lahrmann, Bernd; Gissmann, Lutz; Quint, Wim; Bosch, Francesc X; de Sanjose, Silvia; Pawlita, Michael

2014-12-01

Eight HPV types (HPV26, 53, 66, 67, 68, 70, 73 and 82) that are phylogenetically closely related to 12 WHO-defined high-risk (HR) HPV have been rarely but consistently identified as single HPV infections in about 3% of cervical cancer (CxCa) tissues. Due to lack of biological data, these types are referred to as probable/possible (p) HR-HPV. To analyse their biological activity in direct comparison to HR-HPV types, we selected 55 formalin-fixed, paraffin-embedded (FFPE) CxCa tissues harbouring single pHR-HPV infections (2-13 cases per type) and 266 tissues harbouring single HR-HPV (7-40 cases per type) from a worldwide, retrospective, cross-sectional study. Single HPV infection was verified by two genotyping methods. Presence of type-specific spliced E6*I mRNA transcripts and expression of cellular proteins indicative of HPV transformation were assessed in all cases. In 55 CxCa tissues with pHR-HPV, E6*I mRNA expression was 100%; high p16(INK4a) , 98%; low pRb, 96%; low CyD1, 93%; and low p53, 84%. Compared to HPV16 tissues as a reference, individual frequencies of these five markers did not differ significantly, either for any of the eight pHR-HPV and the 11 other HR types individually or for the groups of pHR and HR types without HPV16. We conclude that the eight pHR-HPV types, when present as a single infection in CxCa, are biologically active and affect the same cellular pathways as any of the fully recognized carcinogenic HR-HPV types. Therefore we have provided molecular evidence of carcinogenicity for types currently classified as probably/possibly carcinogenic. Although this evidence is crucial for HPV-type carcinogenicity classification, per se it is not sufficient for inclusion of these HPV types into population-wide primary and secondary prevention programmes. Such decisions have to include careful estimation of effectiveness and cost-benefit analyses. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bi-PROF

PubMed Central

Gries, Jasmin; Schumacher, Dirk; Arand, Julia; Lutsik, Pavlo; Markelova, Maria Rivera; Fichtner, Iduna; Walter, Jörn; Sers, Christine; Tierling, Sascha

2013-01-01

The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 102-104 sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer. PMID:23803588

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

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

NASA Astrophysics Data System (ADS)

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

2013-03-01

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

Combining lipophilic dye, in situ hybridization, immunohistochemistry, and histology.

PubMed

Duncan, Jeremy; Kersigo, Jennifer; Gray, Brian; Fritzsch, Bernd

2011-03-17

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

PubMed Central

Duncan, Jeremy; Kersigo, Jennifer; Gray, Brian; Fritzsch, Bernd

2011-01-01

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data. PMID:21445047

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

Nanostructures based on quantum dots for application in promising methods of single- and multiphoton imaging and diagnostics

NASA Astrophysics Data System (ADS)

Nabiev, I. R.

2017-01-01

Molecules recognizing biomarkers of diseases (monoclonal antibodies (monoABs)) are often too large for biomedical applications, and the conditions that are used to bind them with nanolabels lead to disordered orientation of monoABs with respect to the nanoparticle surface. Extremely small nanoprobes, designed via oriented conjugation of quantum dots (QDs) with single-domain antibodies (sdABs) derived from the immunoglobulin of llama and produced in the E. coli culture, have a hydrodynamic diameter less than 12 nm and contain equally oriented sdAB molecules on the surface of each QD. These nanoprobes exhibit excellent specificity and sensitivity in quantitative determination of a small number of cells expressing biomarkers. In addition, the higher diffusion coefficient of sdABs makes it possible to perform immunohistochemical analysis in bulk tissue, inaccessible for conventional monoABs. The necessary conditions for implementing high-quality immunofluorescence diagnostics are a high specificity of labeling and clear differences between the fluorescence of nanoprobes and the autofluorescence of tissues. Multiphoton micros-copy with excitation in the near-IR spectral range, which is remote from the range of tissue autofluorescence excitation, makes it possible to solve this problem and image deep layers in biological tissues. The two-photon absorption cross sections of CdSe/ZnS QDs conjugated with sdABs exceed the corresponding values for organic fluorophores by several orders of magnitude. These nanoprobes provide clear discrimination between the regions of tumor and normal tissues with a ratio of the sdAB fluorescence to the tissue autofluorescence upon two-photon excitation exceeding that in the case of single-photon excitation by a factor of more than 40. The data obtained indicate that the sdAB-QD conjugates used as labels provide the same, or even better, quality as the "gold standard" of immunohistochemical diagnostics. The developed nanoprobes are expected to find wide application in high-efficiency imaging of tumor and multiparameter diagnostics.

Effects of Spirulina on Cyclophosphamide-Induced Ovarian Toxicity in Rats: Biochemical and Histomorphometric Evaluation of the Ovary

PubMed Central

Yener, Nese Arzu; Sinanoglu, Orhun; Ilter, Erdin; Celik, Aygen; Sezgin, Gulbuz; Midi, Ahmet; Aksungar, Fehime

2013-01-01

Cyclophosphamide (Cyc) is known to cause ovotoxicity and infertility in women. Our aim is to investigate the possible ovotoxic effects of Cyc and possible antioxidant and protective effects of blue-green algae, Spirulina (Sp), in rat ovaries. Eighteen rats were given: group I (n = 6, control); group II (n = 6, CP), a single dose Cyc; group III (n = 6, Sp+Cyc), 7 days Sp+single dose Cyc. Tissue malondialdehyde (MDA) levels, superoxide dismutase (SOD), and catalase (CAT) activities are assessed biochemically. Normal and atretic primordial and primary follicle counts for all sections obtained for each ovary are calculated. Mean number of follicle counts for each group are compared. In Sp+Cyc group, tissue MDA levels were significantly lower than those in the CP and higher than those in the C group (CP > Sp+Cyc > C). Tissue SOD activity was significantly higher in Sp+Cyc group than that in the CP group and lower than that in the C group (C > Sp+Cyc > C). No statistically significant difference was found between the ovarian CAT activities in any group. Histomorphometrically, there was also no significant difference between the mean numbers of normal and atretic small follicle counts. Our results suggest that single dose Cyc has adverse effects on oxidant status of the ovaries and Sp has protective effects in Cyc-induced ovotoxicity. PMID:23762559

Single-Stage Resection of Type II Constriction Rings in Limbs on the Basis of Histologic and Magnetic Resonance Imaging Observations: A Retrospective Study of 21 Consecutive Patients.

PubMed

Jiang, Yongkang; Mao, Hailei; Yang, Xi; Zhou, Shengbo; Ni, Feng; Xu, Qiming; Wang, Bin

2016-07-01

The purpose of this study was to determine the feasibility of single-stage resection for type II congenital constriction rings by means of histologic examination of resected specimens and imaging examination of affected extremities, and to evaluate the appearance and function of the extremities after single-stage surgery. The features of the skin on the constriction rings and the subcutaneous tissues were identified through continuous sectioning, hematoxylin and eosin staining, and immunohistologic staining of specimens of type II constriction rings obtained by means of surgery. The relationship between the constriction rings and the deep main blood vessels was evaluated using magnetic resonance imaging. Single-stage resection of the constriction band, reduction of the fascial flap, and triangular flap-plasty were performed for 21 patients. The appearance, lymphedema, and movement of the extremities were compared before and after the operation. Type II constriction rings in the extremities had normal full-layer skin structures. Collagen was found deposited densely at the base of the grooves, but the normal subcutaneous tissue space remained, and the vital nerves and blood vessels were unaffected. Complete resection of the constriction rings was achieved in all 21 patients, and lymphedema subsided 2 months after the operation. No episode of recurrence was found, and limb function was not affected at 26-month follow-up. Type II congenital constriction rings in limbs possess normal subcutaneous tissue spaces. A single-stage operation, which includes complete resection of the rings, fascial flap reduction, and triangular flap-plasty, could achieve a satisfactory appearance and good function. Therapeutic, III.

High-Resolution Ambient MS Imaging of Negative Ions in Positive Ion Mode: Using Dicationic Reagents with the Single-Probe

NASA Astrophysics Data System (ADS)

Rao, Wei; Pan, Ning; Tian, Xiang; Yang, Zhibo

2016-01-01

We have used the Single-probe, a miniaturized sampling device utilizing in-situ surface microextraction for ambient mass spectrometry (MS) analysis, for the high resolution MS imaging (MSI) of negatively charged species in the positive ionization mode. Two dicationic compounds, 1,5-pentanediyl-bis(1-butylpyrrolidinium) difluoride [C5(bpyr)2F2] and 1,3-propanediyl-bis(tripropylphosphonium) difluoride [C3(triprp)2F2], were added into the sampling solvent to form 1+ charged adducts with the negatively charged species extracted from tissues. We were able to detect 526 and 322 negatively charged species this way using [C5(bpyr)2F2] and [C3(triprp)2F2], respectively, including oleic acid, arachidonic acid, and several species of phosphatidic acid, phosphoethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and others. In conjunction with the identification of the non-adduct cations, we have tentatively identified a total number of 1200 and 828 metabolites from mouse brain sections using [C5(bpyr)2F2] and [C3(triprp)2F2], respectively, through high mass accuracy measurements (mass error <5 ppm); MS/MS analyses were also performed to verify the identity of selected species. In addition to the high mass accuracy measurement, we were able to generate high spatial resolution (~17 μm) MS images of mouse brain sections. Our study demonstrated that utilization of dicationic compounds in the surface microextraction with the Single-probe device can perform high mass and spatial resolution ambient MSI measurements of broader types of compounds in tissues. Other reagents can be potentially used with the Single-probe device for a variety of reactive MSI studies to enable the analysis of species that are previously intractable.

Cell Migration

PubMed Central

Trepat, Xavier; Chen, Zaozao; Jacobson, Ken

2015-01-01

Cell migration is fundamental to establishing and maintaining the proper organization of multicellular organisms. Morphogenesis can be viewed as a consequence, in part, of cell locomotion, from large-scale migrations of epithelial sheets during gastrulation, to the movement of individual cells during development of the nervous system. In an adult organism, cell migration is essential for proper immune response, wound repair, and tissue homeostasis, while aberrant cell migration is found in various pathologies. Indeed, as our knowledge of migration increases, we can look forward to, for example, abating the spread of highly malignant cancer cells, retarding the invasion of white cells in the inflammatory process, or enhancing the healing of wounds. This article is organized in two main sections. The first section is devoted to the single-cell migrating in isolation such as occurs when leukocytes migrate during the immune response or when fibroblasts squeeze through connective tissue. The second section is devoted to cells collectively migrating as part of multicellular clusters or sheets. This second type of migration is prevalent in development, wound healing, and in some forms of cancer metastasis. PMID:23720251

Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation

PubMed Central

Yango, Pamela; Altman, Eran; Smith, James F.; Klatsky, Peter C.; Tran, Nam D.

2015-01-01

Objective To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. Design In vitro human testicular tissues. Setting Academic research unit. Patients Adult testicular tissues (n = 4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n = 3). Intervention(s) Testicular tissue vs. single cell suspension cryopreservation. Main Outcome Measures Cell viability, total cell recovery per milligram of tissue, as well as, viable and SSEA-4+ cell recovery. Results Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Conclusions Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient age, type of samples cryopreserved, and end points of therapeutic applications. PMID:25241367

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.

Single-cell metabolomics: analytical and biological perspectives.

PubMed

Zenobi, R

2013-12-06

There is currently much interest in broad molecular profiling of single cells; a cell's metabolome-its full complement of small-molecule metabolites-is a direct indicator of phenotypic diversity of single cells and a nearly immediate readout of how cells react to environmental influences. However, the metabolome is very difficult to measure at the single-cell level because of rapid metabolic dynamics, the structural diversity of the molecules, and the inability to amplify or tag small-molecule metabolites. Measurement techniques including mass spectrometry, capillary electrophoresis, and, to a lesser extent, optical spectroscopy and fluorescence detection have led to impressive advances in single-cell metabolomics. Even though none of these methodologies can currently measure the metabolome of a single cell completely, rapidly, and nondestructively, progress has been sufficient such that the field is witnessing a shift from feasibility studies to investigations that yield new biological insight. Particularly interesting fields of application are cancer biology, stem cell research, and monitoring of xenobiotics and drugs in tissue sections at the single-cell level.

Deep learning and shapes similarity for joint segmentation and tracing single neurons in SEM images

NASA Astrophysics Data System (ADS)

Rao, Qiang; Xiao, Chi; Han, Hua; Chen, Xi; Shen, Lijun; Xie, Qiwei

2017-02-01

Extracting the structure of single neurons is critical for understanding how they function within the neural circuits. Recent developments in microscopy techniques, and the widely recognized need for openness and standardization provide a community resource for automated reconstruction of dendritic and axonal morphology of single neurons. In order to look into the fine structure of neurons, we use the Automated Tape-collecting Ultra Microtome Scanning Electron Microscopy (ATUM-SEM) to get images sequence of serial sections of animal brain tissue that densely packed with neurons. Different from other neuron reconstruction method, we propose a method that enhances the SEM images by detecting the neuronal membranes with deep convolutional neural network (DCNN) and segments single neurons by active contour with group shape similarity. We joint the segmentation and tracing together and they interact with each other by alternate iteration that tracing aids the selection of candidate region patch for active contour segmentation while the segmentation provides the neuron geometrical features which improve the robustness of tracing. The tracing model mainly relies on the neuron geometrical features and is updated after neuron being segmented on the every next section. Our method enables the reconstruction of neurons of the drosophila mushroom body which is cut to serial sections and imaged under SEM. Our method provides an elementary step for the whole reconstruction of neuronal networks.

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.

Breast Cancer Diagnosis Using a Microfluidic Multiplexed Immunohistochemistry Platform

PubMed Central

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

2010-01-01

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

Three-dimensional holographic optical manipulation through a high-numerical-aperture soft-glass multimode fibre

NASA Astrophysics Data System (ADS)

Leite, Ivo T.; Turtaev, Sergey; Jiang, Xin; Šiler, Martin; Cuschieri, Alfred; Russell, Philip St. J.; Čižmár, Tomáš

2018-01-01

Holographic optical tweezers (HOT) hold great promise for many applications in biophotonics, allowing the creation and measurement of minuscule forces on biomolecules, molecular motors and cells. Geometries used in HOT currently rely on bulk optics, and their exploitation in vivo is compromised by the optically turbid nature of tissues. We present an alternative HOT approach in which multiple three-dimensional (3D) traps are introduced through a high-numerical-aperture multimode optical fibre, thus enabling an equally versatile means of manipulation through channels having cross-section comparable to the size of a single cell. Our work demonstrates real-time manipulation of 3D arrangements of micro-objects, as well as manipulation inside otherwise inaccessible cavities. We show that the traps can be formed over fibre lengths exceeding 100 mm and positioned with nanometric resolution. The results provide the basis for holographic manipulation and other high-numerical-aperture techniques, including advanced microscopy, through single-core-fibre endoscopes deep inside living tissues and other complex environments.

Evidence of Nonuniformity in Urothelium Barrier Function between the Upper Urinary Tract and Bladder.

PubMed

Williams, Nicholas A; Barnard, Luke; Allender, Chris J; Bowen, Jenna L; Gumbleton, Mark; Harrah, Tim; Raja, Aditya; Joshi, Hrishi B

2016-03-01

We compared the relative permeability of upper urinary tract and bladder urothelium to mitomycin C. Ex vivo porcine bladder, ureters and kidneys were dissected out and filled with 1 mg ml(-1) mitomycin C. At 60 minutes the organs were emptied and excised tissue samples were sectioned parallel to the urothelium. Sectioned tissue was homogenized and extracted mitomycin C was quantified. Transurothelial permeation across the different urothelia was calculated by normalizing the total amount of drug extracted to the surface area of the tissue sample. Average mitomycin C concentrations at different tissue depths (concentration-depth profiles) were calculated by dividing the total amount of drug recovered by the total weight of tissue. Mitomycin C permeation across the ureteral urothelium was significantly greater than across the bladder and renal pelvis urothelium (9.07 vs 0.94 and 3.61 μg cm(-2), respectively). Concentrations of mitomycin C in the ureter and kidney were markedly higher than those achieved in the bladder at all tissue depths. Average urothelial mitomycin C concentrations were greater than 6.5-fold higher in the ureter and renal pelvis than in the bladder. To our knowledge we report for the first time that the upper urinary tract and bladder show differing permeability to a single drug. Ex vivo porcine ureter is significantly more permeable to mitomycin C than bladder urothelium and consequently higher mitomycin C tissue concentrations can be achieved after topical application. Data in this study correlate with the theory that mammalian upper tract urothelium represents a different cell lineage than that of the bladder and it is innately more permeable to mitomycin C. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2008-01-01

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

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.

MCNP modelling of the wall effects observed in tissue-equivalent proportional counters.

PubMed

Hoff, J L; Townsend, L W

2002-01-01

Tissue-equivalent proportional counters (TEPCs) utilise tissue-equivalent materials to depict homogeneous microscopic volumes of human tissue. Although both the walls and gas simulate the same medium, they respond to radiation differently. Density differences between the two materials cause distortions, or wall effects, in measurements, with the most dominant effect caused by delta rays. This study uses a Monte Carlo transport code, MCNP, to simulate the transport of secondary electrons within a TEPC. The Rudd model, a singly differential cross section with no dependence on electron direction, is used to describe the energy spectrum obtained by the impact of two iron beams on water. Based on the models used in this study, a wall-less TEPC had a higher lineal energy (keV.micron-1) as a function of impact parameter than a solid-wall TEPC for the iron beams under consideration. An important conclusion of this study is that MCNP has the ability to model the wall effects observed in TEPCs.

An analysis of particle track effects on solid mammalian tissues

NASA Technical Reports Server (NTRS)

Todd, P.; Clarkson, T. W. (Principal Investigator)

1992-01-01

Relative biological effectiveness (RBE) and quality factor (Q) at extreme values of linear energy transfer (LET) have been determined on the basis of experiments with single-cell systems and specific tissue responses. In typical single-cell systems, each heavy particle (Ar or Fe) passes through a single cell or no cell. In experiments on animal tissues, however, each heavy particle passes through several cells, and the LET can exceed 200 keV micrometers-1 in every cell. In most laboratory animal tissue systems, however, only a small portion of the hit cells are capable of expressing the end-point being measured, such as cell killing, mutation or carcinogenesis. The following question was therefore addressed: do RBEs and Q factors derived from single-cell experiments properly account for the damage at high LET when multiple cells are hit by HZE tracks? A review is offered in which measured radiation effects and known tissue properties are combined to estimate on the one hand, the number of cells at risk, p3n, per track, where n is the number of cells per track based on tissue and organ geometry, and p3 is the probability that a cell in the track is capable of expressing the experimental end-point. On the other hand, the tissue and single-cell responses are compared by determining the ratio RBE in tissue/RBE in corresponding single cells. Experimental data from the literature indicate that tissue RBEs at very high LET (Fe and Ar ions) are higher than corresponding single-cell RBEs, especially in tissues in which p3n is high.

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

NASA Astrophysics Data System (ADS)

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

2011-09-01

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

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.

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

PubMed

Walton, Katherine D; Kolterud, Asa

2014-09-04

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

Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

PubMed

Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

2016-01-01

Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter.

Localization of sweet potato chlorotic stunt virus (SPCSV) in synergic infection with potyviruses in sweet potato.

PubMed

Nome, Claudia Fernanda; Nome, Sergio Fernando; Guzmán, Fabiana; Conci, Luis; Laguna, Irma Graciela

2007-01-01

Among diseases reported worldwidely for sweet potato (Ipomoea batatas (L) Lam) crop, one of the most frequent is the Sweet potato virus disease (SPVD), caused by sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) co-infection. In Argentina, there exists the sweet potato chlorotic dwarf (SPCD), a sweet potato disease caused by triple co-infection with SPCSV, SPFMV and sweet potato mild speckling virus (SPMSV). Both diseases cause a synergism between the potyviruses (SPFMV and SPMSV) and the crinivirus (SPCSV). Up to date, studies carried out on the interaction among these three viruses have not described their localization in the infected tissues. In single infections, virions of the crinivirus genus are limited to the phloem while potyviral virions are found in most tissues of the infected plant. The purpose of this work was to localize the heat shock protein 70 homolog (HSP70h), a movement protein for genus crinivirus, of an Argentinean SPCSV isolate in its single infection and in its double and triple co-infection with SPFMV and SPMSV. The localization was made by in situ hybridization (ISH) for electron microscopy (EM) on ultrathin sections of sweet potato cv. Morada INTA infected tissues. The results demonstrated that viral RNA coding HSP70h is restricted to phloem cells during crinivirus single infection, while it was detected outside the phloem in infections combined with the potyviruses involved in chlorotic dwarf disease.

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

PubMed

Perruchini, Claire; Pecorari, Frederic; Bourgeois, Jean-Pierre; Duyckaerts, Charles; Rougeon, François; Lafaye, Pierre

2009-11-01

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

Low power laser irradiation stimulates cell proliferation via proliferating cell nuclear antigen and Ki-67 expression during tissue repair

NASA Astrophysics Data System (ADS)

Prabhu, Vijendra; Rao, Bola Sadashiva Satish; Mahato, Krishna Kishore

2015-03-01

Low power laser irradiation (LPLI) is becoming an increasingly popular and fast growing therapeutic modality in dermatology to treat various ailments without any reported side effects. In the present study an attempt was made to investigate the proliferative potential of red laser light during tissue repair in Swiss albino mice. To this end, full thickness excisional wounds of diameter 15 mm created on mice were exposed to single dose of Helium-Neon laser (632.8 nm; 7 mW; 4.02 mWcm-2; Linear polarization) at 2 Jcm-2 and 10 Jcm-2 along with un-illuminated controls. The granulation tissues from all the respective experimental groups were harvested on day 10 post-wounding following euthanization. Subsequently, tissue regeneration potential of these laser doses under study were evaluated by monitoring proliferating cell nuclear antigen and Ki-67 following the laser treatment and comparing it with the un-illuminated controls. The percentages of Ki-67 or PCNA positive cells were determined by counting positive nuclei (Ki-67/PCNA) and total nuclei in five random fields per tissue sections. Animal wounds treated with single exposure of the 2 Jcm-2 indicated significant elevation in PCNA (P<0.01) and Ki-67 (P<0.05 compared to un-illuminated control and P<0.01 compared to 10 Jcm-2) expression as compared to other tested experimental groups as evidenced by the microscopy results in the study. In summary, the findings of the present study have clearly demonstrated the regulation of cell proliferation by LPLI via PCNA and Ki-67 expression during tissue regeneration.

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.

Tissue distribution, metabolism and hepatic tissue injury in Chinese lizards (Eremias argus) after a single oral administration of lambda-cyhalothrin.

PubMed

Chang, Jing; Li, Jitong; Wang, Huili; Wang, Yinghuan; Guo, Baoyuan; Yin, Jing; Hao, Weiyu; Li, Wei; Li, Jianzhong; Xu, Peng

2016-11-01

Lambda-cyhalothrin (LCT) is a widely used pyrethroid with neurotoxicity. However, little is known about the toxicokinetics of LCT in reptiles. In this study, the absorption, distribution, metabolism and excretion of LCT in Chinese lizards (Eremias Argus) were determined following a single dose (10 mg kg -1 ) treatment. In the liver, brain, gonads and skin, LCT levels peaked within several hours and then decreased rapidly. However, the concentration of LCT gradually increased in the fat tissue. More than 90% of the LCT dose was excreted in the faeces. One LCT metabolite, 3-phenoxybenzoic acid (PBA), was detected in lizard plasma and tissues. PBA preferentially accumulates in the brain and plasma. The half-life of PBA in the brain was 3.2 days, which was 35.4-fold greater than that of LCT. In the plasma, the concentration of PBA was significantly higher than that of LCT. The bioaccumulation of LCT in tissues was enantioselective, and the enantiomeric fractions (EF) ranged from 0.72 to 0.26. The preferential accumulation of enantiomers changed according to exposure time, but the reasons behind this phenomenon were not clear. For pathological analysis, vacuolation of the cytoplasm and large areas of necrosis were observed in the liver sections after 168 h of dosing. The liver tissues exhibited both decreases in the hepatosomatic index and histopathological lesions during the exposure period. In this study, the effect concentration of LCT in lizards was 200-fold lower than its LD 50 value used in risk assessments for birds. These results may provide additional information for the risk assessment of LCT for reptiles and indicate that birds may not be an ideal surrogate for reptile toxicity evaluation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Design of an Optically Controlled MR-Compatible Active Needle

PubMed Central

Ryu, Seok Chang; Quek, Zhan Fan; Koh, Je-Sung; Renaud, Pierre; Black, Richard J.; Moslehi, Behzad; Daniel, Bruce L.; Cho, Kyu-Jin; Cutkosky, Mark R.

2015-01-01

An active needle is proposed for the development of magnetic resonance imaging (MRI)-guided percutaneous procedures. The needle uses a low-transition-temperature shape memory alloy (LT SMA) wire actuator to produce bending in the distal section of the needle. Actuation is achieved with internal optical heating using laser light transported via optical fibers and side coupled to the LT SMA. A prototype, with a size equivalent to a standard 16-gauge biopsy needle, exhibits significant bending, with a tip deflection of more than 14° in air and 5° in hard tissue. A single-ended optical sensor with a gold-coated tip is developed to measure the curvature independently of temperature. The experimental results in tissue phantoms show that human tissue causes fast heat dissipation from the wire actuator; however, the active needle can compensate for typical targeting errors during prostate biopsy. PMID:26512231

Irradiation Does Not Increase the Penetration Depth of Doxorubicin in Normal Tissue After Pressurized Intra-peritoneal Aerosol Chemotherapy (PIPAC) in an Ex Vivo Model.

PubMed

Khosrawipour, Veria; Bellendorf, Alexander; Khosrawipour, Carolina; Hedayat-Pour, Yousef; Diaz-Carballo, David; Förster, Eckart; Mücke, Ralph; Kabakci, Burak; Adamietz, Irenäus Anton; Fakhrian, Khashayar

To compare the impact of single fractional with bi-fractional irradiation on the depth of doxorubicin penetration into the normal tissue after pressurized intra-peritoneal aerosol chemotherapy (PIPAC) in our ex vivo model. Fresh post mortem swine peritoneum was cut into 12 proportional sections. Two control samples were treated with PIPAC only (no irradiation), one sample on day 1, the other on day 2. Five samples were irradiated with 1, 2, 4, 7 or 14 Gy followed by PIPAC. Four samples were treated on day one with 0.5, 1, 2, 3.5 or 7 Gy and with the same radiation dose 24 h later followed by PIPAC. Doxorubicin was aerosolized in an ex vivo PIPAC model at 12 mmHg/36°C. In-tissue doxorubicin penetration was measured using fluorescence microscopy on frozen thin sections. Doxorubicin penetration (DP) after PIPAC for the control samples was 407 μm and 373 μm, respectively. DP for samples with single fraction irradiation was 396 μm after 1 Gy, 384 μm after 2 Gy, 327 μm after 4 Gy, 280 μm after 7 Gy and 243 μm after 14 Gy. DP for samples with 2 fractions of irradiation was 376 μm after 0.5+0.5 Gy, 363 μm after 1+1 Gy, 372 μm after 2+2 Gy, 341 μm after 3.5+3.5 and 301 μm after 7+7 Gy irradiation. Fractionating of the irradiation did not significantly change DP into normal tissue. Irradiation does not increase the penetration depth of doxorubicin into the normal tissue but might have a limiting impact on penetration and distribution of doxorubicin. Further studies are warranted to investigate the impact of addition of irradiation to PIPAC of tumor cells and to find out if irradiation can be used safely as chemopotenting agent for patients with peritoneal metastases treated with PIPAC. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Human immunodeficiency viruses appear compartmentalized to the female genital tract in cross-sectional analyses but genital lineages do not persist over time.

PubMed

Bull, Marta E; Heath, Laura M; McKernan-Mullin, Jennifer L; Kraft, Kelli M; Acevedo, Luis; Hitti, Jane E; Cohn, Susan E; Tapia, Kenneth A; Holte, Sarah E; Dragavon, Joan A; Coombs, Robert W; Mullins, James I; Frenkel, Lisa M

2013-04-15

Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood. Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests. In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences. The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood.

Light-patterning of synthetic tissues with single droplet resolution.

PubMed

Booth, Michael J; Restrepo Schild, Vanessa; Box, Stuart J; Bayley, Hagan

2017-08-24

Synthetic tissues can be generated by forming networks of aqueous droplets in lipid-containing oil. Each droplet contains a cell-free expression system and is connected to its neighbor through a lipid bilayer. In the present work, we have demonstrated precise external control of such networks by activating protein expression within single droplets, by using light-activated DNA to encode either a fluorescent or a pore-forming protein. By controlling the extent of activation, synthetic tissues were generated with graded levels of protein expression in patterns of single droplets. Further, we have demonstrated reversible activation within individual compartments in synthetic tissues by turning a fluorescent protein on-and-off. This is the first example of the high-resolution patterning of droplet networks, following their formation. Single-droplet control will be essential to power subsets of compartments within synthetic tissues or to stimulate subsets of cells when synthetic tissues are interfaced with living tissues.

Determination of age, sex, and blood group from a single tooth.

PubMed

Nayar, Amit K; Parhar, Swati; Thind, Gagandeep; Sharma, Aman; Sharma, Divya

2017-01-01

Human identification is one of the most challenging subjects that human has been confronted with. Through the ages, odontological examinations have been a critical determinant in the search of human identity. Data in the form of age, gender, and blood group might provide vital clues in such investigations. In the recent times, it has been often desirable to preserve tissues for further investigations following the unfolding of certain events or discovery of new data. Hence, it is important to gather as much data as possible using less tissue. The purpose of this study was to determine age, sex, and ABO blood group of individual from a single tooth, to determine the effect of different environmental conditions, and to extract maximum information also at the same time preserving some tissue for the further investigation whenever needed. The study sample consisted of sixty teeth divided into four groups under different environmental conditions and time. The teeth were sectioned longitudinally in the buccolingual plane along the midline. Longitudinal ground sections of each tooth were prepared for age determination from cemental lines. Pulp removed was divided into two halves thereafter sex and blood group was determined. For correlation of age between estimated age and actual age, using cemental lines Pearson's correlation coefficient was applied. Further for determination of both sex and blood group between groups, Chi-square test was applied. A strong positive correlation was found between the estimated age and actual age of the study groups. Moreover, there was no significant difference between the actual and determined sex and blood group of the study groups. Although age, sex, and blood group are more reliably determined in freshly extracted teeth, these variables may be of significant help in identification even after a period of 6 weeks postextraction.

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.

In Situ Detection of MicroRNA Expression with RNAscope Probes.

PubMed

Yin, Viravuth P

2018-01-01

Elucidating the spatial resolution of gene transcripts provides important insight into potential gene function. MicroRNAs are short, singled-stranded noncoding RNAs that control gene expression through base-pair complementarity with target mRNAs in the 3' untranslated region (UTR) and inhibiting protein expression. However, given their small size of ~22- to 24-nt and low expression levels, standard in situ hybridization detection methods are not amendable for microRNA spatial resolution. Here, I describe a technique that employs RNAscope probe design and propriety amplification technology that provides simultaneous single molecule detection of individual microRNA and its target gene. This method allows for rapid and sensitive detection of noncoding RNA transcripts in frozen tissue sections.

Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry: In Situ Molecular Mapping

PubMed Central

Angel, Peggi M.; Caprioli, Richard M.

2013-01-01

Matrix-assisted laser desorption ionization imaging mass spectrometry (IMS) is a relatively new imaging modality that allows mapping of a wide range of biomolecules within a thin tissue section. The technology uses a laser beam to directly desorb and ionize molecules from discrete locations on the tissue that are subsequently recorded in a mass spectrometer. IMS is distinguished by the ability to directly measure molecules in situ ranging from small metabolites to proteins, reporting hundreds to thousands of expression patterns from a single imaging experiment. This article reviews recent advances in IMS technology, applications, and experimental strategies that allow it to significantly aid in the discovery and understanding of molecular processes in biological and clinical samples. PMID:23259809

Increasing the number of unloading/reambulation cycles does not adversely impact body composition and lumbar bone mineral density but reduces tissue sensitivity

NASA Astrophysics Data System (ADS)

Gupta, Shikha; Manske, Sarah L.; Judex, Stefan

2013-11-01

A single exposure to hindlimb unloading leads to changes in body mass, body composition and bone, but the consequences of multiple exposures are not yet understood. Within a 18 week period, adult C57BL/6 male mice were exposed to 1 (1x-HLU), 2 (2x-HLU) or 3 (3x-HLU) cycles of 2 weeks of hindlimb unloading (HLU) followed by 4 weeks of reambulation (RA), or served as ambulatory age-matched controls. In vivo μCT longitudinally tracked changes in abdominal adipose and lean tissues, lumbar vertebral apparent volumetric bone mineral density (vBMD) and upper hindlimb muscle cross-sectional area before and after the final HLU and RA cycle. During the final HLU cycle, significant decreases in total adipose tissue and vertebral vBMD in the three experimental groups occurred such that there were no significant between-group differences at the beginning of the final RA cycle. However, the magnitude of the HLU induced losses diminished in mice undergoing their 2nd or 3rd HLU cycle. Irrespective of the number of HLU/RA cycles, total adipose tissue and vertebral vBMD recovered and were no different from age-matched controls after the final RA period. In contrast, upper hindlimb muscle cross-sectional area was significantly lower than controls in all unloaded groups after the final RA period. These results suggest that tissues in the abdominal region are more resilient to multiple bouts of unloading and more amenable to recovery during reambulation than the peripheral musculoskeletal system.

Programmed cell death-ligand 1 (PD-L1) expression is associated with RAS/TP53 mutations in lung adenocarcinoma.

PubMed

Serra, Pierre; Petat, Arthur; Maury, Jean-Michel; Thivolet-Bejui, Françoise; Chalabreysse, Lara; Barritault, Marc; Ebran, Nathalie; Milano, Gérard; Girard, Nicolas; Brevet, Marie

2018-04-01

The systematic assessment of anti-programmed cell death ligand 1 (PD-L1) expression by immunohistochemistry (IHC) in lung adenocarcinomas is becoming standard practice. However, the assessment of PD-L1 expression on small tissue specimens needs to be evaluated and the association with other features more thoroughly analyzed. This retrospective single center study evaluated the immunohistochemical expression of the SP263 anti-PD-L1 antibody on tissue microarrays (TMA) of 152 surgically resected lung adenocarcinomas, using a 25% positivity threshold. The positive cases and 50 randomly chosen negative cases in tissue microarray (TMA) were reassessed on whole tissue sections. The results were correlated to clinical, histopathological and to molecular data obtained through the screening of 214 mutations in 26 genes (LungCarta panel, Agena Biosciences). Among 152 primary lung adenocarcinomas, 19 cases (13%) showed PD-L1 expression. The agreement between TMA and whole tissue sections was 89%, specificity was 97%. PD-L1 expression was correlated to RAS mutations (p = .04), RAS/TP53 co-mutations (p = .01) and to the solid or acinar subtype (p = .048). With the SP263 PD-L1 antibody, small samples appear as a reliable means to evaluate the PD-L1 status in lung adenocarcinoma. The association between PD-L1 expression and RAS/TP53 mutations may have clinical relevance to predict the efficacy of PD-1/PD-L1 immune checkpoints inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

Monoclonal antibody specific for IDH1 R132H mutation.

PubMed

Capper, David; Zentgraf, Hanswalter; Balss, Jörg; Hartmann, Christian; von Deimling, Andreas

2009-11-01

IDH1 R132H mutations occur in approximately 70% of astrocytomas and oligodendroglial tumors. We developed a mouse monoclonal antibody targeting the IDH1 R132H mutation. Here, we show the high specificity and sensitivity of this antibody on Western blots and tissue sections from formalin fixed paraffin embedded tumor specimens. This antibody is highly useful for tumor classification, in detecting single infiltrating tumor cells and for the characterization of the cellular role of mutant IDH1 protein.

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

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

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.

Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications

PubMed Central

Peña-Llopis, Samuel; Brugarolas, James

2014-01-01

Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), messenger RNA (mRNA), noncoding RNA (ncRNA; enriched in microRNA (miRNA)) and protein from the same tissues. After tissue selection, about 12–16 extractions of DNA/RNA/protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins. PMID:24136348

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

PubMed Central

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

2016-01-01

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

3-dimensional digital reconstruction of the murine coronary system for the evaluation of chronic allograft vasculopathy.

PubMed

Fónyad, László; Shinoda, Kazunobu; Farkash, Evan A; Groher, Martin; Sebastian, Divya P; Szász, A Marcell; Colvin, Robert B; Yagi, Yukako

2015-03-28

Chronic allograft vasculopathy (CAV) is a major mechanism of graft failure of transplanted organs in humans. Morphometric analysis of coronary arteries enables the quantitation of CAV in mouse models of heart transplantation. However, conventional histological procedures using single 2-dimensional sections limit the accuracy of CAV quantification. The aim of this study is to improve the accuracy of CAV quantification by reconstructing the murine coronary system in 3-dimensions (3D) and using virtual reconstruction and volumetric analysis to precisely assess neointimal thickness. Mouse tissue samples, native heart and transplanted hearts with chronic allograft vasculopathy, were collected and analyzed. Paraffin embedded samples were serially sectioned, stained and digitized using whole slide digital imaging techniques under normal and ultraviolet lighting. Sophisticated software tools were used to generate and manipulate 3D reconstructions of the major coronary arteries and branches. The 3D reconstruction provides not only accurate measurements but also exact volumetric data of vascular lesions. This virtual coronary arteriography demonstrates that the vasculopathy lesions in this model are localized to the proximal coronary segments. In addition, virtual rotation and volumetric analysis enabled more precise measurements of CAV than single, randomly oriented histologic sections, and offer an improved readout for this important experimental model. We believe 3D reconstruction of 2D histological slides will provide new insights into pathological mechanisms in which structural abnormalities play a role in the development of a disease. The techniques we describe are applicable to the analysis of arteries, veins, bronchioles and similar sized structures in a variety of tissue types and disease model systems. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3772457541477230 .

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

NASA Astrophysics Data System (ADS)

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

2017-06-01

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

Human Immunodeficiency Viruses Appear Compartmentalized to the Female Genital Tract in Cross-Sectional Analyses but Genital Lineages Do Not Persist Over Time

PubMed Central

Bull, Marta E.; Heath, Laura M.; McKernan-Mullin, Jennifer L.; Kraft, Kelli M.; Acevedo, Luis; Hitti, Jane E.; Cohn, Susan E.; Tapia, Kenneth A.; Holte, Sarah E.; Dragavon, Joan A.; Coombs, Robert W.; Mullins, James I.; Frenkel, Lisa M.

2013-01-01

Background. Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood. Methods. Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests. Results. In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences. Conclusions. The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood. PMID:23315326

Distribution of bemitil in organs and tissues of rats after single or repeated administration.

PubMed

Sergeeva, S A; Gulyaeva, I L

2006-05-01

After single and repeated peroral administration of bemitil to rats this drug was found in the liver, brain, kidneys, spleen, heart, skeletal muscles, lungs, adipose tissue, and testicles. After single treatment accumulation of bemitil was most pronounced in the liver. After repeated treatment the decrease in bemitil concentration in the liver was probably associated with increased elimination of the drug from liver tissue due to intensification of its biotransformation. We conclude that bemitil can accumulate in the blood, but not in tissues.

Biophysical interpretation and ex-vivo characterization of scattered light from tumor-associated breast stroma

NASA Astrophysics Data System (ADS)

Laughney, Ashley; Krishnaswamy, Venkat; Schwab, Mary; Wells, Wendy A.; Paulsen, Keith D.; Pogue, Brian W.

2009-02-01

The purpose of this study was to extract scatter parameters related to tissue ultra-structures from freshly excised breast tissue and to assess whether evident changes in scatter across diagnostic categories is primarily influenced by variation in the composition of each tissues subtypes or by physical remodeling of the extra-cellular environment. Pathologists easily distinguish between epithelium, stroma and adipose tissues, so this classification was adopted for macroscopic subtype classification. Micro-sampling reflectance spectroscopy was used to characterize single-backscattered photons from fresh, excised tumors and normal reduction specimens with sub-millimeter resolution. Phase contrast microscopy (sub-micron resolution) was used to characterize forward-scattered light through frozen tissue from the DHMC Tissue Bank, representing normal, benign and malignant breast tissue, sectioned at 10 microns. The packing density and orientation of collagen fibers in the extracellular matrix (ECM) associated with invasive, normal and benign epithelium was evaluated using transmission electron microscopy (TEM). Regions of interest (ROIs) in the H&E stained tissues were identified for analysis, as outlined by a pathologist as the gold standard. We conclude that the scatter parameters associated with tumor specimens (Npatients=6, Nspecimens=13) significantly differs from that of normal reductions (Npatients=6, Nspecimens=10). Further, tissue subtypes may be identified by their scatter spectra at sub-micron resolution. Stromal tissue scatters significantly more than the epithelial cells embedded in its ECM and adipose tissue scatters much less. However, the scatter signature of the stroma at the sub-micron level is not particularly differentiating in terms of a diagnosis.

Highly multiplexed subcellular RNA sequencing in situ

PubMed Central

Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Yang, Joyce L.; Terry, Richard; Jeanty, Sauveur S. F.; Li, Chao; Amamoto, Ryoji; Peters, Derek T.; Turczyk, Brian M.; Marblestone, Adam H.; Inverso, Samuel A.; Bernard, Amy; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M.

2014-01-01

Understanding the spatial organization of gene expression with single nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using a simulated wound healing assay. FISSEQ is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ. PMID:24578530

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

Radiation-induced impairment in lung lymphatic vasculature.

PubMed

Cui, Ye; Wilder, Julie; Rietz, Cecilia; Gigliotti, Andrew; Tang, Xiaomeng; Shi, Yuanyuan; Guilmette, Raymond; Wang, Hao; George, Gautam; Nilo de Magaldi, Eduarda; Chu, Sarah G; Doyle-Eisele, Melanie; McDonald, Jacob D; Rosas, Ivan O; El-Chemaly, Souheil

2014-12-01

The lymphatic vasculature has been shown to play important roles in lung injury and repair, particularly in lung fibrosis. The effects of ionizing radiation on lung lymphatic vasculature have not been previously reported. C57Bl/6 mice were immobilized in a lead shield exposing only the thoracic cavity, and were irradiated with a single dose of 14 Gy. Animals were sacrificed and lungs collected at different time points (1, 4, 8, and 16 weeks) following radiation. To identify lymphatic vessels in lung tissue sections, we used antibodies that are specific for lymphatic vessel endothelial receptor 1 (LYVE-1), a marker of lymphatic endothelial cells (LEC). To evaluate LEC cell death and oxidative damage, lung tissue sections were stained for LYVE-1 and with TUNEL staining, or 8-oxo-dG respectively. Images were imported into ImageJ v1.36b and analyzed. Compared to a non-irradiated control group, we observed a durable and progressive decrease in the density, perimeter, and area of lymphatic vessels over the study period. The decline in the density of lymphatic vessels was observed in both subpleural and interstitial lymphatics. Histopathologically discernible pulmonary fibrosis was not apparent until 16 weeks after irradiation. Furthermore, there was significantly increased LEC apoptosis and oxidative damage at one week post-irradiation that persisted at 16 weeks. There is impairment of lymphatic vasculature after a single dose of ionizing radiation that precedes architectural distortion and fibrosis, suggesting important roles for the lymphatic circulation in the pathogenesis of the radiation-induced lung injury.

Refractive index measurements of single, spherical cells using digital holographic microscopy.

PubMed

Schürmann, Mirjam; Scholze, Jana; Müller, Paul; Chan, Chii J; Ekpenyong, Andrew E; Chalut, Kevin J; Guck, Jochen

2015-01-01

In this chapter, we introduce digital holographic microscopy (DHM) as a marker-free method to determine the refractive index of single, spherical cells in suspension. The refractive index is a conclusive measure in a biological context. Cell conditions, such as differentiation or infection, are known to yield significant changes in the refractive index. Furthermore, the refractive index of biological tissue determines the way it interacts with light. Besides the biological relevance of this interaction in the retina, a lot of methods used in biology, including microscopy, rely on light-tissue or light-cell interactions. Hence, determining the refractive index of cells using DHM is valuable in many biological applications. This chapter covers the main topics that are important for the implementation of DHM: setup, sample preparation, and analysis. First, the optical setup is described in detail including notes and suggestions for the implementation. Following that, a protocol for the sample and measurement preparation is explained. In the analysis section, an algorithm for the determination of quantitative phase maps is described. Subsequently, all intermediate steps for the calculation of the refractive index of suspended cells are presented, exploiting their spherical shape. In the last section, a discussion of possible extensions to the setup, further measurement configurations, and additional analysis methods are given. Throughout this chapter, we describe a simple, robust, and thus easily reproducible implementation of DHM. The different possibilities for extensions show the diverse fields of application for this technique. Copyright © 2015 Elsevier Inc. All rights reserved.

Clustering Single-Cell Expression Data Using Random Forest Graphs.

PubMed

Pouyan, Maziyar Baran; Nourani, Mehrdad

2017-07-01

Complex tissues such as brain and bone marrow are made up of multiple cell types. As the study of biological tissue structure progresses, the role of cell-type-specific research becomes increasingly important. Novel sequencing technology such as single-cell cytometry provides researchers access to valuable biological data. Applying machine-learning techniques to these high-throughput datasets provides deep insights into the cellular landscape of the tissue where those cells are a part of. In this paper, we propose the use of random-forest-based single-cell profiling, a new machine-learning-based technique, to profile different cell types of intricate tissues using single-cell cytometry data. Our technique utilizes random forests to capture cell marker dependences and model the cellular populations using the cell network concept. This cellular network helps us discover what cell types are in the tissue. Our experimental results on public-domain datasets indicate promising performance and accuracy of our technique in extracting cell populations of complex tissues.

Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds

NASA Astrophysics Data System (ADS)

Wu, Tsai-Jung; Tzeng, Yan-Kai; Chang, Wei-Wei; Cheng, Chi-An; Kuo, Yung; Chien, Chin-Hsiang; Chang, Huan-Cheng; Yu, John

2013-09-01

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45-CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells' properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation.

A waveform diversity method for optimizing 3-d power depositions generated by ultrasound phased arrays.

PubMed

Zeng, Xiaozheng Jenny; Li, Jian; McGough, Robert J

2010-01-01

A waveform-diversity-based approach for 3-D tumor heating is compared to spot scanning for hyperthermia applications. The waveform diversity method determines the excitation signals applied to the phased array elements and produces a beam pattern that closely matches the desired power distribution. The optimization algorithm solves the covariance matrix of the excitation signals through semidefinite programming subject to a series of quadratic cost functions and constraints on the control points. A numerical example simulates a 1444-element spherical-section phased array that delivers heat to a 3-cm-diameter spherical tumor located 12 cm from the array aperture, and the results show that waveform diversity combined with mode scanning increases the heated volume within the tumor while simultaneously decreasing normal tissue heating. Whereas standard single focus and multiple focus methods are often associated with unwanted intervening tissue heating, the waveform diversity method combined with mode scanning shifts energy away from intervening tissues where hotspots otherwise accumulate to improve temperature localization in deep-seated tumors.

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

PubMed Central

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

2012-01-01

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

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.

Quantitative assessment of protein activity in orphan tissues and single cells using the metaVIPER algorithm. | Office of Cancer Genomics

Cancer.gov

We and others have shown that transition and maintenance of biological states is controlled by master regulator proteins, which can be inferred by interrogating tissue-specific regulatory models (interactomes) with transcriptional signatures, using the VIPER algorithm. Yet, some tissues may lack molecular profiles necessary for interactome inference (orphan tissues), or, as for single cells isolated from heterogeneous samples, their tissue context may be undetermined.

Scholte wave generation during single tracking location shear wave elasticity imaging of engineered tissues.

PubMed

Mercado, Karla P; Langdon, Jonathan; Helguera, María; McAleavey, Stephen A; Hocking, Denise C; Dalecki, Diane

2015-08-01

The physical environment of engineered tissues can influence cellular functions that are important for tissue regeneration. Thus, there is a critical need for noninvasive technologies capable of monitoring mechanical properties of engineered tissues during fabrication and development. This work investigates the feasibility of using single tracking location shear wave elasticity imaging (STL-SWEI) for quantifying the shear moduli of tissue-mimicking phantoms and engineered tissues in tissue engineering environments. Scholte surface waves were observed when STL-SWEI was performed through a fluid standoff, and confounded shear moduli estimates leading to an underestimation of moduli in regions near the fluid-tissue interface.

Data-driven sampling method for building 3D anatomical models from serial histology

NASA Astrophysics Data System (ADS)

Salunke, Snehal Ulhas; Ablove, Tova; Danforth, Theresa; Tomaszewski, John; Doyle, Scott

2017-03-01

In this work, we investigate the effect of slice sampling on 3D models of tissue architecture using serial histopathology. We present a method for using a single fully-sectioned tissue block as pilot data, whereby we build a fully-realized 3D model and then determine the optimal set of slices needed to reconstruct the salient features of the model objects under biological investigation. In our work, we are interested in the 3D reconstruction of microvessel architecture in the trigone region between the vagina and the bladder. This region serves as a potential avenue for drug delivery to treat bladder infection. We collect and co-register 23 serial sections of CD31-stained tissue images (6 μm thick sections), from which four microvessels are selected for analysis. To build each model, we perform semi-automatic segmentation of the microvessels. Subsampled meshes are then created by removing slices from the stack, interpolating the missing data, and re-constructing the mesh. We calculate the Hausdorff distance between the full and subsampled meshes to determine the optimal sampling rate for the modeled structures. In our application, we found that a sampling rate of 50% (corresponding to just 12 slices) was sufficient to recreate the structure of the microvessels without significant deviation from the fullyrendered mesh. This pipeline effectively minimizes the number of histopathology slides required for 3D model reconstruction, and can be utilized to either (1) reduce the overall costs of a project, or (2) enable additional analysis on the intermediate slides.

One Size Fits All: Evaluation of the Transferability of a New "Learning" Histologic Image Analysis Application.

PubMed

Arlt, Janine; Homeyer, André; Sänger, Constanze; Dahmen, Uta; Dirsch, Olaf

2016-01-01

Quantitative analysis of histologic slides is of importance for pathology and also to address surgical questions. Recently, a novel application was developed for the automated quantification of whole-slide images. The aim of this study was to test and validate the underlying image analysis algorithm with respect to user friendliness, accuracy, and transferability to different histologic scenarios. The algorithm splits the images into tiles of a predetermined size and identifies the tissue class of each tile. In the training procedure, the user specifies example tiles of the different tissue classes. In the subsequent analysis procedure, the algorithm classifies each tile into the previously specified classes. User friendliness was evaluated by recording training time and testing reproducibility of the training procedure of users with different background. Accuracy was determined with respect to single and batch analysis. Transferability was demonstrated by analyzing tissue of different organs (rat liver, kidney, small bowel, and spleen) and with different stainings (glutamine synthetase and hematoxylin-eosin). Users of different educational background could apply the program efficiently after a short introduction. When analyzing images with similar properties, accuracy of >90% was reached in single images as well as in batch mode. We demonstrated that the novel application is user friendly and very accurate. With the "training" procedure the application can be adapted to novel image characteristics simply by giving examples of relevant tissue structures. Therefore, it is suitable for the fast and efficient analysis of high numbers of fully digitalized histologic sections, potentially allowing "high-throughput" quantitative "histomic" analysis.

Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

PubMed

Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan; He, Ting-Fang; Greenbaum, Alon; Sohn, Chang Ho; Lignell, Antti; Choi, Harry M T; Gradinaru, Viviana; Pierce, Niles A; Cai, Long

2016-08-01

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. © 2016. Published by The Company of Biologists Ltd.

Transcriptome In Vivo Analysis (TIVA) of spatially defined single cells in intact live mouse and human brain tissue

PubMed Central

Lovatt, Ditte; Ruble, Brittani K.; Lee, Jaehee; Dueck, Hannah; Kim, Tae Kyung; Fisher, Stephen; Francis, Chantal; Spaethling, Jennifer M.; Wolf, John A.; Grady, M. Sean; Ulyanova, Alexandra V.; Yeldell, Sean B.; Griepenburg, Julianne C.; Buckley, Peter T.; Kim, Junhyong; Sul, Jai-Yoon; Dmochowski, Ivan J.; Eberwine, James

2014-01-01

Transcriptome profiling is an indispensable tool in advancing the understanding of single cell biology, but depends upon methods capable of isolating mRNA at the spatial resolution of a single cell. Current capture methods lack sufficient spatial resolution to isolate mRNA from individual in vivo resident cells without damaging adjacent tissue. Because of this limitation, it has been difficult to assess the influence of the microenvironment on the transcriptome of individual neurons. Here, we engineered a Transcriptome In Vivo Analysis (TIVA)-tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA-tag in combination with RNA-seq to analyze transcriptome variance among single dispersed cells and in vivo resident mouse and human neurons, we show that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology provides the first noninvasive approach for capturing mRNA from single cells in their natural microenvironment. PMID:24412976

Quantitative single-particle digital autoradiography with α-particle emitters for targeted radionuclide therapy using the iQID camera

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

Miller, Brian W., E-mail: brian.miller@pnnl.gov; Frost, Sofia H. L.; Frayo, Shani L.

2015-07-15

Purpose: Alpha-emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50–80 μm), causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with α emitters may thus inactivate targeted cells with minimal radiation damage to surrounding tissues. Tools are needed to visualize and quantify the radioactivity distribution and absorbed doses to targeted and nontargeted cells for accurate dosimetry of all treatment regimens utilizing α particles, including RIT and others (e.g., Ra-223), especially for organs and tumors with heterogeneous radionuclidemore » distributions. The aim of this study was to evaluate and characterize a novel single-particle digital autoradiography imager, the ionizing-radiation quantum imaging detector (iQID) camera, for use in α-RIT experiments. Methods: The iQID camera is a scintillator-based radiation detection system that images and identifies charged-particle and gamma-ray/x-ray emissions spatially and temporally on an event-by-event basis. It employs CCD-CMOS cameras and high-performance computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, the authors evaluated its characteristics for α-particle imaging, including measurements of intrinsic detector spatial resolutions and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 ({sup 211}At) activity distributions in cryosections of murine and canine tissue samples. Results: The highest spatial resolution was measured at ∼20 μm full width at half maximum and the α-particle background was measured at a rate as low as (2.6 ± 0.5) × 10{sup −4} cpm/cm{sup 2} (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the {sup 211}At activity distribution was demonstrated at mBq/μg-levels. Conclusions: Single-particle digital autoradiography of α emitters has advantages over traditional film-based autoradiographic techniques that use phosphor screens, in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that the iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using α emitters.« less

Quantitative single-particle digital autoradiography with α-particle emitters for targeted radionuclide therapy using the iQID camera.

PubMed

Miller, Brian W; Frost, Sofia H L; Frayo, Shani L; Kenoyer, Aimee L; Santos, Erlinda; Jones, Jon C; Green, Damian J; Hamlin, Donald K; Wilbur, D Scott; Fisher, Darrell R; Orozco, Johnnie J; Press, Oliver W; Pagel, John M; Sandmaier, Brenda M

2015-07-01

Alpha-emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50-80 μm), causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with α emitters may thus inactivate targeted cells with minimal radiation damage to surrounding tissues. Tools are needed to visualize and quantify the radioactivity distribution and absorbed doses to targeted and nontargeted cells for accurate dosimetry of all treatment regimens utilizing α particles, including RIT and others (e.g., Ra-223), especially for organs and tumors with heterogeneous radionuclide distributions. The aim of this study was to evaluate and characterize a novel single-particle digital autoradiography imager, the ionizing-radiation quantum imaging detector (iQID) camera, for use in α-RIT experiments. The iQID camera is a scintillator-based radiation detection system that images and identifies charged-particle and gamma-ray/x-ray emissions spatially and temporally on an event-by-event basis. It employs CCD-CMOS cameras and high-performance computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, the authors evaluated its characteristics for α-particle imaging, including measurements of intrinsic detector spatial resolutions and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 ((211)At) activity distributions in cryosections of murine and canine tissue samples. The highest spatial resolution was measured at ∼20 μm full width at half maximum and the α-particle background was measured at a rate as low as (2.6 ± 0.5) × 10(-4) cpm/cm(2) (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the (211)At activity distribution was demonstrated at mBq/μg-levels. Single-particle digital autoradiography of α emitters has advantages over traditional film-based autoradiographic techniques that use phosphor screens, in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that the iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using α emitters.

Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients.

PubMed

Onsum, Matthew D; Geretti, Elena; Paragas, Violette; Kudla, Arthur J; Moulis, Sharon P; Luus, Lia; Wickham, Thomas J; McDonagh, Charlotte F; MacBeath, Gavin; Hendriks, Bart S

2013-11-01

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

3D confocal reconstruction of gene expression in mouse.

PubMed

Hecksher-Sørensen, J; Sharpe, J

2001-01-01

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

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

NASA Astrophysics Data System (ADS)

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

2013-03-01

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

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

PubMed

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

2017-09-01

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

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.

Evaluation of a menstrual cup to collect shed endometrium for in vitro studies.

PubMed

Koks, C A; Dunselman, G A; de Goeij, A F; Arends, J W; Evers, J L

1997-09-01

To evaluate whether a menstrual cup is a suitable instrument to collect antegradely shed endometrium for in vitro studies. A prospective, descriptive, cell biological and immunohistochemical study. Tertiary care university medical center. Nine female volunteers with regular cycles. Menstrual effluent was collected with a menstrual cup. Experience with the menstrual cup was described. Cytospin specimens, frozen sections, and cultures were prepared from the obtained menstrual tissue. The acceptability of the menstrual cup. The presence and viability of endometrial tissue was evaluated using immunohistochemical staining and culture outcome. All women except one described the menstrual cup as acceptable. Menstrual effluent contained single cells, clumps of cells, and glandlike structures. After 5 days of culture, the endometrial tissue appeared to be viable. Immunohistochemistry showed positive staining for vimentin in most cytospin specimens, in all cryostat specimens, and in 10 of 17 cultures. Cytokeratin 18 stained most cytospin specimens, all cryostat specimens, and 10 of 17 cultures. Positive staining for BW495/36 was observed in most cytospin specimens, all cryostat specimens, and 11 of 17 cultures. A menstrual cup in an acceptable instrument to collect antegradely shed menstrual tissue. Menstruum contains viable endometrial tissue that can be used for in vitro studies of endometrium and endometriosis.

A theoretical framework for jamming in confluent biological tissues

NASA Astrophysics Data System (ADS)

Manning, M. Lisa

2015-03-01

For important biological functions such as wound healing, embryonic development, and cancer tumorogenesis, cells must initially rearrange and move over relatively large distances, like a liquid. Subsequently, these same tissues must undergo buckling and support shear stresses, like a solid. Our work suggests that biological tissues can accommodate these disparate requirements because the tissues are close to glass or jamming transition. While recent self propelled particle models generically predict a glass/jamming transition that is driven by packing density φ and happens at some critical φc less than unity, many biological tissues that are confluent with no gaps between cells appear to undergo a jamming transition at a constant density (φ = 1). I will discuss a new theoretical framework for predicting energy barriers and rates of cell migration in 2D tissue monolayers, and show that this model predicts a novel type of rigidity transition, which takes place at constant φ = 1 and depends only on single cell properties such as cell-cell adhesion, cortical tension and cell elasticity. This model additionally predicts that an experimentally observable parameter, the ratio between a cell's perimeter and the square root of its cross-sectional area, attains a specific, critical value at the jamming transition. We show that this prediction is precisely realized in primary epithelial cultures from human patients, with implications for asthma pathology.

Immunodetection of 11 beta-hydroxysteroid dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear localization.

PubMed

Shimojo, M; Ricketts, M L; Petrelli, M D; Moradi, P; Johnson, G D; Bradwell, A R; Hewison, M; Howie, A J; Stewart, P M

1997-03-01

11 beta-Hydroxysteroid dehydrogenase (11 beta HSI) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11 beta HSD2 gene, suggest that it is the 11 beta HSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11 beta HSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11 beta HSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11 beta HSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11 beta HSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 micron indicated significant 11 beta HSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11 beta HSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11 beta HSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the interaction between the MR and aldosterone or cortisol is in part a nuclear event.

Development and proof-of-concept of three-dimensional lung histology volumes

NASA Astrophysics Data System (ADS)

Mathew, Lindsay; Alabousi, Mostafa; Wheatley, Andrew; Aladl, Usaf; Slipetz, Deborah; Hogg, James C.; Fenster, Aaron; Parraga, Grace

2012-03-01

Most medical imaging is inherently three-dimensional (3D) but for validation of pathological findings, histopathology is commonly used and typically histopathology images are acquired as twodimensional slices with quantitative analysis performed in a single dimension. Histopathology is invasive, labour-intensive, and the analysis cannot be performed in real time, yet it remains the gold standard for the pathological diagnosis and validation of clinical or radiological diagnoses of disease. A major goal worldwide is to improve medical imaging resolution, sensitivity and specificity to better guide therapy and biopsy and to one day delay or replace biopsy. A key limitation however is the lack of tools to directly compare 3D macroscopic imaging acquired in patients with histopathology findings, typically provided in a single dimension (1D) or in two dimensions (2D). To directly address this, we developed methods for 2D histology slice visualization/registration to generate 3D volumes and quantified tissue components in the 3D volume for direct comparison to volumetric micro-CT and clinical CT. We used the elastase-instilled mouse emphysema lung model to evaluate our methods with murine lungs sectioned (5 μm thickness/10 μm gap) and digitized with 2μm in-plane resolution. 3D volumes were generated for wildtype and elastase mouse lung sections after semi-automated registration of all tissue slices. The 1D mean linear intercept (Lm) for wildtype (WT) (47.1 μm +/- 9.8 μm) and elastase mouse lung (64.5 μm +/- 14.0 μm) was significantly different (p<.001). We also generated 3D measurements based on tissue and airspace morphometry from the 3D volumes and all of these were significantly different (p<.0001) when comparing elastase and WT mouse lung. The ratio of the airspace-to-lung volume for the entire lung volume was also significantly and strongly correlated with Lm.

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens.

PubMed

Kim, Dong-Young; Sato, Ayuko; Fukuyama, Satoshi; Sagara, Hiroshi; Nagatake, Takahiro; Kong, Il Gyu; Goda, Kaoru; Nochi, Tomonori; Kunisawa, Jun; Sato, Shintaro; Yokota, Yoshifumi; Lee, Chul Hee; Kiyono, Hiroshi

2011-04-01

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

Detection, mapping, and quantification of single walled carbon nanotubes in histological specimens with photoacoustic microscopy.

PubMed

Avti, Pramod K; Hu, Song; Favazza, Christopher; Mikos, Antonios G; Jansen, John A; Shroyer, Kenneth R; Wang, Lihong V; Sitharaman, Balaji

2012-01-01

In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Optical-resolution (OR) and acoustic-resolution (AR)--Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs.

Planar small-angle x-ray scattering imaging of phantoms and biological samples

NASA Astrophysics Data System (ADS)

Choi, M.; Badano, A.

2017-04-01

Coherent small-angle x-ray scattering (SAXS) provides molecular and nanometer-scale structural information. By capturing SAXS data at multiple locations across a sample, we obtained planar images and observed improved contrast given by the difference in the material scattering cross sections. We use phantoms made with 3D printing techniques, with tissue-mimicking plastic (PMMA), and with a highly scattering reference material (AgBe), which were chosen because of their well characterized scattering cross section to demonstrate and characterize the planar imaging of a laboratory SAXS system. We measure 1.07 and 2.14 nm-1 angular intensity maps for AgBe, 9.5 nm-1 for PMMA, and 12.3 nm-1 for Veroclear. The planar SAXS images show material discrimination based on their cross sectional features. The image signal-to-noise ratio (SNR) of each q image was dependent on exposure time and x-ray flux. We observed a lower SNR (91 ± 48) at q angles where no characteristic peaks for either material exist. To improve the visualization of the acquired data by utilizing all q-binned data, we describe a weighted-sum presentation method with a priori knowledge of relevant cross sections to improve the SNR (10 000 ± 6400) over the SNR from a single q-image at 1.07 nm-1 (1100 ± 620). In addition, we describe planar SAXS imaging of a mouse brain slice showing differentiation of tissue types as compared to a conventional absorption-based x-ray imaging technique.

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.

Research on terahertz properties of rat brain tissue sections during dehydration

NASA Astrophysics Data System (ADS)

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

2018-01-01

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

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...

Handling, storage, and preparation of human tissues.

PubMed

Dressler, L G; Visscher, D

2001-05-01

Human tissue for flow cytometry must be prepared as an adequate single-cell suspension. The appropriate methods for tissue collection, transport, storage, and dissociation depend on the cell parameters being measured and the localization of the markers. This unit includes a general method for collecting and transporting human tissue and preparing a tissue imprint. Protocols are supplied for tissue disaggregation by either mechanical or enzymatic means and for preparation of single-cell suspensions of whole cells from fine-needle aspirates, pleural effusions, abdominal fluids, or other body fluids. Other protocols detail preparation of intact nuclei from fresh, frozen, or paraffin-embedded tissue. Support protocols cover fixation, cryospin preparation, cryopreservation, and removal of debris.

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.

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.

Inert gas clearance from tissue by co-currently and counter-currently arranged microvessels

PubMed Central

Lu, Y.; Michel, C. C.

2012-01-01

To elucidate the clearance of dissolved inert gas from tissues, we have developed numerical models of gas transport in a cylindrical block of tissue supplied by one or two capillaries. With two capillaries, attention is given to the effects of co-current and counter-current flow on tissue gas clearance. Clearance by counter-current flow is compared with clearance by a single capillary or by two co-currently arranged capillaries. Effects of the blood velocity, solubility, and diffusivity of the gas in the tissue are investigated using parameters with physiological values. It is found that under the conditions investigated, almost identical clearances are achieved by a single capillary as by a co-current pair when the total flow per tissue volume in each unit is the same (i.e., flow velocity in the single capillary is twice that in each co-current vessel). For both co-current and counter-current arrangements, approximate linear relations exist between the tissue gas clearance rate and tissue blood perfusion rate. However, the counter-current arrangement of capillaries results in less-efficient clearance of the inert gas from tissues. Furthermore, this difference in efficiency increases at higher blood flow rates. At a given blood flow, the simple conduction-capacitance model, which has been used to estimate tissue blood perfusion rate from inert gas clearance, underestimates gas clearance rates predicted by the numerical models for single vessel or for two vessels with co-current flow. This difference is accounted for in discussion, which also considers the choice of parameters and possible effects of microvascular architecture on the interpretation of tissue inert gas clearance. PMID:22604885

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

PubMed

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

2016-06-01

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

Irreversible Electroporation of the Pancreas Using Parallel Plate Electrodes in a Porcine Model: A Feasibility Study.

PubMed

Rombouts, Steffi J E; Nijkamp, Maarten W; van Dijck, Willemijn P M; Brosens, Lodewijk A A; Konings, Maurits; van Hillegersberg, R; Borel Rinkes, Inne H M; Hagendoorn, Jeroen; Wittkampf, Fred H; Molenaar, I Quintus

2017-01-01

Irreversible electroporation (IRE) with needle electrodes is being explored as treatment option in locally advanced pancreatic cancer. Several studies have shown promising results with IRE needles, positioned around the tumor to achieve tumor ablation. Disadvantages are the technical difficulties for needle placement, the time needed to achieve tumor ablation, the risk of needle track seeding and most important the possible occurrence of postoperative pancreatic fistula via the needle tracks. The aim of this experimental study was to evaluate the feasibility of a new IRE-technique using two parallel plate electrodes, in a porcine model. Twelve healthy pigs underwent laparotomy. The pancreas was mobilized to enable positioning of the paddles. A standard monophasic external cardiac defibrillator was used to perform an ablation in 3 separate parts of the pancreas; either a single application of 50 or 100J or a serial application of 4x50J. After 6 hours, pancreatectomy was performed for histology and pigs were terminated. Histology showed necrosis of pancreatic parenchyma with neutrophil influx in 5/12, 11/12 and 12/12 of the ablated areas at 50, 100, and 4x50J respectively. The electric current density threshold to achieve necrosis was 4.3, 5.1 and 3.4 A/cm2 respectively. The ablation threshold was significantly lower for the serial compared to the single applications (p = 0.003). The content of the ablated areas differed between the applications: areas treated with a single application of 50 J often contained vital areas without obvious necrosis, whereas half of the sections treated with 100 J showed small islands of normal looking cells surrounded by necrosis, while all sections receiving 4x 50 J showed a homogeneous necrotic lesion. Pancreatic tissue can be successfully ablated using two parallel paddles around the tissue. A serial application of 4x50J was most effective in creating a homogeneous necrotic lesion.

An investigation into the effects of temporal resolution on hepatic dynamic contrast-enhanced MRI in volunteers and in patients with hepatocellular carcinoma

NASA Astrophysics Data System (ADS)

Gill, Andrew B.; Black, Richard T.; Bowden, David J.; Priest, Andrew N.; Graves, Martin J.; Lomas, David J.

2014-06-01

This study investigated the effect of temporal resolution on the dual-input pharmacokinetic (PK) modelling of dynamic contrast-enhanced MRI (DCE-MRI) data from normal volunteer livers and from patients with hepatocellular carcinoma. Eleven volunteers and five patients were examined at 3 T. Two sections, one optimized for the vascular input functions (VIF) and one for the tissue, were imaged within a single heart-beat (HB) using a saturation-recovery fast gradient echo sequence. The data was analysed using a dual-input single-compartment PK model. The VIFs and/or uptake curves were then temporally sub-sampled (at interval ▵t = [2-20] s) before being subject to the same PK analysis. Statistical comparisons of tumour and normal tissue PK parameter values using a 5% significance level gave rise to the same study results when temporally sub-sampling the VIFs to HB < ▵t <4 s. However, sub-sampling to ▵t > 4 s did adversely affect the statistical comparisons. Temporal sub-sampling of just the liver/tumour tissue uptake curves at ▵t ≤ 20 s, whilst using high temporal resolution VIFs, did not substantially affect PK parameter statistical comparisons. In conclusion, there is no practical advantage to be gained from acquiring very high temporal resolution hepatic DCE-MRI data. Instead the high temporal resolution could be usefully traded for increased spatial resolution or SNR.

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

Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

PubMed Central

Simmons, Alan J.; Scurrah, Cherie’ R.; McKinley, Eliot T.; Herring, Charles A.; Irish, Jonathan M.; Washington, Mary K.; Coffey, Robert J.; Lau, Ken S.

2016-01-01

Cellular heterogeneity poses a significant challenge to understanding tissue level phenotypes and confounds conventional bulk analyses. To facilitate the analysis of signaling at the single-cell level in human tissues, we applied mass cytometry using CyTOF (Cytometry Time-of-Flight) to formalin-fixed paraffin-embedded (FFPE) normal and diseased intestinal specimens. We developed and validated a technique called FFPE-DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue), a single-cell approach for characterizing native signaling states from embedded solid tissue samples. We applied FFPE-DISSECT coupled to mass cytometry and found differential signaling by tumor necrosis factor α (TNF-α) in intestinal enterocytes, goblet cells and enteroendocrine cells, implicating the role of the downstream RAS-RAF-MEK-ERK signaling pathway in dictating goblet cell identity. In addition, application of FFPE-DISSECT, mass cytometry, and data-driven computational analyses to human colon specimens confirmed reduced differentiation in colorectal cancer (CRC) compared to normal colon, and revealed quantitative increases in inter- and intra-tissue heterogeneity in CRC with regards to the modular regulation of signaling pathways. Specifically, modular co-regulation of the kinases P38 and ERK, the translation regulator 4EBP1, and the transcription factor CREB in the proliferative compartment of the normal colon was loss in CRC, as evidenced by their impaired coordination over samplings of single cells in tissue. Our data suggest that this single-cell approach, applied in conjunction with genomic annotation, such as microsatellite instability and mutations in KRAS and BRAF, allows rapid and detailed characterization of cellular heterogeneity from clinical repositories of embedded human tissues. FFPE-DISSECT coupled of mass cytometry can be used for deriving cellular landscapes from archived patient samples, beyond CRC, and as a high resolution tool for disease characterization and subtyping. PMID:27729552

Advanced Image Acquisition and Analytical Techniques for Studies of Living Cells and Tissue Sections.

PubMed

Franek, Michal; Suchánková, Jana; Sehnalová, Petra; Krejčí, Jana; Legartová, Soňa; Kozubek, Stanislav; Večeřa, Josef; Sorokin, Dmitry V; Bártová, Eva

2016-04-01

Studies on fixed samples or genome-wide analyses of nuclear processes are useful for generating snapshots of a cell population at a particular time point. However, these experimental approaches do not provide information at the single-cell level. Genome-wide studies cannot assess variability between individual cells that are cultured in vitro or originate from different pathological stages. Immunohistochemistry and immunofluorescence are fundamental experimental approaches in clinical laboratories and are also widely used in basic research. However, the fixation procedure may generate artifacts and prevents monitoring of the dynamics of nuclear processes. Therefore, live-cell imaging is critical for studying the kinetics of basic nuclear events, such as DNA replication, transcription, splicing, and DNA repair. This review is focused on the advanced microscopy analyses of the cells, with a particular focus on live cells. We note some methodological innovations and new options for microscope systems that can also be used to study tissue sections. Cornerstone methods for the biophysical research of living cells, such as fluorescence recovery after photobleaching and fluorescence resonance energy transfer, are also discussed, as are studies on the effects of radiation at the individual cellular level.

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

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

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

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

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

PubMed

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

2014-01-01

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

Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.

PubMed

Yuan, Yinyin; Failmezger, Henrik; Rueda, Oscar M; Ali, H Raza; Gräf, Stefan; Chin, Suet-Feung; Schwarz, Roland F; Curtis, Christina; Dunning, Mark J; Bardwell, Helen; Johnson, Nicola; Doyle, Sarah; Turashvili, Gulisa; Provenzano, Elena; Aparicio, Sam; Caldas, Carlos; Markowetz, Florian

2012-10-24

Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin-stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. To deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy number profiles between samples. We next devised a predictor for survival in estrogen receptor-negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

X-ray micro-tomography for investigations of brain tissues on cellular level

NASA Astrophysics Data System (ADS)

Khimchenko, Anna; Schulz, Georg; Deyhle, Hans; Thalmann, Peter; Zanette, Irene; Zdora, Marie-Christine; Bikis, Christos; Hipp, Alexander; Hieber, Simone E.; Schweighauser, Gabriel; Hench, Jürgen; Müller, Bert

2016-10-01

X-ray imaging in absorption contrast mode is well established for hard tissue visualization. However, performance for lower density materials is limited due to a reduced contrast. Our aim is three-dimensional (3D) characterization of micro-morphology of human brain tissues down to (sub-)cellular resolution within a laboratory environment. Using the laboratory-based microtomography (μCT) system nanotom m (GE Sensing and Inspection Technologies GmbH, Wunstorf, Germany) and synchrotron radiation at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK), we have acquired 3D data with a resolution down to 0.45 μm for visualization of a human cerebellum specimen down to cellular level. We have shown that all selected modalities, namely laboratory-based absorption contrast micro-tomography (LBμCT), synchrotron radiation based in-line single distance phase contrast tomography (SDPR) and synchrotron radiation based single-grating interferometry (GI), can reach cellular resolution for tissue samples with a size in the mm-range. The results are discussed qualitatively in comparison to optical microscopy of haematoxylin and eosin (HE) stained sections. As phase contrast yields to a better data quality for soft tissues and in order to overcome restrictions of limited beamline access for phase contrast measurements, we have equipped the μCT system nanotom m with a double-grating phase contrast set-up. Preliminary experimental results of a knee sample consisting of a bony part and a cartilage demonstrate that phase contrast data exhibits better quality compared to absorption contrast. Currently, the set-up is under adjustment. It is expected that cellular resolution would also be achieved. The questions arise (1) what would be the quality gain of laboratory-based phase contrast in comparison to laboratory-based absorption contrast tomography and (2) could laboratory-based phase contrast data provide comparable results to synchrotron radiation based phase contrast data.

Nanopipettes: probes for local sample analysis.

PubMed

Saha-Shah, Anumita; Weber, Anna E; Karty, Jonathan A; Ray, Steven J; Hieftje, Gary M; Baker, Lane A

2015-06-01

Nanopipettes (pipettes with diameters <1 μm) were explored as pressure-driven fluid manipulation tools for sampling nanoliter volumes of fluids. The fundamental behavior of fluids confined in the narrow channels of the nanopipette shank was studied to optimize sampling volume and probe geometry. This method was utilized to collect nanoliter volumes (<10 nL) of sample from single Allium cepa cells and live Drosophila melanogaster first instar larvae. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was utilized to characterize the collected sample. The use of nanopipettes for surface sampling of mouse brain tissue sections was also explored. Lipid analyses were performed on mouse brain tissues with spatial resolution of sampling as small as 50 μm. Nanopipettes were shown to be a versatile tool that will find further application in studies of sample heterogeneity and population analysis for a wide range of samples.

A Gibbs point field model for the spatial pattern of coronary capillaries

NASA Astrophysics Data System (ADS)

Karch, R.; Neumann, M.; Neumann, F.; Ullrich, R.; Neumüller, J.; Schreiner, W.

2006-09-01

We propose a Gibbs point field model for the pattern of coronary capillaries in transverse histologic sections from human hearts, based on the physiology of oxygen supply from capillaries to tissue. To specify the potential energy function of the Gibbs point field, we draw on an analogy between the equation of steady-state oxygen diffusion from an array of parallel capillaries to the surrounding tissue and Poisson's equation for the electrostatic potential of a two-dimensional distribution of identical point charges. The influence of factors other than diffusion is treated as a thermal disturbance. On this basis, we arrive at the well-known two-dimensional one-component plasma, a system of identical point charges exhibiting a weak (logarithmic) repulsive interaction that is completely characterized by a single dimensionless parameter. By variation of this parameter, the model is able to reproduce many characteristics of real capillary patterns.

Imaging of zymogen granules in fully wet cells: evidence for restricted mechanism of granule growth.

PubMed

Hammel, Ilan; Anaby, Debbie

2007-09-01

The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules.

Co-expression networks reveal the tissue-specific regulation of transcription and splicing

PubMed Central

Saha, Ashis; Kim, Yungil; Gewirtz, Ariel D.H.; Jo, Brian; Gao, Chuan; McDowell, Ian C.; Engelhardt, Barbara E.

2017-01-01

Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues. PMID:29021288

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.

Determination of optical coefficients of biological tissue from a single integrating-sphere

NASA Astrophysics Data System (ADS)

Zhang, Lianshun; Shi, Aijuan; Lu, Hongguang

2012-01-01

The detection of interactions between light and tissue can be used to characterize the optical properties of the tissue. The development is described of a method that determines optical coefficients of biological tissue from a single optical reflectance spectrum measured with an integrating-sphere. The experimental system incorporated a DH-2000 deuterium tungsten halogen light source, a USB4000-VIS-NIR miniature fiber optic spectrometer and an integrating-sphere. Fat emulsion and ink were used to mimic the scattering and absorbing properties of tissue in the tested sample. The measured optical reflectance spectrums with different scattering and absorbing properties were used to train a back-propagation neural network (BPNN). Then the neural network (BPNN) was used to determine the optical coefficients of biological tissue from a single optical reflectance spectrum measured with an integrating-sphere. Tests on tissue-simulation phantoms showed the relative errors of this technique to be 7% for the reduced scattering coefficient and 15% for the absorption coefficients. The optical properties of human skin were also measured in vivo.

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.

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

Controlled surface topography regulates collective 3D migration by epithelial-mesenchymal composite embryonic tissues.

PubMed

Song, Jiho; Shawky, Joseph H; Kim, YongTae; Hazar, Melis; LeDuc, Philip R; Sitti, Metin; Davidson, Lance A

2015-07-01

Cells in tissues encounter a range of physical cues as they migrate. Probing single cell and collective migratory responses to physically defined three-dimensional (3D) microenvironments and the factors that modulate those responses are critical to understanding how tissue migration is regulated during development, regeneration, and cancer. One key physical factor that regulates cell migration is topography. Most studies on surface topography and cell mechanics have been carried out with single migratory cells, yet little is known about the spreading and motility response of 3D complex multi-cellular tissues to topographical cues. Here, we examine the response to complex topographical cues of microsurgically isolated tissue explants composed of epithelial and mesenchymal cell layers from naturally 3D organized embryos of the aquatic frog Xenopus laevis. We control topography using fabricated micropost arrays (MPAs) and investigate the collective 3D migration of these multi-cellular systems in these MPAs. We find that the topography regulates both collective and individual cell migration and that dense MPAs reduce but do not eliminate tissue spreading. By modulating cell size through the cell cycle inhibitor Mitomycin C or the spacing of the MPAs we uncover how 3D topographical cues disrupt collective cell migration. We find surface topography can direct both single cell motility and tissue spreading, altering tissue-scale processes that enable efficient conversion of single cell motility into collective movement. Copyright © 2015 Elsevier Ltd. All rights reserved.

Macrophage Responses to Epithelial Dysfunction Promote Lung Fibrosis in Aging

DTIC Science & Technology

2017-10-01

alveolar macrophages based on single cell molecular classification in patients with pulmonary fibrosis. We have recruited a planned number of patients...biomarkers expressed by human tissue-resident and monocyte-derived alveolar macrophages based on single cell molecular classification in patients with...identify novel biomarkers expressed by human tissue-resident and monocyte- derived alveolar macrophages based on single cell molecular classification

Heavy Proteinuria as a Manifestation of Acute Allograft Rejection Presenting Early after Kidney Transplantation: A Retrospective, Single-Center Case Series

DTIC Science & Technology

2009-01-01

controls were run in parallel with the test tissues. Sections were considered positive when crisp, brown staining was identified in peritubular...Patient 4: Antibody induction with anti-lymphocyte globulin . Maintenance therapy with cyclosporine, azathioprine, and prednisone. Patient 5: No antibody...2.7 5.6 379mg 1.3 2 gm/Kg over 2d 7 39 F AA Deceased Donor 2 SLE 9 days 4.0 gm/d 1.1 2.2 Urine dip negative 1.2 2 gm/Kg over 1d 28 The

Rotational distortion correction in endoscopic optical coherence tomography based on speckle decorrelation

PubMed Central

Uribe-Patarroyo, Néstor; Bouma, Brett E.

2015-01-01

We present a new technique for the correction of nonuniform rotation distortion in catheter-based optical coherence tomography (OCT), based on the statistics of speckle between A-lines using intensity-based dynamic light scattering. This technique does not rely on tissue features and can be performed on single frames of data, thereby enabling real-time image correction. We demonstrate its suitability in a gastrointestinal balloon-catheter OCT system, determining the actual rotational speed with high temporal resolution, and present corrected cross-sectional and en face views showing significant enhancement of image quality. PMID:26625040

Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

PubMed

Ozbun, Michelle A; Patterson, Nicole A

2014-08-01

Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley & Sons, Inc.

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.

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

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

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

1989-04-01

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

Microwave ablation with multiple simultaneously powered small-gauge triaxial antennas: results from an in vivo swine liver model.

PubMed

Brace, Christopher L; Laeseke, Paul F; Sampson, Lisa A; Frey, Tina M; van der Weide, Daniel W; Lee, Fred T

2007-07-01

To prospectively investigate the ability of a single generator to power multiple small-diameter antennas and create large zones of ablation in an in vivo swine liver model. Thirteen female domestic swine (mean weight, 70 kg) were used for the study as approved by the animal care and use committee. A single generator was used to simultaneously power three triaxial antennas at 55 W per antenna for 10 minutes in three groups: a control group where antennas were spaced to eliminate ablation zone overlap (n=6; 18 individual zones of ablation) and experimental groups where antennas were spaced 2.5 cm (n=7) or 3.0 cm (n=5) apart. Animals were euthanized after ablation, and ablation zones were sectioned and measured. A mixed linear model was used to test for differences in size and circularity among groups. Mean (+/-standard deviation) cross-sectional areas of multiple-antenna zones of ablation at 2.5- and 3.0-cm spacing (26.6 cm(2) +/- 9.7 and 32.2 cm(2) +/- 8.1, respectively) were significantly larger than individual ablation zones created with single antennas (6.76 cm(2) +/- 2.8, P<.001) and were 31% (2.5-cm spacing group: multiple antenna mean area, 26.6 cm(2); 3 x single antenna mean area, 20.28 cm(2)) to 59% (3.0-cm spacing group: multiple antenna mean area, 32.2 cm(2); 3 x single antenna mean area, 20.28 cm(2)) larger than 3 times the mean area of the single-antenna zones. Zones of ablation were found to be very circular, and vessels as large as 1.1 cm were completely coagulated with multiple antennas. A single generator may effectively deliver microwave power to multiple antennas. Large volumes of tissue may be ablated and large vessels coagulated with multiple-antenna ablation in the same time as single-antenna ablation. (c) RSNA, 2007.

Compressed single pixel imaging in the spatial frequency domain

PubMed Central

Torabzadeh, Mohammad; Park, Il-Yong; Bartels, Randy A.; Durkin, Anthony J.; Tromberg, Bruce J.

2017-01-01

Abstract. We have developed compressed sensing single pixel spatial frequency domain imaging (cs-SFDI) to characterize tissue optical properties over a wide field of view (35  mm×35  mm) using multiple near-infrared (NIR) wavelengths simultaneously. Our approach takes advantage of the relatively sparse spatial content required for mapping tissue optical properties at length scales comparable to the transport scattering length in tissue (ltr∼1  mm) and the high bandwidth available for spectral encoding using a single-element detector. cs-SFDI recovered absorption (μa) and reduced scattering (μs′) coefficients of a tissue phantom at three NIR wavelengths (660, 850, and 940 nm) within 7.6% and 4.3% of absolute values determined using camera-based SFDI, respectively. These results suggest that cs-SFDI can be developed as a multi- and hyperspectral imaging modality for quantitative, dynamic imaging of tissue optical and physiological properties. PMID:28300272

Fiber optic biofluorometer for physiological research on muscle slices

NASA Astrophysics Data System (ADS)

Belz, Mathias; Dendorfer, Andreas; Werner, Jan; Lambertz, Daniel; Klein, Karl-Friedrich

2016-03-01

A focus of research in cell physiology is the detection of Ca2+, NADH, FAD, ATPase activity or membrane potential, only to name a few, in muscle tissues. In this work, we report on a biofluorometer using ultraviolet light emitting diodes (UV-LEDs), optical fibers and two photomultipliers (PMTs) using synchronized fluorescence detection with integrated background correction to detect free calcium, Ca2+, in cardiac muscle tissue placed in a horizontal tissue bath and a microscope setup. Fiber optic probes with imaging optics have been designed to transport excitation light from the biofluorometer's light output to a horizontal tissue bath and to collect emission light from a tissue sample of interest to two PMTs allowing either single excitation / single emission or ratiometric, dual excitation / single emission or single excitation / dual emission fluorescence detection of indicator dyes or natural fluorophores. The efficient transport of light from the excitation LEDs to the tissue sample, bleaching effects of the excitation light in both, polymer and fused silica-based fibers will be discussed. Furthermore, a new approach to maximize light collection of the emission light using high NA fibers and high NA coupling optics will be shown. Finally, first results on Ca2+ measurements in cardiac muscle slices in a traditional microscope setup and a horizontal tissue bath using fiber optic probes will be introduced and discussed.

Hyperspectral microscopic analysis of normal, benign and carcinoma microarray tissue sections

NASA Astrophysics Data System (ADS)

Maggioni, Mauro; Davis, Gustave L.; Warner, Frederick J.; Geshwind, Frank B.; Coppi, Andreas C.; DeVerse, Richard A.; Coifman, Ronald R.

2006-02-01

We apply a unique micro-optoelectromechanical tuned light source and new algorithms to the hyper-spectral microscopic analysis of human colon biopsies. The tuned light prototype (Plain Sight Systems Inc.) transmits any combination of light frequencies, range 440nm 700nm, trans-illuminating H and E stained tissue sections of normal (N), benign adenoma (B) and malignant carcinoma (M) colon biopsies, through a Nikon Biophot microscope. Hyper-spectral photomicrographs, randomly collected 400X magnication, are obtained with a CCD camera (Sensovation) from 59 different patient biopsies (20 N, 19 B, 20 M) mounted as a microarray on a single glass slide. The spectra of each pixel are normalized and analyzed to discriminate among tissue features: gland nuclei, gland cytoplasm and lamina propria/lumens. Spectral features permit the automatic extraction of 3298 nuclei with classification as N, B or M. When nuclei are extracted from each of the 59 biopsies the average classification among N, B and M nuclei is 97.1%; classification of the biopsies, based on the average nuclei classification, is 100%. However, when the nuclei are extracted from a subset of biopsies, and the prediction is made on nuclei in the remaining biopsies, there is a marked decrement in performance to 60% across the 3 classes. Similarly the biopsy classification drops to 54%. In spite of these classification differences, which we believe are due to instrument and biopsy normalization issues, hyper-spectral analysis has the potential to achieve diagnostic efficiency needed for objective microscopic diagnosis.

An enhanced droplet-based liquid microjunction surface sampling system coupled with HPLC-ESI-MS/MS for spatially resolved analysis

DOE PAGES

Van Berkel, Gary J.; Weiskittel, Taylor M.; Kertesz, Vilmos

2014-11-07

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparatemore » height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. Lastly, the merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue.« less

An enhanced droplet-based liquid microjunction surface sampling system coupled with HPLC-ESI-MS/MS for spatially resolved analysis

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

Van Berkel, Gary J.; Weiskittel, Taylor M.; Kertesz, Vilmos

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparatemore » height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. Lastly, the merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue.« less

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.

Rapid Detection of Necrosis in Breast Cancer with Desorption Electrospray Ionization Mass Spectrometry

PubMed Central

Tata, Alessandra; Woolman, Michael; Ventura, Manuela; Bernards, Nicholas; Ganguly, Milan; Gribble, Adam; Shrestha, Bindesh; Bluemke, Emma; Ginsberg, Howard J.; Vitkin, Alex; Zheng, Jinzi; Zarrine-Afsar, Arash

2016-01-01

Identification of necrosis in tumors is of prognostic value in treatment planning, as necrosis is associated with aggressive forms of cancer and unfavourable outcomes. To facilitate rapid detection of necrosis with Mass Spectrometry (MS), we report the lipid MS profile of necrotic breast cancer with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging validated with statistical analysis and correlating pathology. This MS profile is characterized by (1) the presence of the ion of m/z 572.48 [Cer(d34:1) + Cl]− which is a ceramide absent from the viable cancer subregions; (2) the absence of the ion of m/z 391.25 which is present in small abundance only in viable cancer subregions; and (3) a slight increase in the relative intensity of known breast cancer biomarker ions of m/z 281.25 [FA(18:1)-H]− and 303.23 [FA(20:4)-H]−. Necrosis is accompanied by alterations in the tissue optical depolarization rate, allowing tissue polarimetry to guide DESI-MS analysis for rapid MS profiling or targeted MS imaging. This workflow, in combination with the MS profile of necrosis, may permit rapid characterization of necrotic tumors from tissue slices. Further, necrosis-specific biomarker ions are detected in seconds with single MS scans of necrotic tumor tissue smears, which further accelerates the identification workflow by avoiding tissue sectioning and slide preparation. PMID:27734938

Deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation.

PubMed

Dueck, Hannah; Khaladkar, Mugdha; Kim, Tae Kyung; Spaethling, Jennifer M; Francis, Chantal; Suresh, Sangita; Fisher, Stephen A; Seale, Patrick; Beck, Sheryl G; Bartfai, Tamas; Kuhn, Bernhard; Eberwine, James; Kim, Junhyong

2015-06-09

Differentiation of metazoan cells requires execution of different gene expression programs but recent single-cell transcriptome profiling has revealed considerable variation within cells of seeming identical phenotype. This brings into question the relationship between transcriptome states and cell phenotypes. Additionally, single-cell transcriptomics presents unique analysis challenges that need to be addressed to answer this question. We present high quality deep read-depth single-cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within-cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single-cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved. Single-cell RNA-sequencing data provide a unique view of transcriptome function; however, careful analysis is required in order to use single-cell RNA-sequencing measurements for this purpose. Technical variation must be considered in single-cell RNA-sequencing studies of expression variation. For a subset of genes, biological variability within each cell type appears to be regulated in order to perform dynamic functions, rather than solely molecular noise.

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.

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

Miller, Brian W.; Frost, Sophia; Frayo, Shani

Abstract Alpha emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50–80 μm) causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with alpha emitters may inactivate targeted cells with minimal radiation damage to surrounding tissues. For accurate dosimetry in alpha-RIT, tools are needed to visualize and quantify the radioactivity distribution and absorbed dose to targeted and non-targeted cells, especially for organs and tumors with heterogeneous radionuclide distributions. The aim of this study was to evaluate and characterizemore » a novel single-particle digital autoradiography imager, iQID (ionizing-radiation Quantum Imaging Detector), for use in alpha-RIT experiments. Methods: The iQID camera is a scintillator-based radiation detection technology that images and identifies charged-particle and gamma-ray/X-ray emissions spatially and temporally on an event-by-event basis. It employs recent advances in CCD/CMOS cameras and computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, we evaluated this system’s characteristics for alpha particle imaging including measurements of spatial resolution and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 (211At) activity distributions in cryosections of murine and canine tissue samples. Results: The highest spatial resolution was measured at ~20 μm full width at half maximum (FWHM) and the alpha particle background was measured at a rate of (2.6 ± 0.5) × 10–4 cpm/cm2 (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the 211At activity distribution was demonstrated at mBq/μg levels. Conclusion: Single-particle digital autoradiography of alpha emitters has advantages over traditional autoradiographic techniques in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using alpha emitters.« less

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

NASA Astrophysics Data System (ADS)

Al-Faouri, Tamara

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

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

NASA Astrophysics Data System (ADS)

Yamada, Kengo; Yoshida, Katsuyuki; Sonoda, Ryoichi

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

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

PubMed

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

2009-07-01

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

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.

Co-expression networks reveal the tissue-specific regulation of transcription and splicing.

PubMed

Saha, Ashis; Kim, Yungil; Gewirtz, Ariel D H; Jo, Brian; Gao, Chuan; McDowell, Ian C; Engelhardt, Barbara E; Battle, Alexis

2017-11-01

Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues. © 2017 Saha et al.; Published by Cold Spring Harbor Laboratory Press.

Endophytic colonization of olive roots by the biocontrol strain Pseudomonas fluorescens PICF7.

PubMed

Prieto, Pilar; Mercado-Blanco, Jesús

2008-05-01

Confocal microscopy combined with three-dimensional olive root tissue sectioning was used to provide evidence of the endophytic behaviour of Pseudomonas fluorescens PICF7, an effective biocontrol strain against Verticillium wilt of olive. Two derivatives of the green fluorescent protein (GFP), the enhanced green and the red fluorescent proteins, have been used to visualize simultaneously two differently fluorescently tagged populations of P. fluorescens PICF7 within olive root tissues at the single cell level. The time-course of colonization events of olive roots cv. Arbequina by strain PICF7 and the localization of tagged bacteria within olive root tissues are described. First, bacteria rapidly colonized root surfaces and were predominantly found in the differentiation zone. Thereafter, microscopy observations showed that PICF7-tagged populations eventually disappeared from the root surface, and increasingly colonized inner root tissues. Localized and limited endophytic colonization by the introduced bacteria was observed over time. Fluorescent-tagged bacteria were always visualized in the intercellular spaces of the cortex region, and no colonization of the root xylem vessels was detected at any time. To the best of our knowledge, this is the first time this approach has been used to demonstrate endophytism of a biocontrol Pseudomonas spp. strain in a woody host such as olive using a nongnotobiotic system.

Detection, Mapping, and Quantification of Single Walled Carbon Nanotubes in Histological Specimens with Photoacoustic Microscopy

PubMed Central

Mikos, Antonios G.; Jansen, John A.; Shroyer, Kenneth R.; Wang, Lihong V.; Sitharaman, Balaji

2012-01-01

Aims In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Materials and Methods Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Results Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. Conclusions The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs. PMID:22496892

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

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

Design and implementation of coded aperture coherent scatter spectral imaging of cancerous and healthy breast tissue samples

PubMed Central

Lakshmanan, Manu N.; Greenberg, Joel A.; Samei, Ehsan; Kapadia, Anuj J.

2016-01-01

Abstract. A scatter imaging technique for the differentiation of cancerous and healthy breast tissue in a heterogeneous sample is introduced in this work. Such a technique has potential utility in intraoperative margin assessment during lumpectomy procedures. In this work, we investigate the feasibility of the imaging method for tumor classification using Monte Carlo simulations and physical experiments. The coded aperture coherent scatter spectral imaging technique was used to reconstruct three-dimensional (3-D) images of breast tissue samples acquired through a single-position snapshot acquisition, without rotation as is required in coherent scatter computed tomography. We perform a quantitative assessment of the accuracy of the cancerous voxel classification using Monte Carlo simulations of the imaging system; describe our experimental implementation of coded aperture scatter imaging; show the reconstructed images of the breast tissue samples; and present segmentations of the 3-D images in order to identify the cancerous and healthy tissue in the samples. From the Monte Carlo simulations, we find that coded aperture scatter imaging is able to reconstruct images of the samples and identify the distribution of cancerous and healthy tissues (i.e., fibroglandular, adipose, or a mix of the two) inside them with a cancerous voxel identification sensitivity, specificity, and accuracy of 92.4%, 91.9%, and 92.0%, respectively. From the experimental results, we find that the technique is able to identify cancerous and healthy tissue samples and reconstruct differential coherent scatter cross sections that are highly correlated with those measured by other groups using x-ray diffraction. Coded aperture scatter imaging has the potential to provide scatter images that automatically differentiate cancerous and healthy tissue inside samples within a time on the order of a minute per slice. PMID:26962543

Design and implementation of coded aperture coherent scatter spectral imaging of cancerous and healthy breast tissue samples.

PubMed

Lakshmanan, Manu N; Greenberg, Joel A; Samei, Ehsan; Kapadia, Anuj J

2016-01-01

A scatter imaging technique for the differentiation of cancerous and healthy breast tissue in a heterogeneous sample is introduced in this work. Such a technique has potential utility in intraoperative margin assessment during lumpectomy procedures. In this work, we investigate the feasibility of the imaging method for tumor classification using Monte Carlo simulations and physical experiments. The coded aperture coherent scatter spectral imaging technique was used to reconstruct three-dimensional (3-D) images of breast tissue samples acquired through a single-position snapshot acquisition, without rotation as is required in coherent scatter computed tomography. We perform a quantitative assessment of the accuracy of the cancerous voxel classification using Monte Carlo simulations of the imaging system; describe our experimental implementation of coded aperture scatter imaging; show the reconstructed images of the breast tissue samples; and present segmentations of the 3-D images in order to identify the cancerous and healthy tissue in the samples. From the Monte Carlo simulations, we find that coded aperture scatter imaging is able to reconstruct images of the samples and identify the distribution of cancerous and healthy tissues (i.e., fibroglandular, adipose, or a mix of the two) inside them with a cancerous voxel identification sensitivity, specificity, and accuracy of 92.4%, 91.9%, and 92.0%, respectively. From the experimental results, we find that the technique is able to identify cancerous and healthy tissue samples and reconstruct differential coherent scatter cross sections that are highly correlated with those measured by other groups using x-ray diffraction. Coded aperture scatter imaging has the potential to provide scatter images that automatically differentiate cancerous and healthy tissue inside samples within a time on the order of a minute per slice.

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.

Highly directional transurethral ultrasound applicators with rotational control for MRI-guided prostatic thermal therapy

NASA Astrophysics Data System (ADS)

Ross, Anthony B.; Diederich, Chris J.; Nau, William H.; Gill, Harcharan; Bouley, Donna M.; Daniel, Bruce; Rieke, Viola; Butts, R. Kim; Sommer, Graham

2004-01-01

Transurethral ultrasound applicators with highly directional energy deposition and rotational control were investigated for precise treatment of benign prostatic hyperplasia (BPH) and adenocarcinoma of the prostate (CaP). Two types of catheter-based applicators were fabricated, using either 90° sectored tubular (3.5 mm OD × 10 mm) or planar transducers (3.5 mm × 10 mm). They were constructed to be MRI compatible, minimally invasive and allow for manual rotation of the transducer array within a 10 mm cooling balloon. In vivo evaluations of the applicators were performed in canine prostates (n = 3) using MRI guidance (0.5 T interventional magnet). MR temperature imaging (MRTI) utilizing the proton resonance frequency shift method was used to acquire multiple-slice temperature overlays in real time for monitoring and guiding the thermal treatments. Post-treatment T1-weighted contrast-enhanced imaging and triphenyl tetrazolium chloride stained tissue sections were used to define regions of tissue coagulation. Single sonications with the 90° tubular applicator (9-15 W, 12 min, 8 MHz) produced coagulated zones covering an 80° wedge of the prostate extending from 1-2 mm outside the urethra to the outer boundary of the gland (16 mm radial coagulation). Single sonications with the planar applicator (15-20 W, 10 min, ~8 MHz) generated thermal lesions of ~30° extending to the prostate boundary. Multiple sequential sonications (sweeping) of a planar applicator (12 W with eight rotations of 30° each) demonstrated controllable coagulation of a 270° contiguous section of the prostate extending to the capsule boundary. The feasibility of using highly directional transurethral ultrasound applicators with rotational capabilities to selectively coagulate regions of the prostate while monitoring and controlling the treatments with MRTI was demonstrated in this study.

Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons

NASA Technical Reports Server (NTRS)

Chalmers, G. R.; Edgerton, V. R.

1989-01-01

The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

A single slide multiplex assay for the evaluation of classical Hodgkin lymphoma.

PubMed

Hollman-Hewgley, Denise; Lazare, Michael; Bordwell, Alex; Zebadua, Emily; Tripathi, Pinky; Ross, Alexander S; Fisher, Deanna; Adams, Alisha; Bouman, Derek; O'Malley, Dennis P; Weiss, Lawrence M

2014-09-01

Classical Hodgkin lymphoma can be diagnosed with confidence in the majority of cases, but there is a significant subset that remains a diagnostic challenge. The authors have investigated the utility of a novel hyperplexing technology, MultiOmyx™, which may be applied to stain with >60 antibodies on single tissue sections from formalin-fixed paraffin-embedded tissue as an aid to the diagnosis of classical Hodgkin lymphoma. The multiplexing protocol included CD30, CD15, PAX-5, CD20, CD79a, CD45, BOB.1, OCT-2, and CD3 antibodies. The technology showed a high degree of sensitivity, specificity, and precision. Comparison studies with routine hematoxylin and eosin and immunohistochemical assessment of hematopathology cases in which classical Hodgkin lymphoma was included in the differential diagnosis showed concordance in 54 of 56 cases, with the 2 discordant cases illustrating the potential of this multiplexed immunofluorescence technology to improve on traditional immunohistochemistry for classical Hodgkin lymphoma diagnosis. This technology is practical for routine diagnosis and may be particularly useful in cases in which the sample size is limited, few Hodgkin-like cells are present, or in CD30-positive lymphoma cases with difficult morphology. MultiOmyx may potentially benefit other areas of research and diagnostic pathology.

Reduction of renal uptake of 111In-DOTA-labeled and A700-labeled RAFT-RGD during integrin αvβ3 targeting using single photon emission computed tomography and optical imaging.

PubMed

Briat, Arnaud; Wenk, Christiane H F; Ahmadi, Mitra; Claron, Michael; Boturyn, Didier; Josserand, Véronique; Dumy, Pascal; Fagret, Daniel; Coll, Jean-Luc; Ghezzi, Catherine; Sancey, Lucie; Vuillez, Jean-Philippe

2012-06-01

Integrin α(v)β(3) expression is upregulated during tumor growth and invasion in newly formed endothelial cells in tumor neovasculature and in some tumor cells. A tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK])4 (RAFT-RGD), specifically targets integrin α(v)β(3) in vitro and in vivo. When labeled with indium-111, the RAFT-RGD is partially reabsorbed and trapped in the kidneys, limiting its use for further internal targeted radiotherapy and imaging investigations. We studied the effect of Gelofusine on RAFT-RGD renal retention in tumor-bearing mice. Mice were imaged using single photon emission computed tomography and optical imaging 1 and 24 h following tracer injection. Distribution of RAFT-RGD was further investigated by tissue removal and direct counting of the tracer. Kidney sections were analyzed by confocal microscopy. Gelofusine significantly induced a >50% reduction of the renal reabsorption of (111)In-DOTA-RAFT-RGD and A700-RAFT-RGD, without affecting tumor uptake. Injection of Gelofusine significantly reduced the renal retention of labeled RAFT-RGD, while increasing the tumor over healthy tissue ratio. These results will lead to the development of future therapeutic approaches. © 2012 Japanese Cancer Association.

Soft tissue contour changes at immediate postextraction single-tooth implants with immediate restoration: a 12-month prospective cohort study.

PubMed

Cardaropoli, Daniele; Tamagnone, Lorenzo; Roffredo, Alessandro; Gaveglio, Lorena

2015-01-01

In the maxillary arch from premolar to premolar, 26 single dental implants were inserted in fresh extraction sockets and immediately provisionalized. The bone-to-implant gap was grafted with a bovine bone mineral. After 3 months, definitive ceramic crowns were placed. At baseline and after 1 year, the soft tissue horizontal width, mesiodistal papillary level, midfacial gingival level, and pink esthetic score were evaluated. No statistical differences were found between baseline and 1 year for all parameters. Immediate single-tooth implants, with immediate restoration, are capable of maintaining the soft tissue contour and esthetics compared to the pretreatment status.

Non-contact single shot elastography using line field low coherence holography

PubMed Central

Liu, Chih-Hao; Schill, Alexander; Wu, Chen; Singh, Manmohan; Larin, Kirill V.

2016-01-01

Optical elastic wave imaging is a powerful technique that can quantify local biomechanical properties of tissues. However, typically long acquisition times make this technique unfeasible for clinical use. Here, we demonstrate non-contact single shot elastographic holography using a line-field interferometer integrated with an air-pulse delivery system. The propagation of the air-pulse induced elastic wave was imaged in real time, and required a single excitation for a line-scan measurement. Results on tissue-mimicking phantoms and chicken breast muscle demonstrated the feasibility of this technique for accurate assessment of tissue biomechanical properties with an acquisition time of a few milliseconds using parallel acquisition. PMID:27570694

5S ribosomal RNA gene repeat sequences define at least eight groups of plant trypanosomatids (Phytomonas spp.): phloem-restricted pathogens form a distinct section.

PubMed

Dollet, M; Sturm, N R; Sánchez-Moreno, M; Campbell, D A

2000-01-01

Trypanosomatids isolated from plants have been assigned typically into the genus Phytomonas. Such designations do not reflect the biology of the diverse isolates; confusion may arise due to the transient presence in plants of monogenetic (insect) trypanosomatids deposited by phytophagous bugs. To develop further molecular markers for the plant kinetoplastids, we have obtained the DNA sequence of the 5S ribosomal RNA gene from 24 isolates harvested from phloem, latex, and fruit. Small, distinct sequence differences were found at the 3'-ends of the transcribed regions; substantial sequence and size differences were found in the non-transcribed regions. Alignment of the gene sequences from all the isolates suggested the presence of eight groupings. While six groups contained isolates from single plant tissues, groups C and A contained isolates from both fruit and latex. The DNA sequences of the 10 phloem-restricted pathogenic isolates from South America and the Carribean were highly conserved and thus comprised a single group (H). The conserved nature of the 5S ribosomal RNA genes in these plant pathogens supports the proposal that they be considered as a distinct section, the phloemicola.

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

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.

A mutational approach for the detection of genetic factors affecting seed size in maize.

PubMed

Sangiorgio, Stefano; Carabelli, Laura; Gabotti, Damiano; Manzotti, Priscilla Sofia; Persico, Martina; Consonni, Gabriella; Gavazzi, Giuseppe

2016-12-01

Genes influencing seed size. The designation emp (empty pericarp) refers to a group of defective kernel mutants that exhibit a drastic reduction in endosperm tissue production. They allow the isolation of genes controlling seed development and affecting seed size. Nine independently isolated emp mutants have been analyzed in this study and in all cases longitudinal sections of mature seeds revealed the absence of morphogenesis in the embryo proper, an observation that correlates with their failure to germinate. Complementation tests with the nine emp mutants, crossed inter se in all pairwise combinations, identified complementing and non-complementing pairs in the F 1 progenies. Data were then validated in the F 2 /F 3 generations. Mutant chromosomal location was also established. Overall our study has identified two novel emp genes and a novel allele at the previously identified emp4 gene. The introgression of single emp mutants in a different genetic background revealed the existence of a cryptic genetic variation (CGV) recognizable as a variable increase in the endosperm tissue. The unmasking of CGV by introducing single mutants in different genetic backgrounds is the result of the interaction of the emp mutants with a suppressor that has no obvious phenotype of its own and is present in the genetic background of the inbred lines into which the emp mutants were transferred. On the basis of these results, emp mutants could be used as tools for the detection of genetic factors that enhance the amount of endosperm tissue in the maize kernel and which could thus become valuable targets to exploit in future breeding programs.

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.

Laser dissection sampling modes for direct mass spectral analysis [using a hybrid optical microscopy/laser ablation liquid vortex capture/electrospray ionization system

DOE PAGES

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

2016-02-01

Here, laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis ofmore » single cells and tissue. Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (4-15 m) even when agglomerated together. Turbid Allium Cepa cells (150 m) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. In conclusion, laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.« less

Laser dissection sampling modes for direct mass spectral analysis [using a hybrid optical microscopy/laser ablation liquid vortex capture/electrospray ionization system

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

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

Here, laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis ofmore » single cells and tissue. Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (4-15 m) even when agglomerated together. Turbid Allium Cepa cells (150 m) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. In conclusion, laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.« less

Cellular comparison of sinus mucosa vs polyp tissue from a single sinus cavity in chronic rhinosinusitis.

PubMed

Ho, Jacqueline; Bailey, Michelle; Zaunders, John; Mrad, Nadine; Sacks, Raymond; Sewell, William; Harvey, Richard J

2015-01-01

Nasal polyposis is a common development in chronic rhinosinusitis (CRS), and sinus mucosa and polyp tissue have been used interchangeably in studies investigating CRS. However, potential differences may exist between these 2 tissue types, which have not been entirely characterized. A cross-sectional study of CRS with nasal polyposis (CRSwNP) patients undergoing endoscopic sinus surgery was conducted. Sinus mucosal biopsies and corresponding polyp tissue were obtained from the same sinus cavity via flow cytometry, single-cell suspensions identified type 2 innate lymphoid cells (ILC2s), CD4 and CD8 T cells, activated CD4 and CD8 T cells, plasma cells, plasmacytoid dendritic cells (pDCs), regulatory T cells, T follicular helper cells, B cells, and immunoglobulin A (IgA)(+) and IgG(+) B cells. Cells were measured as a percentage of CD45(+) cells. Paired nonparametric comparisons between sinus and polyp tissue were performed. Ten patients (50% female; age 48 ± 16 years) were recruited. Significantly elevated ILC2 levels were found in polyp tissue compared to sinus mucosa (0.12 [0.07 to 0.23] vs 0.07 [0.04 to 0.16], p = 0.02), as well as plasma cells (2.25 [0.84 to 3.68] vs 1.18 [0.74 to 2.41], p = 0.01); pDCs (0.15 [0.12 to 0.50[ vs 0.04 [0.02 to 0.17], p = 0.03); activated CD8 T cells (29.22 [17.60 to 41.43] vs 16.32 [10.07 to 36.16], p = 0.04) and IgG(+) B cells (6.96 [0.06 to 11.82] vs 1.51 [0.38 to 5.13], p = 0.04). Other cell populations showed no significant differences. Polyps have a similar cellular composition to that of mucosa. Higher levels of ILC2s, plasma cells, pDCs, activated CD8 T cells, and IgG(+) B cells in polyp tissue may be reflective of cell populations driving nasal polyp development. The cellular machinery of CRS is present in polyps and representative of the disease process. This pilot study strongly suggests that a larger study would provide significant insights into the relationship of sinus mucosa to pathogenesis of nasal polyps. © 2014 ARS-AAOA, LLC.

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.

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...

Single-stage Reconstruction of Elbow Flexion Associated with Massive Soft-Tissue Defect Using the Latissimus Dorsi Muscle Bipolar Rotational Transfer

PubMed Central

Cuéllar, Vanessa G.; Ghiassi, Alidad; Sharpe, Frances

2016-01-01

Introduction: In the upper extremity, the latissimus dorsi muscle can be used as an ipsilateral rotational muscle flap for soft-tissue coverage or functional reconstruction of arm and elbow. Patients who have both major soft-tissue loss and functional deficits can be successfully treated with a single-stage functional latissimus dorsi rotational muscle transfer that provides simultaneous soft-tissue coverage and functional reconstruction. Methods: Our data base was queried for all patients undergoing a rotational latissimus dorsi muscle transfer for simultaneous soft-tissue coverage and functional reconstruction of elbow flexion. Four patients were identified. A chart review documented the mechanism of injury, associated injuries, soft-tissue defect size, number of surgical procedures, length of follow-up, last elbow range of motion, and flexion strength. Results: Four patients with loss of elbow flexion due to traumatic loss of the anterior compartment muscles and the overlying soft tissue underwent simultaneous soft-tissue coverage and elbow flexorplasty using the ipsilateral latissimus dorsi as a bipolar muscle rotational tissue transfer. All flaps survived and had a recovery of Medical Research Council Grade 4/5 elbow flexion strength. No additional procedures were required for elbow flexion. The surgical technique is described and supplemented with surgical technique video and patient outcome. Conclusions: This patient series augments the data provided in other series supporting the safety and efficacy of this procedure which provides both soft-tissue coverage and functional restoration of elbow flexion as a single-stage procedure in the setting of massive traumatic soft-tissue loss of the arm. PMID:27757363

Single-stage Reconstruction of Elbow Flexion Associated with Massive Soft-Tissue Defect Using the Latissimus Dorsi Muscle Bipolar Rotational Transfer.

PubMed

Stevanovic, Milan V; Cuéllar, Vanessa G; Ghiassi, Alidad; Sharpe, Frances

2016-09-01

In the upper extremity, the latissimus dorsi muscle can be used as an ipsilateral rotational muscle flap for soft-tissue coverage or functional reconstruction of arm and elbow. Patients who have both major soft-tissue loss and functional deficits can be successfully treated with a single-stage functional latissimus dorsi rotational muscle transfer that provides simultaneous soft-tissue coverage and functional reconstruction. Our data base was queried for all patients undergoing a rotational latissimus dorsi muscle transfer for simultaneous soft-tissue coverage and functional reconstruction of elbow flexion. Four patients were identified. A chart review documented the mechanism of injury, associated injuries, soft-tissue defect size, number of surgical procedures, length of follow-up, last elbow range of motion, and flexion strength. Four patients with loss of elbow flexion due to traumatic loss of the anterior compartment muscles and the overlying soft tissue underwent simultaneous soft-tissue coverage and elbow flexorplasty using the ipsilateral latissimus dorsi as a bipolar muscle rotational tissue transfer. All flaps survived and had a recovery of Medical Research Council Grade 4/5 elbow flexion strength. No additional procedures were required for elbow flexion. The surgical technique is described and supplemented with surgical technique video and patient outcome. This patient series augments the data provided in other series supporting the safety and efficacy of this procedure which provides both soft-tissue coverage and functional restoration of elbow flexion as a single-stage procedure in the setting of massive traumatic soft-tissue loss of the arm.

Overlapping MALDI-Mass Spectrometry Imaging for In-Parallel MS and MS/MS Data Acquisition without Sacrificing Spatial Resolution

NASA Astrophysics Data System (ADS)

Hansen, Rebecca L.; Lee, Young Jin

2017-09-01

Metabolomics experiments require chemical identifications, often through MS/MS analysis. In mass spectrometry imaging (MSI), this necessitates running several serial tissue sections or using a multiplex data acquisition method. We have previously developed a multiplex MSI method to obtain MS and MS/MS data in a single experiment to acquire more chemical information in less data acquisition time. In this method, each raster step is composed of several spiral steps and each spiral step is used for a separate scan event (e.g., MS or MS/MS). One main limitation of this method is the loss of spatial resolution as the number of spiral steps increases, limiting its applicability for high-spatial resolution MSI. In this work, we demonstrate multiplex MS imaging is possible without sacrificing spatial resolution by the use of overlapping spiral steps, instead of spatially separated spiral steps as used in the previous work. Significant amounts of matrix and analytes are still left after multiple spectral acquisitions, especially with nanoparticle matrices, so that high quality MS and MS/MS data can be obtained on virtually the same tissue spot. This method was then applied to visualize metabolites and acquire their MS/MS spectra in maize leaf cross-sections at 10 μm spatial resolution. [Figure not available: see fulltext.

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.

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

PubMed Central

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

2011-01-01

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

Whole slide imaging of unstained tissue using lensfree microscopy

NASA Astrophysics Data System (ADS)

Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Cioni, Olivier; Delon, Antoine; Fromentin, Catherine; Dinten, Jean-Marc; Allier, Cédric

2016-04-01

Pathologist examination of tissue slides provides insightful information about a patient's disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2-4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.

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.

[Comparison of basic carboxypeptidases activity in male rats tissues at a single injection of haloperidol].

PubMed

Pravosudova, N A; Bykova, I O

2014-01-01

The influence of a single injection of haloperidol on basic carboxypeptidases (biologically active peptide processing enzymes) activity in rat tissues was studied. Acute exposure to haloperidol increased the activity of carboxypeptidases H (CP H) in hypothalamic-pituitary-adrenal system and cerebellum and reduced such activity in testes. Multidirectional changes of PMSF-inhibited carboxypeptidases activity (PMSF-CP) were observed after a single haloperidol injection in all studied tissues except testes. It is suggested that changes of CP H and PMSF-CP activity might affect levels of regulatory peptides in the brain and blood and thus may be involved in general and side effects of haloperidol on the organism.

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...

Ex vivo detection of macrophages in atherosclerotic plaques using intravascular ultrasonic-photoacoustic imaging

NASA Astrophysics Data System (ADS)

Quang Bui, Nhat; Hlaing, Kyu Kyu; Lee, Yong Wook; Kang, Hyun Wook; Oh, Junghwan

2017-01-01

Macrophages are excellent imaging targets for detecting atherosclerotic plaques as they are involved in all the developmental stages of atherosclerosis. However, no imaging technique is currently capable of visualizing macrophages inside blood vessel walls. The current study develops an intravascular ultrasonic-photoacoustic (IVUP) imaging system combined with indocyanine green (ICG) as a contrast agent to provide morphological and compositional information about the targeted samples. Both tissue-mimicking vessel phantoms and atherosclerotic plaque-mimicking porcine arterial tissues are used to demonstrate the feasibility of mapping macrophages labeled with ICG by endoscopically applying the proposed hybrid technique. A delay pulse triggering technique is able to sequentially acquire photoacoustic (PA) and ultrasound (US) signals from a single scan without using any external devices. The acquired PA and US signals are used to reconstruct 2D cross-sectional and 3D volumetric images of the entire tissue with the ICG-loaded macrophages injected. Due to high imaging contrast and sensitivity, the IVUP imaging vividly reveals structural information and detects the spatial distribution of the ICG-labeled macrophages inside the samples. ICG-assisted IVUP imaging can be a feasible imaging modality for the endoscopic detection of atherosclerotic plaques.

Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

PubMed

Garcia, I; Rodgers, M; Pepin, R; Hsieh, T F; Matringe, M

1999-04-01

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

Quantitative analysis of drug distribution by ambient mass spectrometry imaging method with signal extinction normalization strategy and inkjet-printing technology.

PubMed

Luo, Zhigang; He, Jingjing; He, Jiuming; Huang, Lan; Song, Xiaowei; Li, Xin; Abliz, Zeper

2018-03-01

Quantitative mass spectrometry imaging (MSI) is a robust approach that provides both quantitative and spatial information for drug candidates' research. However, because of complicated signal suppression and interference, acquiring accurate quantitative information from MSI data remains a challenge, especially for whole-body tissue sample. Ambient MSI techniques using spray-based ionization appear to be ideal for pharmaceutical quantitative MSI analysis. However, it is more challenging, as it involves almost no sample preparation and is more susceptible to ion suppression/enhancement. Herein, based on our developed air flow-assisted desorption electrospray ionization (AFADESI)-MSI technology, an ambient quantitative MSI method was introduced by integrating inkjet-printing technology with normalization of the signal extinction coefficient (SEC) using the target compound itself. The method utilized a single calibration curve to quantify multiple tissue types. Basic blue 7 and an antitumor drug candidate (S-(+)-deoxytylophorinidine, CAT) were chosen to initially validate the feasibility and reliability of the quantitative MSI method. Rat tissue sections (heart, kidney, and brain) administered with CAT was then analyzed. The quantitative MSI analysis results were cross-validated by LC-MS/MS analysis data of the same tissues. The consistency suggests that the approach is able to fast obtain the quantitative MSI data without introducing interference into the in-situ environment of the tissue sample, and is potential to provide a high-throughput, economical and reliable approach for drug discovery and development. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

NASA Astrophysics Data System (ADS)

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-04-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

PubMed Central

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-01-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue. PMID:27063397

[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.

Imaging plasmodesmata with high-resolution scanning electron microscopy.

PubMed

Barton, Deborah A; Overall, Robyn L

2015-01-01

High-resolution scanning electron microscopy (HRSEM) is an effective tool to investigate the distribution of plasmodesmata within plant cell walls as well as to probe their complex, three-dimensional architecture. It is a useful alternative to traditional transmission electron microscopy (TEM) in which plasmodesmata are sectioned to reveal their internal substructures. Benefits of adopting an HRSEM approach to studies of plasmodesmata are that the specimen preparation methods are less complex and time consuming than for TEM, many plasmodesmata within a large region of tissue can be imaged in a single session, and three-dimensional information is readily available without the need for reconstructing TEM serial sections or employing transmission electron tomography, both of which are lengthy processes. Here we describe methods to prepare plant samples for HRSEM using pre- or postfixation extraction of cellular material in order to visualize plasmodesmata embedded within plant cell walls.

Investigation of factors affecting hypothermic pelvic tissue cooling using bio-heat simulation based on MRI-segmented anatomic models.

PubMed

Lin, Yuting; Lin, Wei-Ching; Fwu, Peter T; Shih, Tzu-Ching; Yeh, Lee-Ren; Su, Min-Ying; Chen, Jeon-Hor

2015-10-01

This study applied a simulation method to map the temperature distribution based on magnetic resonance imaging (MRI) of individual patients, and investigated the influence of different pelvic tissue types as well as the choice of thermal property parameters on the efficiency of endorectal cooling balloon (ECB). MR images of four subjects with different prostate sizes and pelvic tissue compositions, including fatty tissue and venous plexus, were analyzed. The MR images acquired using endorectal coil provided a realistic geometry of deformed prostate that resembled the anatomy in the presence of ECB. A single slice with the largest two-dimensional (2D) cross-sectional area of the prostate gland was selected for analysis. The rectal wall, prostate gland, peri-rectal fatty tissue, peri-prostatic fatty tissue, peri-prostatic venous plexus, and urinary bladder were manually segmented. Pennes' bioheat thermal model was used to simulate the temperature distribution dynamics, by using an in-house finite element mesh based solver written in MATLAB. The results showed that prostate size and periprostatic venous plexus were two major factors affecting ECB cooling efficiency. For cases with negligible amount of venous plexus and small prostate, the average temperature in the prostate and neurovascular bundles could be cooled down to 25 °C within 30 min. For cases with abundant venous plexus and large prostate, the temperature could not reach 25 °C at the end of 3 h cooling. Large prostate made the cooling difficult to propagate through. The impact of fatty tissue on cooling effect was small. The filling of bladder with warm urine during the ECB cooling procedure did not affect the temperature in the prostate or NVB. In addition to the 2D simulation, in one case a 3D pelvic model was constructed for volumetric simulation. It was found that the 2D slice with the largest cross-sectional area of prostate had the most abundant venous plexus, and was the most difficult slice to cool, thus it may provide a conservative prediction of the cooling effect. This feasibility study demonstrated that the simulation tool could potentially be used for adjusting the setting of ECB for individual patients during hypothermic radical prostatectomy. Further studies using MR thermometry are required to validate the in silico results obtained using simulation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Investigation of factors affecting hypothermic pelvic tissue cooling using bio-heat simulation based on MRI-segmented anatomic models

PubMed Central

Lin, Yuting; Lin, Wei-Ching; Fwu, Peter T.; Shih, Tzu-Ching; Yeh, Lee-Ren; Su, Min-Ying; Chen, Jeon-Hor

2015-01-01

This study applied a simulation method to map the temperature distribution based on magnetic resonance imaging (MRI) of individual patients, and investigated the influence of different pelvic tissue types as well as the choice of thermal property parameters on the efficiency of endorectal cooling balloon (ECB). MR images of four subjects with different prostate sizes and pelvic tissue compositions, including fatty tissue and venous plexus, were analyzed. The MR images acquired using endorectal coil provided a realistic geometry of deformed prostate that resembled the anatomy in the presence of ECB. A single slice with the largest two-dimensional (2D) cross-sectional area of the prostate gland was selected for analysis. The rectal wall, prostate gland, peri-rectal fatty tissue, peri-prostatic fatty tissue, peri-prostatic venous plexus, and urinary bladder were manually segmented. Pennes’ bioheat thermal model was used to simulate the temperature distribution dynamics, by using an in-house finite element mesh based solver written in Matlab. The results showed that prostate size and periprostatic venous plexus were two major factors affecting ECB cooling efficiency. For cases with negligible amount of venous plexus and small prostate, the averaged temperature in the prostate and neurovascular bundles could be cooled down to 25°C within 30 minutes. For cases with abundant venous plexus and large prostate, the temperature could not reach 25°C at the end of 3 hours cooling. Large prostate made the cooling difficult to propagate through. The impact of fatty tissue on cooling effect was small. The filling of bladder with warm urine during the ECB cooling procedure did not affect the temperature in the prostate or NVB. In addition to the 2D simulation, in one case a 3D pelvic model was constructed for volumetric simulation. It was found that the 2D slice with the largest cross-sectional area of prostate had the most abundant venous plexus, and was the most difficult slice to cool, thus it may provide a conservative prediction of the cooling effect. This feasibility study demonstrated that the simulation tool could potentially be used for adjusting the setting of ECB for individual patients during hypothermic radical prostatectomy. Further studies using MR thermometry are required to validate the in silico results obtained using simulation. PMID:26198131

Cytologic features of the normal pineal gland on squash preparations.

PubMed

Murro, Diana; Alsadi, Alaa; Nag, Sukriti; Arvanitis, Leonidas; Gattuso, Paolo

2014-11-01

As primary pineal lesions are extremely rare, many surgical pathologists are unfamiliar with normal pineal cytologic features. We describe cytologic features of the normal pineal gland in patients of varying ages and identify common diagnostic pitfalls. We performed a retrospective review of pineal gland biopsies performed at our institution, where approximately 30,000 surgical specimens are accessioned yearly, for the last 23 years. Only two pineal gland biopsies were found. Although both cases were initially diagnosed as low-grade gliomas on frozen section, the final diagnosis was benign pineal tissue based on light microscopy and immunohistochemistry results. Additionally, we performed squash preparations of five normal pineal gland autopsy specimens with Papanicolaou and Diff-Quik® (Dade Behring, Newark, DE) stains. Infant preparations were highly cellular smears composed of numerous, uniform, single cells with indistinct cytoplasm, small round-to-oval nuclei, fine chromatin, and absent nucleoli and calcifications. The vague microfollicular pattern mimicked a pineocytoma and the fine fibrillary background mimicked a glial neoplasm. Young adult smears were similar; however, microcalcifications were present with fewer background single cells. Older patients had much less cellular smears composed of small clusters of cells with fusiform-to-spindle nuclei, a fine chromatin pattern, and indistinct cytoplasmic borders. There were fewer background single cells and more microcalcifications. The cytologic features of the native pineal gland vary with age. Normal pineal tissue can be confused with a pineocytoma or low-grade glioma. Familiarity with normal pineal gland cytological features will help to avoid a potential misdiagnosis. © 2014 Wiley Periodicals, Inc.

The diversity of the effects of sulfur mustard gas inhalation on respiratory system 10 years after a single, heavy exposure: analysis of 197 cases.

PubMed

Emad, A; Rezaian, G R

1997-09-01

To find out the late pulmonary sequelae of sulfur mustard gas inhalation in 197 veterans, 10 years after their exposure. Cross-sectional clinical study. University hospital. One hundred ninety-seven veterans with a single, heavy exposure to sulfur mustard gas in 1986 and 86 nonexposed veterans as their control group. Pulmonary function tests, carbon monoxide diffusion capacity, bronchoscopy, and high-resolution CT of the chest were performed in all patients. Transbronchial lung biopsy was done in 24 suspected cases of pulmonary fibrosis. Asthma was diagnosed in 21 (10.65%), chronic bronchitis in 116 (58.88%), bronchiectasis in 17 (8.62%), airway narrowing due to searing or granulation tissue in 19 (9.64%), and pulmonary fibrosis in 24 (12.18%) cases. None of these were found among the control group except for a single case of chronic bronchitis. Although the respiratory symptoms of an acute sulfur mustard gas inhalation are usually transient and nonspecific, it can lead to the development of a series of chronic destructive pulmonary sequelae in such cases.

Microscopic neural image registration based on the structure of mitochondria

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.

PubMed

Volovitz, Ilan; Shapira, Netanel; Ezer, Haim; Gafni, Aviv; Lustgarten, Merav; Alter, Tal; Ben-Horin, Idan; Barzilai, Ori; Shahar, Tal; Kanner, Andrew; Fried, Itzhak; Veshchev, Igor; Grossman, Rachel; Ram, Zvi

2016-06-01

Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 °C to overnight dissociation at ambient temperature. The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient-temperature shipping of tumor pieces in multi-center clinical trials, meanwhile being dissociated. As clinical grade NP is commercially available it can be easily integrated into cell-therapy clinical trials in neuro-oncology. The high quality viable cells produced may enable investigators to conduct more consistent research by avoiding the experimental artifacts associated with the presence dead cells or cellular debris.

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.

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.

Nanopipettes: probes for local sample analysis† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc00668f Click here for additional data file.

PubMed Central

Saha-Shah, Anumita; Weber, Anna E.; Karty, Jonathan A.; Ray, Steven J.; Hieftje, Gary M.

2015-01-01

Nanopipettes (pipettes with diameters <1 μm) were explored as pressure-driven fluid manipulation tools for sampling nanoliter volumes of fluids. The fundamental behavior of fluids confined in the narrow channels of the nanopipette shank was studied to optimize sampling volume and probe geometry. This method was utilized to collect nanoliter volumes (<10 nL) of sample from single Allium cepa cells and live Drosophila melanogaster first instar larvae. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was utilized to characterize the collected sample. The use of nanopipettes for surface sampling of mouse brain tissue sections was also explored. Lipid analyses were performed on mouse brain tissues with spatial resolution of sampling as small as 50 μm. Nanopipettes were shown to be a versatile tool that will find further application in studies of sample heterogeneity and population analysis for a wide range of samples. PMID:28706697

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

PubMed

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

1999-08-27

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

High-pulse energy supercontinuum laser for high-resolution spectroscopic photoacoustic imaging of lipids in the 1650-1850 nm region.

PubMed

Dasa, Manoj Kumar; Markos, Christos; Maria, Michael; Petersen, Christian R; Moselund, Peter M; Bang, Ole

2018-04-01

We propose a cost-effective high-pulse energy supercontinuum (SC) source based on a telecom range diode laser-based amplifier and a few meters of standard single-mode optical fiber, with a pulse energy density as high as ~25 nJ/nm in the 1650-1850 nm regime (factor >3 times higher than any SC source ever used in this wavelength range). We demonstrate how such an SC source combined with a tunable filter allows high-resolution spectroscopic photoacoustic imaging and the spectroscopy of lipids in the first overtone transition band of C-H bonds (1650-1850 nm). We show the successful discrimination of two different lipids (cholesterol and lipid in adipose tissue) and the photoacoustic cross-sectional scan of lipid-rich adipose tissue at three different locations. The proposed high-pulse energy SC laser paves a new direction towards compact, broadband and cost-effective source for spectroscopic photoacoustic imaging.

Shedding Light on the Molecular Pathology of Amyloid Plaques in Transgenic Alzheimer's Disease Mice Using Multimodal MALDI Imaging Mass Spectrometry.

PubMed

Kaya, Ibrahim; Zetterberg, Henrik; Blennow, Kaj; Hanrieder, Jörg

2018-05-04

Senile plaques formed by aggregated amyloid β peptides are one of the major pathological hallmarks of Alzheimer's disease (AD) which have been suggested to be the primary influence triggering the AD pathogenesis and the rest of the disease process. However, neurotoxic Aβ aggregation and progression are associated with a wide range of enigmatic biochemical, biophysical and genetic processes. MALDI imaging mass spectrometry (IMS) is a label-free method to elucidate the spatial distribution patterns of intact molecules in biological tissue sections. In this communication, we utilized multimodal MALDI-IMS analysis on 18 month old transgenic AD mice (tgArcSwe) brain tissue sections to enhance molecular information correlated to individual amyloid aggregates on the very same tissue section. Dual polarity MALDI-IMS analysis of lipids on the same pixel points revealed high throughput lipid molecular information including sphingolipids, phospholipids, and lysophospholipids which can be correlated to the ion images of individual amyloid β peptide isoforms at high spatial resolutions (10 μm). Further, multivariate image analysis was applied in order to probe the multimodal MALDI-IMS data in an unbiased way which verified the correlative accumulations of lipid species with dual polarity and Aβ peptides. This was followed by the lipid fragmentation obtained directly on plaque aggregates at higher laser pulse energies which provided tandem MS information useful for structural elucidation of several lipid species. Majority of the amyloid plaque-associated alterations of lipid species are for the first time reported here. The significance of this technique is that it allows correlating the biological discussion of all detected plaque-associated molecules to the very same individual amyloid plaques which can give novel insights into the molecular pathology of even a single amyloid plaque microenvironment in a specific brain region. Therefore, this allowed us to interpret the possible roles of lipids and amyloid peptides in amyloid plaque-associated pathological events such as focal demyelination, autophagic/lysosomal dysfunction, astrogliosis, inflammation, oxidative stress, and cell death.

A comparison study of different excitation wavelengths to determine the relative content of key biomolecules in breast cancer and breast normal tissue

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Sordillo, Peter P.; Budansky, Yury; Pu, Yang; Alfano, R. R.

2015-03-01

Fluorescence profiles from breast cancer and breast normal tissue samples with excitation wavelengths at 280 nm and 340 nm were obtained using the conventional LS-50 Perkin-Elmer spectrometer. Fluorescence ratios from these tissue samples, demonstrated by emission peaks at 340 nm, 440 nm and 460 nm and likely representing tryptophan and NADH, show increased relative content of tryptophan in malignant samples. Double ratio (DR) techniques were used to measure the severity of disease. The single excitation double ratio (Single-DR) method utilizes the emission intensity peaks from the spectrum acquired using a single excitation of 280 nm; while the dual excitation double ratio (dual-DR) method utilizes the emission intensity peaks from the spectra acquired using an excitation of 280 nm and 340 nm. Single-DR and dual-DR from 13 patients with breast carcinoma were compared in terms of their efficiency to distinguish high from low/intermediate tumors. Similar results were found with both methods. Results suggest that dual excitation wavelengths may be as effective as single excitation wavelength in calculating the relative content of biomolecules in breast cancer tissue, as well as for the assessment of the malignant potential of these tumors.

Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low-cost microfluidic instrumentation.

PubMed

Stephenson, William; Donlin, Laura T; Butler, Andrew; Rozo, Cristina; Bracken, Bernadette; Rashidfarrokhi, Ali; Goodman, Susan M; Ivashkiv, Lionel B; Bykerk, Vivian P; Orange, Dana E; Darnell, Robert B; Swerdlow, Harold P; Satija, Rahul

2018-02-23

Droplet-based single-cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While this approach offers the exciting promise to deconvolute cellular heterogeneity in diseased tissues, the lack of cost-effective and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we developed a 3D-printed, low-cost droplet microfluidic control instrument and deploy it in a clinical environment to perform single-cell transcriptome profiling of disaggregated synovial tissue from five rheumatoid arthritis patients. We sequence 20,387 single cells revealing 13 transcriptomically distinct clusters. These encompass an unsupervised draft atlas of the autoimmune infiltrate that contribute to disease biology. Additionally, we identify previously uncharacterized fibroblast subpopulations and discern their spatial location within the synovium. We envision that this instrument will have broad utility in both research and clinical settings, enabling low-cost and routine application of microfluidic techniques.

76 FR 13404 - Cancer Therapy Evaluation Program Intellectual Property Option to Collaborator

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-11

... with the Agent (including specimens obtained from NCI CTEP-funded tissue banks) (``Section B Inventions... tissue banks) (``Section B Inventions''): Institution agrees to grant to Collaborator(s): (i) a paid-up...

Tissue-Specific and Genetic Regulation of Insulin Sensitivity-Associated Transcripts in African Americans

PubMed Central

Sharma, Neeraj K.; Sajuthi, Satria P.; Chou, Jeff W.; Calles-Escandon, Jorge; Demons, Jamehl; Rogers, Samantha; Ma, Lijun; Palmer, Nicholette D.; McWilliams, David R.; Beal, John; Comeau, Mary E.; Cherry, Kristina; Hawkins, Gregory A.; Menon, Lata; Kouba, Ethel; Davis, Donna; Burris, Marcie; Byerly, Sara J.; Easter, Linda; Bowden, Donald W.; Freedman, Barry I.; Langefeld, Carl D.

2016-01-01

Context: Compared with European Americans, African Americans (AAs) are more insulin resistant, have a higher insulin secretion response to glucose, and develop type 2 diabetes more often. Molecular processes and/or genetic variations contributing to altered glucose homeostasis in high-risk AAs remain uncharacterized. Objective: Adipose and muscle transcript expression profiling and genotyping were performed in 260 AAs to identify genetic regulatory mechanisms associated with insulin sensitivity (SI). We hypothesized that: 1) transcription profiles would reveal tissue-specific modulation of physiologic pathways with SI, and 2) a subset of SI-associated transcripts would be controlled by DNA sequence variants as expression quantitative traits, and these variants in turn would be associated with SI. Design and Settings: The cross-sectional research study was performed in a clinical research unit. Participants: Unrelated nondiabetic AAs were recruited for the study. Main Outcome Measures: SI was measured by frequently sampled iv glucose tolerance test. Results: The expression levels of 2212 transcripts in adipose and 145 transcripts in muscle were associated with SI. Genes involved in eIF2, eIF4-p70S6K, and mTOR signaling were modulated with SI in both tissues. Genes involved in leukocyte extravasation signaling showed adipose-specific regulation, and genes involved in oxidative phosphorylation had discordant regulation between tissues. Intersecting cis-expression quantitative trait loci results with data from transcript-SI association analysis identified cis-regulatory single nucleotide polymorphisms for 363 and 42 SI-associated transcripts in adipose and muscle, respectively. Cis-eSNPs for three SI-associated adipose transcripts, NINJ1, AGA, and CLEC10A were associated with SI. Abrogation of NINJ1 induction in THP1 macrophages modulated expression of genes in chemokine signaling, cell adhesion, and angiogenesis pathways. Conclusion: This study identified multiple pathways associated with SI; particularly discordant tissue-specific regulation of the oxidative phosphorylation pathway, and adipose-specific regulation of transcripts in the leukocyte extravasation signaling pathway that seem to be important in insulin resistance. Identification of single nucleotide polymorphisms associated with SI and with modulation of expression of SI-associated transcripts, including NINJ1, reveals novel genetic regulatory mechanisms of insulin resistance in AAs. PMID:26789776

Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations.

PubMed

Bull, Marta; Learn, Gerald; Genowati, Indira; McKernan, Jennifer; Hitti, Jane; Lockhart, David; Tapia, Kenneth; Holte, Sarah; Dragavon, Joan; Coombs, Robert; Mullins, James; Frenkel, Lisa

2009-09-22

Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons. Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage. In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.

Sequencing thousands of single-cell genomes with combinatorial indexing.

PubMed

Vitak, Sarah A; Torkenczy, Kristof A; Rosenkrantz, Jimi L; Fields, Andrew J; Christiansen, Lena; Wong, Melissa H; Carbone, Lucia; Steemers, Frank J; Adey, Andrew

2017-03-01

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

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

Implementation of the Single European Code in a Multi-Tissue Bank.

PubMed

Schroeter, Jan; Schulz, Tino; Schroeter, Bernard; Fleischhauer, Katrin; Pruß, Axel

2017-11-01

The traceability of tissue and cells transplants is important to ensure a high level of safety for the recipients. With the final introduction of the Single European Code (SEC) in April 2017 in the EU a consistent system among all member states became mandatory. The regulations for the SEC on EU and national level were evaluated. An overview on the different parts of the SEC with detailed explanations is given. Our own experiences with the implementation of the SEC in our multi-tissue bank are reported in addition. The implementation of the SEC in our multi-tissue bank could be successfully realized. However, it revealed a number of difficulties, especially the sterile labeling of certain tissue transplants and the complex update of the existing database. The introduction of the SEC has made a contribution to the safety of recipients of tissue and cells transplants through a system of comprehensive and transparent traceability.

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.

Single-stage soft tissue reconstruction and orbital fracture repair for complex facial injuries.

PubMed

Wu, Peng Sen; Matoo, Reshvin; Sun, Hong; Song, Li Yuan; Kikkawa, Don O; Lu, Wei

2017-02-01

Orbital fractures with open periorbital wounds cause significant morbidity. Timing of debridement with fracture repair and soft tissue reconstruction is controversial. This study focuses on the efficacy of early single-stage repair in combined bony and soft tissue injuries. Retrospective review. Twenty-three patients with combined open soft tissue wounds and orbital fractures were studied for single-stage orbital reconstruction and periorbital soft tissue repair. Inclusion criteria were open soft tissue wounds with clinical and radiographic evidence of orbital fractures and repair performed within 48 h after injury. Surgical complications and reconstructive outcomes were assessed over 6 months. The main outcome measures were enophthalmos, pre- and post-CT imaging of orbits, scar evaluation, presence of diplopia, and eyelid position. Enophthalmos was corrected in 16/19 cases and improved in 3/19 cases. 3D reconstruction of CT images showed markedly improved orbital alignment with objective measurements of the optic foramen to cornea distance (mm) in reconstructed orbits relative to intact orbits of 0.66, 95% confidence interval [CI] (lower 0.33, upper 0.99) mm. The mean baseline of Stony Brook Scar Evaluation Scale was 0.6, 95%CI (0.30-0.92), and for 6 months, the mean score was 3.4, 95%CI (3.05-3.73). Residual diplopia in secondary gazes was present in two patients; one patient had ectropion. Complications included one case of local wound infection. An early single-stage repair of combined soft tissue and orbital fractures yields satisfactory functional and aesthetic outcomes. Complications are low and likely related to trauma severity. Copyright © 2016 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Multimodal autofluorescence detection of cancer: from single cells to living organism

NASA Astrophysics Data System (ADS)

Horilova, J.; Cunderlikova, B.; Cagalinec, M.; Chorvat, D.; Marcek Chorvatova, A.

2018-02-01

Multimodal optical imaging of suspected tissues is showing to be a promising method for distinguishing suspected cancerous tissues from healthy ones. In particular, the combination of steady-state spectroscopic methods with timeresolved fluorescence provides more precise insight into native metabolism when focused on tissue autofluorescence. Cancer is linked to specific metabolic remodelation detectable spectroscopically. In this work, we evaluate possibilities and limitations of multimodal optical cancer detection in single cells, collagen-based 3D cell cultures and in living organisms (whole mice), as a representation of gradually increasing complexity of model systems.

[Report on notifications according to section 8d of the German Transplantation Act (TPG) for the years 2009-2011].

PubMed

Schilling-Leiß, D; Witt, F; Tönjes, R R

2014-01-01

In Germany, the Tissue Act came into effect on 1 August 2007. Since then, every tissue establishment is legally obligated to keep a record of its activities according to section 8d subsection 3 of the Transplantation Act (TPG). An annual report must be submitted to the Paul Ehrlich Institute once a year up to 1 March of the subsequent year. The report should include the types and quantities of tissues procured, conditioned, processed, stored, distributed or otherwise disposed of, imported, and exported. The report should be made on a TPG-based notification form published on the Internet by the Paul Ehrlich Institute. The present report according to section 8d subsection 3 of the TPG is based on data of the reporting years 2009-2011. Six years after implementation of the TPG's reporting obligation for tissue establishments, the number of tissue establishments known by the Paul Ehrlich Institute has increased from 349 in 2007 to 949 in 2011. In the course of continuous optimization of the notification forms, including tissue-specific glossaries, the reported data of most of the tissues and tissue preparations have become more conclusive.

Therapeutic Applications of PARP Inhibitors: Anticancer Therapy and Beyond

PubMed Central

Curtin, Nicola; Szabo, Csaba

2013-01-01

The aim of this article is to describe the current and potential clinical translation of pharmacological inhibitors of poly(ADP-ribose) polymerase (PARP) for the therapy of various diseases. The first section of the present review summarizes the available preclinical and clinical data with PARP inhibitors in various forms of cancer. In this context, the role of PARP in single-strand DNA break repair is relevant, leading to replication-associated lesions that cannot be repaired if homologous recombination (HRR) repair is defective, and the synthetic lethality of PARP inhibitors in HRR-defective cancer. HRR defects are classically associated with BRCA1 and 2 mutations associated with familial breast and ovarian cancer, but there may be many other causes of HRR defects. Thus, PARP inhibitors may be the drugs of choice for BRCA mutant breast and ovarian cancers, and extend beyond these tumors if appropriate biomarkers can be developed to identify HRR defects. Multiple lines of preclinical data demonstrate that PARP inhibition increases cytotoxicity and tumor growth delay in combination with temozolomide, topoisomerase inhibitors and ionizing radiation. Both single agent and combination clinical trials are underway. The final part of the first section of the present review summarizes the current status of the various PARP inhibitors that are in various stages of clinical development. The second section of the present review summarizes the role of PARP in selected non-oncologic indications. In a number of severe, acute diseases (such as stroke, neurotrauma, circulatory shock and acute myocardial infarction) the clinical translatability of PARP inhibition is supported by multiple lines of preclinical data, as well as observational data demonstrating PARP activation in human tissue samples. In these disease indications, PARP overactivation due to oxidative and nitrative stress drives cell necrosis and pro-inflammatory gene expression, which contributes to disease pathology. Accordingly, multiple lines of preclinical data indicate the efficacy of PARP inhibitors to preserve viable tissue and to down-regulate inflammatory responses. As the clinical trials with PARP inhibitors in various forms of cancer progress, it is hoped that a second line of clinical investigations, aimed at testing of PARP inhibitors for various non-oncologic indications, will be initiated, as well. PMID:23370117

Multi-class biological tissue classification based on a multi-classifier: Preliminary study of an automatic output power control for ultrasonic surgical units.

PubMed

Youn, Su Hyun; Sim, Taeyong; Choi, Ahnryul; Song, Jinsung; Shin, Ki Young; Lee, Il Kwon; Heo, Hyun Mu; Lee, Daeweon; Mun, Joung Hwan

2015-06-01

Ultrasonic surgical units (USUs) have the advantage of minimizing tissue damage during surgeries that require tissue dissection by reducing problems such as coagulation and unwanted carbonization, but the disadvantage of requiring manual adjustment of power output according to the target tissue. In order to overcome this limitation, it is necessary to determine the properties of in vivo tissues automatically. We propose a multi-classifier that can accurately classify tissues based on the unique impedance of each tissue. For this purpose, a multi-classifier was built based on single classifiers with high classification rates, and the classification accuracy of the proposed model was compared with that of single classifiers for various electrode types (Type-I: 6 mm invasive; Type-II: 3 mm invasive; Type-III: surface). The sensitivity and positive predictive value (PPV) of the multi-classifier by cross checks were determined. According to the 10-fold cross validation results, the classification accuracy of the proposed model was significantly higher (p<0.05 or <0.01) than that of existing single classifiers for all electrode types. In particular, the classification accuracy of the proposed model was highest when the 3mm invasive electrode (Type-II) was used (sensitivity=97.33-100.00%; PPV=96.71-100.00%). The results of this study are an important contribution to achieving automatic optimal output power adjustment of USUs according to the properties of individual tissues. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas.

PubMed

Haugg, Anke M; Rennspiess, Dorit; zur Hausen, Axel; Speel, Ernst-Jan M; Cathomas, Gieri; Becker, Jürgen C; Schrama, David

2014-12-15

The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumor-specific mutations in the large T antigen are strong arguments that MCPyV is a human tumor virus. However, the relationship between viral presence and cancer induction remains discussed controversially. Since almost all studies on virus prevalence are based on PCR techniques, we performed MCPyV fluorescence in situ hybridization (FISH) on MCC to gain information about the quality of the viral presence on the single cell level. MCPyV-FISH was performed on tissue microarrays containing 62 formalin-fixed and paraffin-embedded tissue samples including all tumor grades of 42 patients. The hybridization patterns were correlated to the qPCR data determined on corresponding whole tissue sections. Indeed, MCPyV-FISH and qPCR data were highly correlated, i.e. 83% for FISH-positive and 93% for FISH-negative cores. Accordingly, the mean of the qPCR values of all MCPyV-positive cores differed significantly from the mean of the negative cores (p = 0.0076). Importantly, two hybridization patterns were definable in the MCPyV-FISH: a punctate pattern (85%) indicating viral integration, which correlated with a moderate viral abundance and a combination of the punctate with a diffuse pattern (15%), suggesting a possible coexistence of integrated and episomal virus which was associated with very high viral load and VP1 expression. Thus, MCPyV-FISH adds important information on the single cell level within the histomorphological context and could therefore be an important tool to further elucidate MCPyV related carcinogenesis. © 2014 UICC.

Spatially coordinated dynamic gene transcription in living pituitary tissue

PubMed Central

Featherstone, Karen; Hey, Kirsty; Momiji, Hiroshi; McNamara, Anne V; Patist, Amanda L; Woodburn, Joanna; Spiller, David G; Christian, Helen C; McNeilly, Alan S; Mullins, John J; Finkenstädt, Bärbel F; Rand, David A; White, Michael RH; Davis, Julian RE

2016-01-01

Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary ‘on-off’ process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour. DOI: http://dx.doi.org/10.7554/eLife.08494.001 PMID:26828110

Molecular testing of Klebsiella pneumoniae contaminating tissue allografts recovered from deceased donors.

PubMed

Ghalavand, Zohreh; Heidary Rouchi, Alireza; Bahraminasab, Hassan; Ravanasa, Elham; Mirsamadi, Elnaz Sadat; Nodeh Farahani, Narges; Nikmanesh, Bahram

2018-02-03

Microbiological screening of tissue allografts is crucial to prevent the transmission of bacterial and fungal infections to transplant recipients. Klebsiella was the most prevalent and resistant contaminating microorganism observed in our setting in the Iranian Tissue Bank. This study was conducted to determine the presence of extended-spectrum β-lactamase (ESBL) genes, antimicrobial resistance patterns of Klebsiella pneumoniae isolates, and their clonal relationships in allograft materials. K. pneumoniae contaminating bone and other tissue allografts recovered from deceased donors were identified and ESBL isolates were detected using a phenotypic confirmatory method. Antimicrobial susceptibility testing was carried out using the disk diffusion method. Distribution of ESBL genes and molecular typing were performed using polymerase chain reaction (PCR) and Repetitive-element (rep-PCR) methods. Of 3828 donated tissues, 51 (1.3%) were found contaminated by K. pneumoniae isolates. Compared to tissue allografts from brain-dead, heart-beating tissue donors, allografts from donors with circulatory cessation were associated with a higher risk of K. pneumoniae contamination [odds ratio (OR), 1.2 (CI 95% 0.9-2.3) (P value < 0.001)]. Half of the isolates produced ESBL, and the rate of susceptibility to cephalosporins was 51%. Among isolates, 22 (43.1%) harbored CTX-M, 31 (60.8%) SHV, and 9 (17.6%) harbored TEM types. The rep-dendrogram indicated that clones having identical or related strains with a similar antibiotype were isolated in the same period. This study provides evidence that a single clone of K. pneumoniae contaminated tissue allografts recovered from many different donors. A single clone found on tissues from several donors suggests contamination of tissues from a single source such as the tissue recovery process and environment. Genomic DNA testing and clonality of contaminating bacteria using molecular methods can focus the epidemiologic investigation on the tissue allograft recovery process including a search for contamination of the tissue recovery room environment, recovery staff, recovery equipment, reagents, solutions and supplies.

Selection and validation of suitable reference genes for miRNA expression normalization by quantitative RT-PCR in citrus somatic embryogenic and adult tissues.

PubMed

Kou, Shu-Jun; Wu, Xiao-Meng; Liu, Zheng; Liu, Yuan-Long; Xu, Qiang; Guo, Wen-Wu

2012-12-01

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30 °C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14 + U6 for SE tissues and snoR14 + U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants. Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14 + U6, snoR14 + U5 were identified.

Metabolite mapping by consecutive nanostructure and silver-assisted mass spectrometry imaging on tissue sections.

PubMed

Gustafsson, O J R; Guinan, T M; Rudd, D; Kobus, H; Benkendorff, K; Voelcker, N H

2017-06-30

Nanostructure-based mass spectrometry imaging (MSI) is a promising technology for molecular imaging of small molecules, without the complex chemical background typically encountered in matrix-assisted molecular imaging approaches. Here, we have enhanced these surfaces with silver (Ag) to provide a second tier of MSI data from a single sample. MSI data was acquired through the application of laser desorption/ionization mass spectrometry to biological samples imprinted onto desorption/ionization on silicon (DIOS) substrates. Following initial analysis, ultra-thin Ag layers were overlaid onto the followed by MSI analysis (Ag-DIOS MSI). This approach was first demonstrated for fingermark small molecules including environmental contaminants and sebum components. Subsequently, this bimodal method was translated to lipids and metabolites in fore-stomach sections from a 6-bromoisatin chemopreventative murine mouse model. DIOS MSI allowed mapping of common ions in fingermarks as well as 6-bromoisatin metabolites and lipids in murine fore-stomach. Furthermore, DIOS MSI was complemented by the Ag-DIOS MSI of Ag-adductable lipids such as wax esters in fingermarks and cholesterol in murine fore-stomach. Gastrointestinal acid condensation products of 6-bromoisatin, such as the 6,6'-dibromoindirubin mapped herein, are very challenging to isolate and characterize. By re-analyzing the same tissue imprints, this metabolite was readily detected by DIOS, placed in a tissue-specific spatial context, and subsequently overlaid with additional lipid distributions acquired using Ag-DIOS MSI. The ability to place metabolite and lipid classes in a tissue-specific context makes this novel method suited to MSI analyses where the collection of additional information from the same sample maximises resource use, and also maximises the number of annotated small molecules, in particular for metabolites that are typically undetectable with traditional platforms. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Probability of cavitation for single ultrasound pulses applied to tissues and tissue-mimicking materials

PubMed Central

Maxwell, Adam D.; Cain, Charles A.; Hall, Timothy L.; Fowlkes, J. Brian; Xu, Zhen

2012-01-01

In this article, the negative pressure values at which inertial cavitation consistently occurs in response to a single, 2-cycle, focused ultrasound pulse were measured in several media relevant to cavitation-based ultrasound therapy. The pulse was focused into a chamber containing one of the media, which included liquids, tissue-mimicking materials, and ex-vivo canine tissue. Focal waveforms were measured by two separate techniques using a fiber-optic hydrophone. Inertial cavitation was identified by high-speed photography in optically transparent media and an acoustic passive cavitation detector. The probability of cavitation (Pcav) for a single pulse as a function of peak negative pressure (p−) followed a sigmoid curve, with the probability approaching 1 when the pressure amplitude was sufficient. The statistical threshold (defined as Pcav = 0.5) was between p− = 26.0–30.0 MPa in all samples with a high water content, but varied between p− = 13.7 to > 36 MPa for other media. A model for radial cavitation bubble dynamics was employed to evaluate the behavior of cavitation nuclei at these pressure levels. A single bubble nucleus with an inertial cavitation threshold of p− = 28.2 MPa was estimated to have a 2.5 nm radius in distilled water. These data may be valuable for cavitation-based ultrasound therapy to predict the likelihood of cavitation at different pressure levels and dimensions of cavitation-induced lesions in tissue. PMID:23380152

Single-strand conformation polymorphism analysis of ribosomal DNA for detection of Phytophthora ramorum directly from plant tissues

Treesearch

Ping Kong; Patricia A. Richardson; Chuanxue Hong; Thomas L. Kubisiak

2006-01-01

At the first Sudden Oak Death Science Symposium, we reported on the use of a single strand conformation polymorphism (SSCP) analysis for rapid identification of Phytophthora ramorum in culture. We have since assessed and improved the fingerprinting technique for detecting this pathogen directly from plant tissues. The improved SSCP protocol uses a...

Rapid ultrasonic stimulation of inflamed tissue with diagnostic intent

PubMed Central

McClintic, Abbi M.; Dickey, Trevor C.; Gofeld, Michael; Ray Illian, P.; Kliot, Michel; Kucewicz, John C.; Loeser, John D.; Richebe, Philippe G.; Mourad, Pierre D.

2013-01-01

Previous studies have observed that individual pulses of intense focused ultrasound (iFU) applied to inflamed and normal tissue can generate sensations, where inflamed tissue responds at a lower intensity than normal tissue. It was hypothesized that successively applied iFU pulses will generate sensation in inflamed tissue at a lower intensity and dose than application of a single iFU pulse. This hypothesis was tested using an animal model of chronic inflammatory pain, created by injecting an irritant into the rat hind paw. Ultrasound pulses were applied in rapid succession or individually to rats' rear paws beginning at low peak intensities and progressing to higher peak intensities, until the rats withdrew their paws immediately after iFU application. Focused ultrasound protocols consisting of successively and rapidly applied pulses elicited inflamed paw withdrawal at lower intensity and estimated tissue displacement values than single pulse protocols. However, both successively applied pulses and single pulses produced comparable threshold acoustic dose values and estimates of temperature increases. This raises the possibility that temperature increase contributed to paw withdrawal after rapid iFU stimulation. While iFU-induction of temporal summation may also play a role, electrophysiological studies are necessary to tease out these potential contributors to iFU stimulation. PMID:23927192

Collagen fibre characterisation in arterial tissue under load using SALS.

PubMed

Gaul, R T; Nolan, D R; Lally, C

2017-11-01

The collagen fibre architecture of arterial tissue is known to play a key role in its resultant mechanical behaviour, while maladaptive remodelling of this architecture may be linked to disease. Many of the techniques currently used to analyse collagen fibre architecture require time consuming tissue preparation procedures and are destructive in nature. The aim of this study is to fully explore Small Angle Light Scattering (SALS) as a means to non-destructively assess collagen fibre architecture in arterial tissue and subsequently gain insights into load induced reorientation. The optimised configuration of the SALS system for arterial tissue was determined using quantitative comparisons to histological analyses of porcine carotid artery as its basis. Once established, layer specific fibre orientation and the influence of tissue loading was determined for thin sections of carotid artery using SALS. This process was subsequently repeated for intact carotid artery layers. A single family of circumferentially orientated collagen fibres were found in the intima (- 0.1 ± 1.4° (5.5°)) and media (- 1.7 ± 1.9° (4.7°)) while two perpendicular families of fibres were identified in the adventitia (- 6.4 ± 0.7° (37.7°)) and (118.3 ± 2.7 (39.9°)). An increase in fibre alignment in response to a 20% circumferential strain was also identified using SALS, characterised by an increase in scattered light eccentricity. determined using SALS agreed with those found using traditional destructive techniques, however SALS has the important benefits of allowing vessel layers to remain intact, and has a fast processing time. SALS unique ability to identify load induced reorganisation in intact arterial layers offers an efficient means to gain crucial insights into arterial disease and its development over time. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of Low-Cost Instrumentation for Single Point Autofluorescence Lifetime Measurements.

PubMed

Lagarto, João; Hares, Jonathan D; Dunsby, Christopher; French, Paul M W

2017-09-01

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a "gold-standard" instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.

A novel method for single sample multi-axial nanoindentation of hydrated heterogeneous tissues based on testing great white shark jaws.

PubMed

Ferrara, Toni L; Boughton, Philip; Slavich, Eve; Wroe, Stephen

2013-01-01

Nanomechanical testing methods that are suitable for a range of hydrated tissues are crucial for understanding biological systems. Nanoindentation of tissues can provide valuable insights into biology, tissue engineering and biomimetic design. However, testing hydrated biological samples still remains a significant challenge. Shark jaw cartilage is an ideal substrate for developing a method to test hydrated tissues because it is a unique heterogeneous composite of both mineralized (hard) and non-mineralized (soft) layers and possesses a jaw geometry that is challenging to test mechanically. The aim of this study is to develop a novel method for obtaining multidirectional nanomechanical properties for both layers of jaw cartilage from a single sample, taken from the great white shark (Carcharodon carcharias). A method for obtaining multidirectional data from a single sample is necessary for examining tissue mechanics in this shark because it is a protected species and hence samples may be difficult to obtain. Results show that this method maintains hydration of samples that would otherwise rapidly dehydrate. Our study is the first analysis of nanomechanical properties of great white shark jaw cartilage. Variation in nanomechanical properties were detected in different orthogonal directions for both layers of jaw cartilage in this species. The data further suggest that the mineralized layer of shark jaw cartilage is less stiff than previously posited. Our method allows multidirectional nanomechanical properties to be obtained from a single, small, hydrated heterogeneous sample. Our technique is therefore suitable for use when specimens are rare, valuable or limited in quantity, such as samples obtained from endangered species or pathological tissues. We also outline a method for tip-to-optic calibration that facilitates nanoindentation of soft biological tissues. Our technique may help address the critical need for a nanomechanical testing method that is applicable to a variety of hydrated biological materials whether soft or hard.

Single-stage interpolation flaps in facial reconstruction.

PubMed

Hollmig, S Tyler; Leach, Brian C; Cook, Joel

2014-09-01

Relatively deep and complex surgical defects, particularly when adjacent to or involving free margins, present significant reconstructive challenges. When the use of local flaps is precluded by native anatomic restrictions, interpolation flaps may be modified to address these difficult wounds in a single operative session. To provide a framework to approach difficult soft tissue defects arising near or involving free margins and to demonstrate appropriate design and execution of single-stage interpolation flaps for reconstruction of these wounds. Examination of our utilization of these flaps based on an anatomic region and surgical approach. A region-based demonstration of flap conceptualization, design, and execution is provided. Tunneled, transposed, and deepithelialized variations of single-stage interpolation flaps provide versatile options for reconstruction of a variety of defects encroaching on or involving free margins. The inherently robust vascularity of these flaps supports importation of necessary tissue bulk while allowing aggressive contouring to restore an intricate native topography. Critical flap design allows access to distant tissue reservoirs and placement of favorable incision lines while preserving the inherent advantages of a single operative procedure.

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.

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.

Single dose filgastrim in cytotoxic-induced neutropaenia in children.

PubMed

Abdallah, F K

2008-01-01

To document the impact of fixed dose weight adjusted filgastrim (G-CSF) in cytotoxic-induced neutropaenia. A descriptive cross-sectional study. Paediatric Oncology Unit at Kenyatta National Hospital, Nairobi, Kenya. All paediatric oncology patients who had developed cytotoxic-induced neutropaenia. The following were documented for every tissue proven case of malignancy; age, sex, type of malignancy, treatment regimen and schedule, initial blood count at the time of neutropaenia; subsequent blood counts daily for five days from day one of single dose filgastrim, and the calculated neutrophil incremental count. Initially eight patients with solid tumours previously treated with filgastrim revealed that cytotoxic induced neutropaenia could be ameliorated by a single dose of filgastrim. Subsequently, the study listed thirty patients. This cohort consisted of; 37% rhabdomyosarcoma, 30% Burkitts, 27% acute lymphoblastic leukaemia and 6% Hodgkin's lymphoma. Increased neutrophil count after 48 hours was documented in 26 (87%) patients, with absolute neutrophil counts range of 0.5 to 31.5 x 10(9)/L. This response was significantly influenced by gender (p>0.0001), malignancy type and chemotherapy regimen (p>0.001). The study shows that chemotherapy induced neutropaenia can be alleviated by a single dose of filgastrim without adverse effects on lymphoblastic leukaemia. This study suggests that a single dose of filgastrim should be first tried in cytotoxic induced neutropaenia in the paediatric age group.

MICROSCOPIC DETERMINATION OF BONE PHOSPHORUS BY QUANTITATIVE AUTORADIOGRAPHY OF NEUTRON-ACTIVATED SECTIONS

PubMed Central

Vincent, Jacques; Haumont, Stanislas; Roels, Joseph

1965-01-01

Longitudinal sections of human cortical bone were submitted to thermal neutrons. γ-ray spectra were recorded repeatedly during 15 days following irradiation. They showed that Na24 is predominant as early as 3 hours after activation and that all the γ-emitters have decayed on the 15th day. When the γ-rays have disappeared, β-rays are still produced by the sections. It was proved by the absorption curve in aluminium that all these β-rays are issued from the P32 induced in the sections by activation of P31. Therefore autoradiograms registered 15 days after activation reveal the distribution of P32 in the sections. γ-ray spectra and β-ray absorption curves of neutron activated sections of ivory demonstrated a mineral composition similar to that of bone. Autoradiograms of ivory sections activated for various times were used to establish the relation between the optical density of the autoradiograms and the radioactivity in P32. When the bone autoradiograms are compared with the ivory standards of known radioactivity, the optical densities of single osteons (Haversian systems), can be related to their phosphorus contents. Autoradiograms and microradiograms of the same sections were examined side by side. The least calcified osteons, that contain 80 per cent of the calcium of the fully calcified osteons, also contain about 80 per cent of the phosphorus of the fully mineralized osteons. It is concluded that the Ca:P ratio remains constant while mineralization of bone tissue is being completed. PMID:14286295

Investigation of optimal method for inducing harmonic motion in tissue using a linear ultrasound phased array--a simulation study.

PubMed

Heikkilä, Janne; Hynynen, Kullervo

2006-04-01

Many noninvasive ultrasound techniques have been developed to explore mechanical properties of soft tissues. One of these methods, Localized Harmonic Motion Imaging (LHMI), has been proposed to be used for ultrasound surgery monitoring. In LHMI, dynamic ultrasound radiation-force stimulation induces displacements in a target that can be measured using pulse-echo imaging and used to estimate the elastic properties of the target. In this initial, simulation study, the use of a one-dimensional phased array is explored for the induction of the tissue motion. The study compares three different dual-frequency and amplitude-modulated single-frequency methods for the inducing tissue motion. Simulations were computed in a homogeneous soft-tissue volume. The Rayleigh integral was used in the simulations of the ultrasound fields and the tissue displacements were computed using a finite-element method (FEM). The simulations showed that amplitude-modulated sonication using a single frequency produced the largest vibration amplitude of the target tissue. These simulations demonstrate that the properties of the tissue motion are highly dependent on the sonication method and that it is important to consider the full three-dimensional distribution of the ultrasound field for controlling the induction of tissue motion.

Biosafety of Non-Surface Modified Carbon Nanocapsules as a Potential Alternative to Carbon Nanotubes for Drug Delivery Purposes

PubMed Central

Tang, Alan C. L.; Hwang, Gan-Lin; Chang, Min-Yao; Tang, Zack C. W.; Tsai, Meng-Da; Luo, Chwan-Yao; Hoffman, Allan S.; Hsieh, Patrick C. H.

2012-01-01

Background Carbon nanotubes (CNTs) have found wide success in circuitry, photovoltaics, and other applications. In contrast, several hurdles exist in using CNTs towards applications in drug delivery. Raw, non-modified CNTs are widely known for their toxicity. As such, many have attempted to reduce CNT toxicity for intravenous drug delivery purposes by post-process surface modification. Alternatively, a novel sphere-like carbon nanocapsule (CNC) developed by the arc-discharge method holds similar electric and thermal conductivities, as well as high strength. This study investigated the systemic toxicity and biocompatibility of different non-surface modified carbon nanomaterials in mice, including multi-walled carbon nanotubes (MWCNTs), single-walled carbon nanotubes (SWCNTs), carbon nanocapsules (CNCs), and C60 fullerene (C60). The retention of the nanomaterials and systemic effects after intravenous injections were studied. Methodology and Principal Findings MWCNTs, SWCNTs, CNCs, and C60 were injected intravenously into FVB mice and then sacrificed for tissue section examination. Inflammatory cytokine levels were evaluated with ELISA. Mice receiving injection of MWCNTs or SWCNTs at 50 µg/g b.w. died while C60 injected group survived at a 50% rate. Surprisingly, mortality rate of mice injected with CNCs was only at 10%. Tissue sections revealed that most carbon nanomaterials retained in the lung. Furthermore, serum and lung-tissue cytokine levels did not reveal any inflammatory response compared to those in mice receiving normal saline injection. Conclusion Carbon nanocapsules are more biocompatible than other carbon nanomaterials and are more suitable for intravenous drug delivery. These results indicate potential biomedical use of non-surface modified carbon allotrope. Additionally, functionalization of the carbon nanocapsules could further enhance dispersion and biocompatibility for intravenous injection. PMID:22457723

Development and characterization of a handheld hyperspectral Raman imaging probe system for molecular characterization of tissue on mesoscopic scales.

PubMed

St-Arnaud, Karl; Aubertin, Kelly; Strupler, Mathias; Madore, Wendy-Julie; Grosset, Andrée-Anne; Petrecca, Kevin; Trudel, Dominique; Leblond, Frédéric

2018-01-01

Raman spectroscopy is a promising cancer detection technique for surgical guidance applications. It can provide quantitative information relating to global tissue properties associated with structural, metabolic, immunological, and genetic biochemical phenomena in terms of molecular species including amino acids, lipids, proteins, and nucleic acid (DNA). To date in vivo Raman spectroscopy systems mostly included probes and biopsy needles typically limited to single-point tissue interrogation over a scale between 100 and 500 microns. The development of wider field handheld systems could improve tumor localization for a range of open surgery applications including brain, ovarian, and skin cancers. Here we present a novel Raman spectroscopy implementation using a coherent imaging bundle of fibers to create a probe capable of reconstructing molecular images over mesoscopic fields of view. Detection is performed using linear scanning with a rotation mirror and an imaging spectrometer. Different slits widths were tested at the entrance of the spectrometer to optimize spatial and spectral resolution while preserving sufficient signal-to-noise ratios to detect the principal Raman tissue features. The nonbiological samples, calcite and polytetrafluoroethylene (PTFE), were used to characterize the performance of the system. The new wide-field probe was tested on ex vivo samples of calf brain and swine tissue. Raman spectral content of both tissue types were validated with data from the literature and compared with data acquired with a single-point Raman spectroscopy probe. The single-point probe was used as the gold standard against which the new instrument was benchmarked as it has already been thoroughly validated for biological tissue characterization. We have developed and characterized a practical noncontact handheld Raman imager providing tissue information at a spatial resolution of 115 microns over a field of view >14 mm 2 and a spectral resolution of 6 cm -1 over the whole fingerprint region. Typical integration time to acquire an entire Raman image over swine tissue was set to approximately 100 s. Spectra acquired with both probes (single-point and wide-field) showed good agreement, with a Pearson correlation factor >0.85 over different tissue categories. Protein and lipid content of imaged tissue were manifested into the measured spectra which correlated well with previous findings in the literature. An example of quantitative molecular map is presented for swine tissue and calf brain based on the ratio of protein-to-lipid content showing clear delineations between white and gray matter as well as between adipose and muscle tissue. We presented the development of a Raman imaging probe with a field of view of a few millimeters and a spatial resolution consistent with standard surgical imaging methods using an imaging bundle. Spectra acquired with the newly developed system on swine tissue and calf brain correlated well with an establish single-point probe and observed spectral features agreed with previous finding in the literature. The imaging probe has demonstrated its ability to reconstruct molecular images of soft tissues. The approach presented here has a lot of potential for the development of surgical Raman imaging probe to guide the surgeon during cancer surgery. © 2017 American Association of Physicists in Medicine.

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.

Effect of Antigen Retrieval Methods on Nonspecific Binding of Antibody-Metal Nanoparticle Conjugates on Formalin-Fixed Paraffin-Embedded Tissue.

PubMed

Zhang, Yuying; Wang, Xin-Ping; Perner, Sven; Bankfalvi, Agnes; Schlücker, Sebastian

2018-01-02

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissues provides important diagnostic and prognostic information in pathology. Metal nanoparticles (NPs) and, in particular, surface-enhanced Raman scattering (SERS) nanotags as a new class of labeling reagents are promising to be used for multiplexed protein profiling on tissue sections. However, nonspecific binding of NPs onto the tissue specimens greatly hampers their clinical applications. In this study, we found that the antigen retrieval method strongly influences the extent of nonspecific binding of the antibody-SERS NP conjugates to the tissue. Our SERS labels comprised ca. 70 nm Au nanostars coated with ethylene glycol-modified Raman reporter molecules for hydrophilic stabilization and subsequent covalent bioconjugation to antibodies. We systematically investigated the influence of heat- and protease-induced epitope retrieval (HIER and PIER, respectively) on the immunostaining quality of prostate-specific antigen (PSA) on human prostate tissue sections. The best staining results were obtained with PIER. Pretreatment of the tissue sections by HIER led to selective but nonspecific adsorption of the antibody-Au nanostar conjugates onto epithelial cells, while enzymatic treatment within PIER did not. In addition to gold nanostars, also other types of metal NPs with different shapes and sizes (including ca. 20 nm quasi-spherical Au NPs and ca. 60 nm quasi-spherical Au/Ag nanoshells) as well as tissue sections from different organs (including prostate and breast) were tested; in each case the same tendency was observed, i.e., PIER yielded better results than HIER. Therefore, we recommend PIER for future NP-based tissue immunostaining such as immuno-SERS microscopy. Alternatively, for antigens that can only be unmasked by heating, PEGylation of the NPs is recommended to avoid nonspecific binding.

Mesh electronics: a new paradigm for tissue-like brain probes.

PubMed

Hong, Guosong; Yang, Xiao; Zhou, Tao; Lieber, Charles M

2018-06-01

Existing implantable neurotechnologies for understanding the brain and treating neurological diseases have intrinsic properties that have limited their capability to achieve chronically-stable brain interfaces with single-neuron spatiotemporal resolution. These limitations reflect what has been dichotomy between the structure and mechanical properties of living brain tissue and non-living neural probes. To bridge the gap between neural and electronic networks, we have introduced the new concept of mesh electronics probes designed with structural and mechanical properties such that the implant begins to 'look and behave' like neural tissue. Syringe-implanted mesh electronics have led to the realization of probes that are neuro-attractive and free of the chronic immune response, as well as capable of stable long-term mapping and modulation of brain activity at the single-neuron level. This review provides a historical overview of a 10-year development of mesh electronics by highlighting the tissue-like design, syringe-assisted delivery, seamless neural tissue integration, and single-neuron level chronic recording stability of mesh electronics. We also offer insights on unique near-term opportunities and future directions for neuroscience and neurology that now are available or expected for mesh electronics neurotechnologies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Tissue engineering: state of the art in oral rehabilitation

PubMed Central

SCHELLER, E. L.; KREBSBACH, P. H.; KOHN, D. H.

2009-01-01

SUMMARY More than 85% of the global population requires repair or replacement of a craniofacial structure. These defects range from simple tooth decay to radical oncologic craniofacial resection. Regeneration of oral and craniofacial tissues presents a formidable challenge that requires synthesis of basic science, clinical science and engineering technology. Identification of appropriate scaffolds, cell sources and spatial and temporal signals (the tissue engineering triad) is necessary to optimize development of a single tissue, hybrid organ or interface. Furthermore, combining the understanding of the interactions between molecules of the extracellular matrix and attached cells with an understanding of the gene expression needed to induce differentiation and tissue growth will provide the design basis for translating basic science into rationally developed components of this tissue engineering triad. Dental tissue engineers are interested in regeneration of teeth, oral mucosa, salivary glands, bone and periodontium. Many of these oral structures are hybrid tissues. For example, engineering the periodontium requires growth of alveolar bone, cementum and the periodontal ligament. Recapitulation of biological development of hybrid tissues and interfaces presents a challenge that exceeds that of engineering just a single tissue. Advances made in dental interface engineering will allow these tissues to serve as model systems for engineering other tissues or organs of the body. This review will begin by covering basic tissue engineering principles and strategic design of functional biomaterials. We will then explore the impact of biomaterials design on the status of craniofacial tissue engineering and current challenges and opportunities in dental tissue engineering. PMID:19228277

Tissue engineering: state of the art in oral rehabilitation.

PubMed

Scheller, E L; Krebsbach, P H; Kohn, D H

2009-05-01

More than 85% of the global population requires repair or replacement of a craniofacial structure. These defects range from simple tooth decay to radical oncologic craniofacial resection. Regeneration of oral and craniofacial tissues presents a formidable challenge that requires synthesis of basic science, clinical science and engineering technology. Identification of appropriate scaffolds, cell sources and spatial and temporal signals (the tissue engineering triad) is necessary to optimize development of a single tissue, hybrid organ or interface. Furthermore, combining the understanding of the interactions between molecules of the extracellular matrix and attached cells with an understanding of the gene expression needed to induce differentiation and tissue growth will provide the design basis for translating basic science into rationally developed components of this tissue engineering triad. Dental tissue engineers are interested in regeneration of teeth, oral mucosa, salivary glands, bone and periodontium. Many of these oral structures are hybrid tissues. For example, engineering the periodontium requires growth of alveolar bone, cementum and the periodontal ligament. Recapitulation of biological development of hybrid tissues and interfaces presents a challenge that exceeds that of engineering just a single tissue. Advances made in dental interface engineering will allow these tissues to serve as model systems for engineering other tissues or organs of the body. This review will begin by covering basic tissue engineering principles and strategic design of functional biomaterials. We will then explore the impact of biomaterials design on the status of craniofacial tissue engineering and current challenges and opportunities in dental tissue engineering.

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.

Multiplex coherent anti-Stokes Raman scattering microspectroscopy of brain tissue with higher ranking data classification for biomedical imaging

NASA Astrophysics Data System (ADS)

Pohling, Christoph; Bocklitz, Thomas; Duarte, Alex S.; Emmanuello, Cinzia; Ishikawa, Mariana S.; Dietzeck, Benjamin; Buckup, Tiago; Uckermann, Ortrud; Schackert, Gabriele; Kirsch, Matthias; Schmitt, Michael; Popp, Jürgen; Motzkus, Marcus

2017-06-01

Multiplex coherent anti-Stokes Raman scattering (MCARS) microscopy was carried out to map a solid tumor in mouse brain tissue. The border between normal and tumor tissue was visualized using support vector machines (SVM) as a higher ranking type of data classification. Training data were collected separately in both tissue types, and the image contrast is based on class affiliation of the single spectra. Color coding in the image generated by SVM is then related to pathological information instead of single spectral intensities or spectral differences within the data set. The results show good agreement with the H&E stained reference and spontaneous Raman microscopy, proving the validity of the MCARS approach in combination with SVM.

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

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.

Fetal Tendinous Connection Between the Tensor Tympani and Tensor Veli Palatini Muscles: A Single Digastric Muscle Acting for Morphogenesis of the Cranial Base.

PubMed

Rodríguez-Vázquez, José Francisco; Sakiyama, Koji; Abe, Hiroshi; Amano, Osamu; Murakami, Gen

2016-04-01

Some researchers contend that in adults the tensor tympani muscle (TT) connects with the tensor veli palatini muscle (TVP) by an intermediate tendon, in disagreement with the other researchers. To resolve this controversy, we examined serial sections of 50 human embryos and fetuses at 6-17 weeks of development. At 6 weeks, in the first pharyngeal arch, a mesenchymal connection was found first to divide a single anlage into the TT and TVP. At and after 7 weeks, the TT was connected continuously with the TVP by a definite tendinous tissue mediolaterally crossing the pharyngotympanic tube. At 11 weeks another fascia was visible covering the cranial and lateral sides of the tube. This "gonial fascia" had two thickened borders: the superior one corresponded to a part of the connecting tendon between the TT and TVP; the inferior one was a fibrous band ending at the os goniale near the lateral end of the TVP. In association with the gonial fascia, the fetal TT and TVP seemed to provide a functional complex. The TT-TVP complex might first help elevate the palatal shelves in association with the developing tongue. Next, the tubal passage, maintained by contraction of the muscle complex, seems to facilitate the removal of loose mesenchymal tissues from the tympanic cavity. Third, the muscle complex most likely determined the final morphology of the pterygoid process. Consequently, despite the controversial morphologies in adults, the TT and TVP seemed to make a single digastric muscle acting for the morphogenesis of the cranial base. © 2016 Wiley Periodicals, Inc.

Soft tissue topography and dimensions lateral to single implant-supported restorations. a cross-sectional study.

PubMed

Chang, Moontaek; Wennström, Jan L

2013-05-01

The aim was to evaluate potential relationships between the implant position relative to adjacent teeth and dimensions and topography of the papillae lateral to implant-supported single-tooth restorations. A total of 32 subjects with a single implant-supported restoration in the esthetic zone of the maxilla were consecutively selected for the study. Soft and hard tissues at the proximal sites of the restoration were evaluated by the use of clinical, photographic, diagnostic cast, and radiographic assessments. A questionnaire was used for assessment of the patients' satisfaction with the esthetic outcome of the restorations. Potential factors influencing the papilla level and the presence of a complete papilla fill were investigated with generalized estimation equations (GEE) analysis. The bone level at the adjacent tooth significantly influenced the papilla level (P < 0.001). The distance between the contact point and the bone level at the adjacent tooth was significantly shorter for "complete" (4.3 mm) papillae than that for "deficient" papillae (5.7 mm) (P < 0.001). The GEE logistic model revealed that the chance of a complete papilla fill improved with increased facio-lingual thickness of the papilla (P = 0.004) and decreased distance between the contact point and the bone level at the tooth (P = 0.004). The self-reported satisfaction with the esthetic appearance of the implant-born restoration was not significantly different between patients with "complete" and "deficient" papillae. The probability of a complete papilla fill was significantly affected by the facio-lingual dimension of the papilla base and the distance between the contact point between the crowns and the bone level at the tooth. © 2012 John Wiley & Sons A/S.

THE INCIDENCE AND PATHOGENESIS OF MYOCARDITIS IN RABBITS AFTER GROUP A STREPTOCOCCAL PHARYNGEAL INFECTIONS

PubMed Central

Glaser, Robert J.; Thomas, Wilbur A.; Morse, Stephen I.; Darnell, James E.

1956-01-01

Rabbits subjected to single pharyngeal infections with group A streptococci developed cardiac lesions characterized by myofiber necrosis and a non-granulocytic cellular reaction with histiocytes, lymphocytes, and Anitschkow myocytes. The histopathologic changes were demonstrable in some animals within 24 hours of inoculation, apparently were maximal 72 hours after induction of infection (at which time they were seen in the hearts of all nine rabbits studied), and thereafter healed in the course of the following 2 weeks. The extent of involvement was variable, and with healing the necrotic areas were replaced by fibrous tissue. When intradermal infections with the same organisms were produced in rabbits, cardiac lesions, indistinguishable from those observed in the pharyngeally infected group, appeared in a much smaller number of animals. The hearts of five of six rabbits sacrificed a month or more following the last of a series of streptococcal pharyngeal infections exhibited lesions characterized chiefly by fibrosis, although mononuclear cellular infiltrations were also noted. In these repetitively infected animals the presence of occasional multinucleated giant cells and a few small foci of calcification were features not encountered in the single infection group. In a second series of rabbits sacrificed 3 days after the last of three pharyngeal infections with different strains of streptococci, acute as well as more chronic changes were observed. In none of the lesions in rabbits subjected to single or multiple streptococcal infections were bacteria demonstrable, either in histologic sections or in cultures of myocardial tissue. A large number of control animals was studied concomitantly, and in only one instance was a lesion, considered comparable to those described in the streptococcal series, encountered. The implications of these findings, particularly in terms of the non-suppurative sequelae of streptococcal infections in man, are discussed. PMID:13278463

Cutting performance orthogonal test of single plane puncture biopsy needle based on puncture force

NASA Astrophysics Data System (ADS)

Xu, Yingqiang; Zhang, Qinhe; Liu, Guowei

2017-04-01

Needle biopsy is a method to extract the cells from the patient's body with a needle for tissue pathological examination. Many factors affect the cutting process of soft tissue, including the geometry of the biopsy needle, the mechanical properties of the soft tissue, the parameters of the puncture process and the interaction between them. This paper conducted orthogonal experiment of main cutting parameters based on single plane puncture biopsy needle, and obtained the cutting force curve of single plane puncture biopsy needle by studying the influence of the inclination angle, diameter and velocity of the single plane puncture biopsy needle on the puncture force of the biopsy needle. Stage analysis of the cutting process of biopsy needle puncture was made to determine the main influencing factors of puncture force during the cutting process, which provides a certain theoretical support for the design of new type of puncture biopsy needle and the operation of puncture biopsy.

Automated cell-type classification in intact tissues by single-cell molecular profiling

PubMed Central

2018-01-01

A major challenge in biology is identifying distinct cell classes and mapping their interactions in vivo. Tissue-dissociative technologies enable deep single cell molecular profiling but do not provide spatial information. We developed a proximity ligation in situ hybridization technology (PLISH) with exceptional signal strength, specificity, and sensitivity in tissue. Multiplexed data sets can be acquired using barcoded probes and rapid label-image-erase cycles, with automated calculation of single cell profiles, enabling clustering and anatomical re-mapping of cells. We apply PLISH to expression profile ~2900 cells in intact mouse lung, which identifies and localizes known cell types, including rare ones. Unsupervised classification of the cells indicates differential expression of ‘housekeeping’ genes between cell types, and re-mapping of two sub-classes of Club cells highlights their segregated spatial domains in terminal airways. By enabling single cell profiling of various RNA species in situ, PLISH can impact many areas of basic and medical research. PMID:29319504

Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process.

PubMed

Wen, Xianyu; Jeong, Seorin; Kim, Younghoon; Bae, Jeong Mo; Cho, Nam Yun; Kim, Jung Ho; Kang, Gyeong Hoon

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively. Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer. Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells.

PubMed

Zhu, Ying; Piehowski, Paul D; Zhao, Rui; Chen, Jing; Shen, Yufeng; Moore, Ronald J; Shukla, Anil K; Petyuk, Vladislav A; Campbell-Thompson, Martha; Mathews, Clayton E; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

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

Zhu, Ying; Piehowski, Paul D.; Zhao, Rui

Nanoscale or single cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) platform as a major advance in overall sensitivity. NanoPOTS dramatically enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive LC-MS, nanoPOTS allows identification of ~1500 to ~3,000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runsmore » algorithm of MaxQuant, >3000 proteins were consistently identified from as few as 10 cells. Furthermore, we demonstrate robust quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

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

Zhu, Ying; Piehowski, Paul D.; Zhao, Rui

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

DOE PAGES

Zhu, Ying; Piehowski, Paul D.; Zhao, Rui; ...

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Characterization of the Expression of the Petunia Glycine-Rich Protein-1 Gene Product 1

PubMed Central

Condit, Carol M.; McLean, B. Gail; Meagher, Richard B.

1990-01-01

We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178-181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:16667509

Assessment of collagen changes in ovarian tissue by extracting optical scattering coefficient from OCT images

NASA Astrophysics Data System (ADS)

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

2012-01-01

Optical scattering coefficient from ex-vivo unfixed normal and malignant ovarian tissue was quantitatively extracted by fitting optical coherence tomography (OCT) A-line signals to a single scattering model. 1097 average A-line measurements at a wavelength of 1310nm were performed at 108 sites obtained from 18 ovaries. The average scattering coefficient obtained from normal group consisted of 833 measurements from 88 sites was 2.41 mm-1 (+/-0.59), while the average coefficient obtained from malignant group consisted of 264 measurements from 20 sites was 1.55 mm-1 (+/-0.46). Using a threshold of 2 mm-1 for each ovary, a sensitivity of 100% and a specificity of 100% were achieved. The amount of collagen within OCT imaging depth was analyzed from the tissue histological section stained with Sirius Red. The average collagen area fraction (CAF) obtained from normal group was 48.4% (+/-12.3%), while the average CAF obtained from malignant group was 11.4% (+/-4.7%). Statistical significance of the collagen content was found between the two groups (p < 0.001). The preliminary data demonstrated that quantitative extraction of optical scattering coefficient from OCT images could be a potential powerful method for ovarian cancer detection and diagnosis.

MYELIN IN THE CENTRAL NERVOUS SYSTEM AS OBSERVED IN EXPERIMENTALLY INDUCED EDEMA IN THE RAT

PubMed Central

Hirano, Asao; Zimmerman, H. M.; Levine, Seymour

1966-01-01

The compact arrangement of cells in the normal white matter of the brain makes an analysis of cellular architecture difficult. To overcome this difficulty, cerebral edema was induced in rats by means of the unilateral intracerebral implantation of silver nitrate. Within 48 hr, the brains were fixed by perfusion with glutaraldehyde followed by immersion in Dalton's chrome-osmium. Sections of the callosal radiations were studied in the electron microscope. The untreated hemisphere appeared entirely unaltered, whereas in the edematous hemisphere the edema fluid separated individual cell processes and small groups of them. The myelin sheaths and their relationships to the axons appeared essentially unaltered. In this material, analysis of cellular architecture was relatively easy, and the widely held theory of spiral wrapping could be confirmed. In addition, several other aspects of the myelin and myelin-forming cell relationships became apparent in the edematous tissue. Most of these were later confirmed by extensive and careful study of the nonedematous tissue. These included the presence of occasional isolated cytoplasmic areas in myelin and the presence of two complete sheaths around a single axon. Other observations, such as the appearance of mitochondria and dense bodies within the outer loop and the separation of myelin lamellae, are apparently limited to the edematous tissue. PMID:5971641

Biochemical measurement of bilirubin with an evanescent wave optical sensor

NASA Astrophysics Data System (ADS)

Poscio, Patrick; Depeursinge, Christian D.; Emery, Y.; Parriaux, Olivier M.; Voirin, Guy

1991-09-01

Optical sensing techniques can be considered as powerful information sources on the biochemistry of tissue, blood, and physiological fluids. Various sensing modalities can be considered: spectroscopic determination of the fluorescence or optical absorption of the biological medium itself, or more generally, of a reagent in contact with the biological medium. The principle and realization of the optical sensor developed are based on the use of polished fibers: the cladding of a monomode fiber is removed on a longitudinal section. The device can then be inserted into an hypodermic needle for in-vivo measurements. Using this minute probe, local measurements of the tissue biochemistry or metabolic processes can be obtained. The sensing mechanism is based on the propagation of the evanescent wave in the tissues or reagent: the proximity of the fiber core allows the penetration of the model field tail into the sensed medium, with a uniquely defined field distribution. Single or multi-wavelength analysis of the light collected into the fiber yields the biochemical information. Here an example of this sensing technology is discussed. In-vitro measurement of bilirubin in gastric juice demonstrates that the evanescent wave optical sensor provides a sensitivity which matches the physiological concentrations. A device is proposed for in-vivo monitoring of bilirubin concentration in the gastro-oesophageal tract.

Spatial and molecular resolution of diffuse malignant mesothelioma heterogeneity by integrating label-free FTIR imaging, laser capture microdissection and proteomics

NASA Astrophysics Data System (ADS)

Großerueschkamp, Frederik; Bracht, Thilo; Diehl, Hanna C.; Kuepper, Claus; Ahrens, Maike; Kallenbach-Thieltges, Angela; Mosig, Axel; Eisenacher, Martin; Marcus, Katrin; Behrens, Thomas; Brüning, Thomas; Theegarten, Dirk; Sitek, Barbara; Gerwert, Klaus

2017-03-01

Diffuse malignant mesothelioma (DMM) is a heterogeneous malignant neoplasia manifesting with three subtypes: epithelioid, sarcomatoid and biphasic. DMM exhibit a high degree of spatial heterogeneity that complicates a thorough understanding of the underlying different molecular processes in each subtype. We present a novel approach to spatially resolve the heterogeneity of a tumour in a label-free manner by integrating FTIR imaging and laser capture microdissection (LCM). Subsequent proteome analysis of the dissected homogenous samples provides in addition molecular resolution. FTIR imaging resolves tumour subtypes within tissue thin-sections in an automated and label-free manner with accuracy of about 85% for DMM subtypes. Even in highly heterogeneous tissue structures, our label-free approach can identify small regions of interest, which can be dissected as homogeneous samples using LCM. Subsequent proteome analysis provides a location specific molecular characterization. Applied to DMM subtypes, we identify 142 differentially expressed proteins, including five protein biomarkers commonly used in DMM immunohistochemistry panels. Thus, FTIR imaging resolves not only morphological alteration within tissue but it resolves even alterations at the level of single proteins in tumour subtypes. Our fully automated workflow FTIR-guided LCM opens new avenues collecting homogeneous samples for precise and predictive biomarkers from omics studies.

Spatial and molecular resolution of diffuse malignant mesothelioma heterogeneity by integrating label-free FTIR imaging, laser capture microdissection and proteomics.

PubMed

Großerueschkamp, Frederik; Bracht, Thilo; Diehl, Hanna C; Kuepper, Claus; Ahrens, Maike; Kallenbach-Thieltges, Angela; Mosig, Axel; Eisenacher, Martin; Marcus, Katrin; Behrens, Thomas; Brüning, Thomas; Theegarten, Dirk; Sitek, Barbara; Gerwert, Klaus

2017-03-30

Diffuse malignant mesothelioma (DMM) is a heterogeneous malignant neoplasia manifesting with three subtypes: epithelioid, sarcomatoid and biphasic. DMM exhibit a high degree of spatial heterogeneity that complicates a thorough understanding of the underlying different molecular processes in each subtype. We present a novel approach to spatially resolve the heterogeneity of a tumour in a label-free manner by integrating FTIR imaging and laser capture microdissection (LCM). Subsequent proteome analysis of the dissected homogenous samples provides in addition molecular resolution. FTIR imaging resolves tumour subtypes within tissue thin-sections in an automated and label-free manner with accuracy of about 85% for DMM subtypes. Even in highly heterogeneous tissue structures, our label-free approach can identify small regions of interest, which can be dissected as homogeneous samples using LCM. Subsequent proteome analysis provides a location specific molecular characterization. Applied to DMM subtypes, we identify 142 differentially expressed proteins, including five protein biomarkers commonly used in DMM immunohistochemistry panels. Thus, FTIR imaging resolves not only morphological alteration within tissue but it resolves even alterations at the level of single proteins in tumour subtypes. Our fully automated workflow FTIR-guided LCM opens new avenues collecting homogeneous samples for precise and predictive biomarkers from omics studies.

Noninvasive Vascular Displacement Estimation for Relative Elastic Modulus Reconstruction in Transversal Imaging Planes

PubMed Central

Hansen, Hendrik H.G.; Richards, Michael S.; Doyley, Marvin M.; de Korte, Chris L.

2013-01-01

Atherosclerotic plaque rupture can initiate stroke or myocardial infarction. Lipid-rich plaques with thin fibrous caps have a higher risk to rupture than fibrotic plaques. Elastic moduli differ for lipid-rich and fibrous tissue and can be reconstructed using tissue displacements estimated from intravascular ultrasound radiofrequency (RF) data acquisitions. This study investigated if modulus reconstruction is possible for noninvasive RF acquisitions of vessels in transverse imaging planes using an iterative 2D cross-correlation based displacement estimation algorithm. Furthermore, since it is known that displacements can be improved by compounding of displacements estimated at various beam steering angles, we compared the performance of the modulus reconstruction with and without compounding. For the comparison, simulated and experimental RF data were generated of various vessel-mimicking phantoms. Reconstruction errors were less than 10%, which seems adequate for distinguishing lipid-rich from fibrous tissue. Compounding outperformed single-angle reconstruction: the interquartile range of the reconstructed moduli for the various homogeneous phantom layers was approximately two times smaller. Additionally, the estimated lateral displacements were a factor of 2–3 better matched to the displacements corresponding to the reconstructed modulus distribution. Thus, noninvasive elastic modulus reconstruction is possible for transverse vessel cross sections using this cross-correlation method and is more accurate with compounding. PMID:23478602

Probability of cell hits in selected organs and tissues by high-LET particles at the ISS orbit

NASA Technical Reports Server (NTRS)

Yasuda, H.; Komiyama, T.; Fujitaka, K.; Badhwar, G. D. (Principal Investigator)

2002-01-01

The fluence of high-LET particles (HLP) with LET infinity H2O greater than 15 keV micrometers-1 in selected organs and tissues were measured with plastic nuclear track detectors using a life-size human phantom on the 9th Shuttle-Mir Mission (STS-91). The planar-track fluence of HLP during the 9.8-day mission ranged from 1.9 x 10(3) n cm-2 (bladder) to 5.1 x 10(3) n cm-2 (brain) by a factor of 2.7. Based on these data, a probability of HLP hits to a matured cell of each organ or tissue was roughly estimated for a 90-day ISS mission. In the calculation, all cells were assumed to be spheres with a geometric cross-sectional area of 500 micrometers2 and the cell-hit frequency from isotropic space radiation can be described by the Poisson-distribution function. As results, the probability of one or more than 1 hit to a single cell by HLP for 90 days ranged from 17% to 38%; that of two or more than 2 hits was estimated to be 1.3-8.2%. c2002 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

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...

Imaging Strategies for Tissue Engineering Applications

PubMed Central

Nam, Seung Yun; Ricles, Laura M.; Suggs, Laura J.

2015-01-01

Tissue engineering has evolved with multifaceted research being conducted using advanced technologies, and it is progressing toward clinical applications. As tissue engineering technology significantly advances, it proceeds toward increasing sophistication, including nanoscale strategies for material construction and synergetic methods for combining with cells, growth factors, or other macromolecules. Therefore, to assess advanced tissue-engineered constructs, tissue engineers need versatile imaging methods capable of monitoring not only morphological but also functional and molecular information. However, there is no single imaging modality that is suitable for all tissue-engineered constructs. Each imaging method has its own range of applications and provides information based on the specific properties of the imaging technique. Therefore, according to the requirements of the tissue engineering studies, the most appropriate tool should be selected among a variety of imaging modalities. The goal of this review article is to describe available biomedical imaging methods to assess tissue engineering applications and to provide tissue engineers with criteria and insights for determining the best imaging strategies. Commonly used biomedical imaging modalities, including X-ray and computed tomography, positron emission tomography and single photon emission computed tomography, magnetic resonance imaging, ultrasound imaging, optical imaging, and emerging techniques and multimodal imaging, will be discussed, focusing on the latest trends of their applications in recent tissue engineering studies. PMID:25012069

Mast cells and angiogenesis in wound healing.

PubMed

Gaber, Mohamed A; Seliet, Iman A; Ehsan, Nermin A; Megahed, Mohamed A

2014-02-01

To investigate the role of mast cells and vascular endothelial growth factor (VEGF) as a mediator of angiogenesis to promote wound healing in surgical and pathological scars. The study was carried out on 40 patients who presented with active scar lesions. They were subdivided into 4 groups. They included granulation tissue (10 cases), surgical scar (10 cases), hypertrophic scar (10 cases), and keloid scar (10 cases). Also 10 healthy volunteers of the same age and sex were selected as a control group. Skin biopsies were taken from the patients and the control group. Skin biopsies from clinically assessed studied groups were processed for routine histology and embedded in paraffin. Four sections were prepared from each paraffin block. The first section was stained with hematoxylin and eosin for histological evaluation. The second and third sections were processed for immunostaining of mast cells that contain chymase (MCCs) and mast cells that contain tryptase (MCTs). The fourth section was processed for immunostaining of VEGF. MCCs exhibited mild expression in normal tissue, granulation tissue, and surgical, hypertrophic and keloid scars. MCTs exhibited mild expression in normal tissue, granulation tissue and keloid, whereas moderate expression was exhibited in hypertrophic and surgical scars. VEGF expression was absent in normal tissue, mild in keloid, surgical and hypertrophic scars, and moderate in keloids and granulation tissue. Mast cell expression variation among different scar types signals the pathological evolution of the lesion, and hence may guide the need for therapeutic intervention.

Implementation of the Single European Code in a Multi-Tissue Bank

PubMed Central

Schroeter, Jan; Schulz, Tino; Schroeter, Bernard; Fleischhauer, Katrin; Pruß, Axel

2017-01-01

Introduction The traceability of tissue and cells transplants is important to ensure a high level of safety for the recipients. With the final introduction of the Single European Code (SEC) in April 2017 in the EU a consistent system among all member states became mandatory. Methods The regulations for the SEC on EU and national level were evaluated. An overview on the different parts of the SEC with detailed explanations is given. Our own experiences with the implementation of the SEC in our multi-tissue bank are reported in addition. Results The implementation of the SEC in our multi-tissue bank could be successfully realized. However, it revealed a number of difficulties, especially the sterile labeling of certain tissue transplants and the complex update of the existing database. Conclusion The introduction of the SEC has made a contribution to the safety of recipients of tissue and cells transplants through a system of comprehensive and transparent traceability. PMID:29344015

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.

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies.

PubMed

Kirkton, Robert D; Bursac, Nenad

2011-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair.

Applications of particle microbeams in space radiation research.

PubMed

Durante, Marco

2009-03-01

Galactic cosmic radiation is acknowledged as one of the major barriers to human space exploration. In space, astronauts are exposed to charged particles from Z = 1 (H) up to Z = 28 (Ni), but the probability of a hit to a specific single cell in the human body is low. Particle microbeams can deliver single charged particles of different charge and energy to single cells from different tissues, and microbeam studies are therefore very useful for improving current risk estimates for long-term space travel. 2D in vitro cell cultures can be very useful for establishing basic molecular mechanisms, but they are not sufficient to extrapolate risk, given the substantial evidence proving tissue effects are key in determining the response to radiation insult. 3D tissue or animal systems represent a more promising target for space radiobiology using microbeams.

Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: support for a genetic bottleneck.

PubMed Central

Marchington, D R; Hartshorne, G M; Barlow, D; Poulton, J

1997-01-01

While mtDNA polymorphisms at single base positions are common, the overwhelming majority of the mitochondrial genomes within a single individual are usually identical. When there is a point-mutation difference between a mother and her offspring, there may be a complete switching of mtDNA type within a single generation. It is generally assumed that there is a genetic bottleneck whereby a single or small number of founder mtDNA(s) populate the organism, but it is not known at which stages the restriction/amplification of mtDNA subtype(s) occur, and this uncertainty impedes antenatal diagnosis for mtDNA disorders. Length polymorphisms in homopolymeric tracts have been demonstrated in the large noncoding region of mtDNA. We have developed a new method, T-PCR (trimmed PCR), to quantitate heteroplasmy for two of these tracts (D310 and D16189). D310 variation is sufficient to indicate clonal origins of tissues and single oocytes. Tissues from normal individuals often possessed more than one length variant (heteroplasmy). However, there was no difference in the pattern of the length variants between somatic tissues in any control individual when bulk samples were taken. Oocytes from normal women undergoing in vitro fertilization were frequently heteroplasmic for length variants, and in two cases the modal length of the D310 tract differed in individual oocytes from the same woman. These data suggest that a restriction/amplification event, which we attribute to clonal expansion of founder mtDNA(s), has occurred by the time oocytes are mature, although further segregation may occur at a later stage. In contrast to controls, the length distribution of the D310 tract varied between tissues in a patient with heteroplasmic mtDNA rearrangements, suggesting that these mutants influence segregation. These findings have important implications for the genetic counselling of patients with pathogenic mtDNA mutations. Images Figure 2 Figure 1 Figure 3 Figure 4 Figure 5 PMID:9012414

RMP-02/MTN-006: A Phase 1 Rectal Safety, Acceptability, Pharmacokinetic, and Pharmacodynamic Study of Tenofovir 1% Gel Compared with Oral Tenofovir Disoproxil Fumarate

PubMed Central

Cranston, Ross D.; Kashuba, Angela; Hendrix, Craig W.; Bumpus, Namandjé N.; Richardson-Harman, Nicola; Elliott, Julie; Janocko, Laura; Khanukhova, Elena; Dennis, Robert; Cumberland, William G.; Ju, Chuan; Carballo-Diéguez, Alex; Mauck, Christine; McGowan, Ian

2012-01-01

Abstract This study was designed to assess the safety, acceptability, pharmacokinetic (PK), and pharmacodynamic (PD) responses to rectal administration of tenofovir (TFV) 1% vaginally formulated gel and oral tenofovir disoproxil fumarate (TDF). This study was designed as a phase 1, randomized, two-site (United States), double-blind, placebo-controlled study of sexually abstinent men and women. Eighteen participants received a single 300-mg exposure of oral TDF and were then randomized 2:1 to receive a single and then seven daily exposures of rectal TFV or hydroxyethyl cellulose (HEC) placebo gel. Safety endpoints included clinical adverse events (AEs) and mucosal safety parameters. Blood and colonic biopsies were collected for PK analyses and ex vivo HIV-1 challenge. No serious AEs were reported. However, AEs were significantly increased with 7-day TFV gel use, most prominently with gastrointestinal AEs (p=0.002). Only 25% of participants liked the TFV gel. Likelihood of use “if somewhat protective” was ∼75% in both groups. Indices of mucosal damage showed minimal changes. Tissue TFV diphosphate (TFV-DP) Cmax 30 min after single rectal exposure was 6–10 times greater than single oral exposure; tissue TFV-DP was 5.7 times greater following 7-day versus single rectal exposure. In vivo exposure correlated with significant ex vivo tissue infectibility suppression [single-rectal: p=0.12, analysis of covariance (ANCOVA) p=0.006; 7-day rectal: p=0.02, ANCOVA p=0.005]. Tissue PK–PD was significantly correlated (p=0.002). We conclude that rectal dosing with TFV 1% gel resulted in greater TFV-DP tissue detection than oral dosing with reduced ex vivo biopsy infectibility, enabling PK–PD correlations. On the basis of increased gastrointestinal AEs, rectally applied, vaginally formulated TFV was not entirely safe or acceptable, suggesting the need for alternative rectal-specific formulations. PMID:22943559

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.

Insulin response in individual tissues of control and gold thioglucose-obese mice in vivo with (1-/sup 14/C)2-deoxyglucose

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

Cooney, G.J.; Astbury, L.D.; Williams, P.F.

The dose-response characteristics of several glucose-utilizing tissues (brain, heart, white adipose tissue, brown adipose tissue, and quadriceps muscle) to a single injection of insulin have been compared in control mice and mice made obese with a single injection of gold thioglucose (GTG). Tissue content of (1-/sup 14/C)2-deoxyglucose 6-phosphate and blood disappearance rate of (1-/sup 14/C)2-deoxyglucose (2-DG) were measured at nine different insulin doses and used to calculate rates of 2-DG uptake and phosphorylation in tissues from control and obese mice. The insulin sensitivity of tissues reflected in the ED50 of insulin response varied widely, and brown adipose tissue was themore » most insulin-sensitive tissue studied. In GTG-obese mice, heart, quadriceps, and brown adipose tissue were insulin resistant (demonstrated by increased ED50), whereas in white adipose tissue, 2-DG phosphorylation was more sensitive to insulin. Brain 2-DG phosphorylation was insulin independent in control and obese animals. The largest decrease in insulin sensitivity in GTG-obese mice was observed in brown adipose tissue. The loss of diet-induced thermogenesis in brown adipose tissue as a result of the hypothalamic lesion in GTG-obese mice could be a major cause of insulin resistance in brown adipose tissue. Because brown adipose tissue can make a major contribution to whole-body glucose utilization, insulin resistance in this tissue may have a significant effect on whole-animal glucose homeostasis in GTG-obese mice.« less

Improvement of ambulatory function with multilevel soft tissue surgery in children with spastic diplegic cerebral palsy.

PubMed

Thamkunanon, Verasak

2011-08-01

Single Event Multilevel soft tissue surgery in spastic diplegic children also was effective for improving ambulatory function obviously as multilevel bone and soft tissue surgery. Just muscle and tendon surgery seem to be enough for better lever arm dysfunction of the lower extremity. It has safe, simple and rapid recovery. Gross Motor Functional Classification System (GMFCS) improvement after single event multilevel soft tissue surgery had been observed in these study groups of patients. Retrospective review in 93 spastic diplegic children who were more than 3 years old, had ability to understand communication, at least leaned sitting and one-hand gross function ability had been operated on by single event multilevel soft tissue surgery. GMFCS was assessed at the time of pre-operation and 6-12 months after operation. Analyzing GMFCS change was performed by statistics. Average 7 site surgery per one patient, 84% GMFCS level improvement and 16% GMFCS level non-improvement were reported. Nine cases (9.7%) were improved 2 level of GMFCS and 74% improved 1 level. GMFCS level compared between pre- and post surgery had changed by the significant statistic (p < 0.001). The average GMFCS level improvement for all groups was 0.93 level. The average age in the improved group (75 months old) was less than the non-improved group (92 month old), was a trend difference in statistic (p = 0.032). Single Event Multilevel Soft tissue surgery was effective in improving the GMFCS level average 1 level. It changed ambulatory function of spastic diplegic CP children obviously, immediately and safely. Younger age might get more benefit than older children.

Absorbed dose estimates from a single measurement one to three days after the administration of 177Lu-DOTATATE/-TOC.

PubMed

Hänscheid, Heribert; Lapa, Constantin; Buck, Andreas K; Lassmann, Michael; Werner, Rudolf A

2017-01-01

To retrospectively analyze the accuracy of absorbed dose estimates from a single measurement of the activity concentrations in tumors and relevant organs one to three days after the administration of 177 Lu-DOTA-TATE/TOC assuming tissue specific effective half-lives. Activity kinetics in 54 kidneys, 30 neuroendocrine tumor lesions, 25 livers, and 27 spleens were deduced from series of planar images in 29 patients. After adaptation of mono- or bi-exponential fit functions to the measured data, it was analyzed for each fit function how precise the time integral can be estimated from fixed tissue-specific half-lives and a single measurement at 24, 48, or 72 h after the administration. For the kidneys, assuming a fixed tissue-specific half-life of 50 h, the deviations of the estimate from the actual integral were median (5 % percentile, 95 % percentile): -3 °% (-15 %>; +16 °%) for measurements after 24 h, +2 %> (-9 %>; +12 %>) for measurements after 48 h, and 0 % (-2 %; +12 %) for measurements after 72 h. The corresponding values for the other tissues, assuming fixed tissue-specific half-lives of 67 h for liver and spleen and 77 h for tumors, were +2 % (-25 %; +20 %) for measurements after 24 h, +2 °% (-16 %>; +17 %>) for measurements after 48 h, and +2 %> (-11 %>; +10 %>) for measurements after 72 h. Especially for the kidneys, which often represent the dose limiting organ, but also for liver, spleen, and neuroendocrine tumors, a meaningful absorbed dose estimate is possible from a single measurement after 2, more preferably 3 days after the administration of 177 Lu-DOTA-TATE/-TOC assuming fixed tissue specific effective half-lives. Schattauer GmbH.

[Application of PLA Method for Detection of p53/p63/p73 Complexes in Situ in Tumour Cells and Tumour Tissue].

PubMed

Hrabal, V; Nekulová, M; Nenutil, R; Holčaková, J; Coates, P J; Vojtěšek, B

2017-01-01

PLA (proximity ligation assay) can be used for detection of protein-protein interactions in situ directly in cells and tissues. Due to its high sensitivity and specificity it is useful for detection, localization and quantification of protein complexes with single molecule resolution. One of the mechanisms of mutated p53 gain of function is formation of proten-protein complexes with other members of p53 family - p63 and p73. These interactions influences chemosensitivity and invasivity of cancer cells and this is why these complexes are potential targets of anti-cancer therapy. The aim of this work is to detect p53/p63/p73 interactions in situ in tumour cells and tumour tissue using PLA method. Unique in-house antibodies for specific detection of p63 and p73 isoforms were developed and characterized. Potein complexes were detected using PLA in established cell lines SVK14, HCC1806 and FaDu and in paraffin sections of colorectal carcinoma tissue. Cell lines were also processed to paraffin blocks. p53/T-antigen and ΔNp63/T-antigen protein complexes were detected in SVK14 cells using PLA. Interactions of ΔNp63 and TAp73 isoforms were found in HCC1806 cell line with endogenous expression of these proteins. In FaDu cell line mut-p53/TAp73 complex was localized but not mut-p53/ΔNp63 complex. p53 tetramer was detected directly in colorectal cancer tissue. During development of PLA method for detection of protein complexes between p53 family members we detected interactions of p53 and p63 with T-antigen and mut-p53 and ΔNp63 with TAp73 tumour suppressor in tumour cell lines and p53 tetramers in paraffin sections of colorectal cancer tissue. PLA will be further used for detection of p53/p63, p53/p73 and p63/p73 interactions in tumour tissues and it could be also used for screening of compounds that can block formation of p53/p63/p73 protein complexes.Key words: p53 protein family - protein interaction mapping - immunofluorescence This work was supported by MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

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.

A comparison of microscopic ink characteristics of 35 commercially available surgical margin inks.

PubMed

Milovancev, Milan; Löhr, Christiane V; Bildfell, Robert J; Gelberg, Howard B; Heidel, Jerry R; Valentine, Beth A

2013-11-01

To compare microscopic characteristics of commercially available surgical margin inks used for surgical pathology specimens. Prospective in vitro study. Thirty-five different surgical margin inks (black, blue, green, orange, red, violet, and yellow from 5 different manufacturers). Inks were applied to uniform, single-source, canine cadaveric full-thickness ventral abdominal tissue blocks. Tissue blocks and ink manufacturers were randomly paired and each color was applied to a length of the cut tissue margin. After drying, tissues were fixed in formalin, and 3 radial slices were obtained from each color section and processed for routine histologic evaluation, yielding 105 randomly numbered slides with each manufacturer's color represented in triplicate. Slides were evaluated by 5 blinded, board-certified veterinary anatomic pathologists using a standardized scoring scheme. Statistical analyses were performed to evaluate for ink manufacturer effects on scores, correlation among different subjective variables, and pathologist agreement. Black and blue had the most consistently high scores whereas red and violet had the most consistently low overall scores, across all manufacturers. All colors tested, except yellow, had statistically significant differences in overall scores among individual manufacturers. Overall score was significantly correlated to all other subjective microscopic scores evaluated. The average Spearman correlation coefficient among the 10 pairwise pathologists overall ink scores was 0.60. There are statistically significant differences in microscopic ink characteristics among manufacturers, with a notable degree of inter-pathologist agreement. © Copyright 2013 by The American College of Veterinary Surgeons.

Technique to Obtain a Predictable Aesthetic Result through Appropriate Placement of the Prosthesis/Soft Tissue Junction in the Edentulous Patient with a Gingival Smile.

PubMed

Demurashvili, Georgy; Davarpanah, Keyvan; Szmukler-Moncler, Serge; Davarpanah, Mithridade; Raux, Didier; Capelle-Ouadah, Nedjoua; Rajzbaum, Philippe

2015-10-01

Treating the edentulous patient with a gingival smile requires securing the prosthesis/soft tissue junction (PSTJ) under the upper lip. To present a simple method that helps achieve a predictable aesthetic result when alveoplasty of the anterior maxilla is needed to place implants apical to the presurgical position of the alveolar ridge. The maximum smile line of the patient is recorded and carved on a thin silicone bite impression as a soft tissue landmark. During the three-dimensional radiographic examination, the patient wears the silicone guide loaded with radiopaque markers. The NobelClinician® software is then used to bring the hard and soft tissue landmarks together in a single reading. Using the software, a line is drawn 5 mm apical to the smile line; it dictates the position of the crestal ridge to be reached following the alveoplasty. Subsequently, the simulated implant position and the simulated residual bone height following alveoplasty can be simultaneously evaluated on each transverse section. An alveoplasty of the anterior maxilla was performed as simulated on the software, and implants were placed accordingly. The PSTJ was always under the upper lip, even during maximum smile events. The aesthetic result was, therefore, fully satisfactory. This simple method permits the placement of the PSTJ under the upper lip with a predictable outcome; it ensures a reliable aesthetic result for the edentulous patient with a gingival smile. © 2013 Wiley Periodicals, Inc.

Role of mesenchymal stem cells versus angiotensin converting enzyme inhibitor in kidney repair.

PubMed

Ahmed, Hanaa H; Toson, Elshahat A; El-Mezayen, Hatem A; Rashed, Laila A; Elsherbiny, Eslam S

2017-07-01

The current study sought to clarify the role of bone marrow derived mesenchymal stem cells (BM-MSCs) and adipose tissue derived mesenchymal stem cells (AD-MSCs) in repressing nephropathy in the experimental model. Moreover, the aim of this work was extended to compare between stem cells role and angiotensin converting enzyme inhibitor in kidney repair. Isolation and preparation of MSCs culture, flow cytometry using CD34, CD44 and CD105 cell surface markers, biochemical analyses for determination of serum creatinine, urea, transforming growth factor β (TGF-β), cystatin C (CYS-C) and urinary N-Acetyl-ß-D-Glucosaminidase (UNAG), and histopathological investigation of kidney tissue sections were performed. The results of the present study revealed that single intravenous infusion of MSCs either derived from bone marrow or adipose tissue was able to enhance renal reparative processes through significantly decreased serum creatinine, urea, TGF-β and CYS-C levels as well as UNAG level and significantly increase glomerular filtration rate. Additionally, the histopathological investigations of kidney tissues showed that MSCs have significant regenerative effects as evidenced by the decrease in focal inflammatory cells infiltration, focal interstitial nephritis and congested glomeruli as well as degenerated tubules. The current data provided distinct evidence about the favourable impact of AD-MSCs and BM-MSCs in attenuation of cyclosporine-induced nephropathy in rats through their ability to promote functional and structural kidney repair via transdifferentiation. © 2016 Asian Pacific Society of Nephrology.

Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies.

PubMed

Sakowski, Stacey A; Geddes, Timothy J; Thomas, David M; Levi, Edi; Hatfield, James S; Kuhn, Donald M

2006-04-26

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy.

PubMed

Moen, Aina E F; Tannæs, Tone M; Vatn, Simen; Ricanek, Petr; Vatn, Morten Harald; Jahnsen, Jørgen

2016-06-28

Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.

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

PubMed

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

2017-01-01

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

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

PubMed

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

2013-04-01

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

Microwave Processing of Crowns from Winter Cereals for Light Microscopy.

USDA-ARS?s Scientific Manuscript database

Microwave processing of tissue considerably shortens the time it takes to prepare samples for light and electron microscopy. However, plant tissues from different species and different regions of the plant respond differently making it impossible to use a single protocol for all plant tissue. The ...

Digital quantification of fibrosis in liver biopsy sections: description of a new method by Photoshop software.

PubMed

Dahab, Gamal M; Kheriza, Mohamed M; El-Beltagi, Hussien M; Fouda, Abdel-Motaal M; El-Din, Osama A Sharaf

2004-01-01

The precise quantification of fibrous tissue in liver biopsy sections is extremely important in the classification, diagnosis and grading of chronic liver disease, as well as in evaluating the response to antifibrotic therapy. Because the recently described methods of digital image analysis of fibrosis in liver biopsy sections have major flaws, including the use of out-dated techniques in image processing, inadequate precision and inability to detect and quantify perisinusoidal fibrosis, we developed a new technique in computerized image analysis of liver biopsy sections based on Adobe Photoshop software. We prepared an experimental model of liver fibrosis involving treatment of rats with oral CCl4 for 6 weeks. After staining liver sections with Masson's trichrome, a series of computer operations were performed including (i) reconstitution of seamless widefield images from a number of acquired fields of liver sections; (ii) image size and solution adjustment; (iii) color correction; (iv) digital selection of a specified color range representing all fibrous tissue in the image and; (v) extraction and calculation. This technique is fully computerized with no manual interference at any step, and thus could be very reliable for objectively quantifying any pattern of fibrosis in liver biopsy sections and in assessing the response to antifibrotic therapy. It could also be a valuable tool in the precise assessment of antifibrotic therapy to other tissue regardless of the pattern of tissue or fibrosis.

Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

PubMed

Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

2016-02-01

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. © 2016 The Histochemical 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.

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

PubMed

Joshi, Molishree; Keith Pittman, H; Haisch, Carl; Verbanac, Kathryn

2008-09-01

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

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

Oxygen Nanobubble Tracking by Light Scattering in Single Cells and Tissues.

PubMed

Bhandari, Pushpak; Wang, Xiaolei; Irudayaraj, Joseph

2017-03-28

Oxygen nanobubbles (ONBs) have significant potential in targeted imaging and treatment in cancer diagnosis and therapy. Precise localization and tracking of single ONBs is demonstrated based on hyperspectral dark-field microscope (HSDFM) to image and track single oxygen nanobubbles in single cells. ONBs were proposed as promising contrast-generating imaging agents due to their strong light scattering generated from nonuniformity of refractive index at the interface. With this powerful platform, we have revealed the trajectories and quantities of ONBs in cells, and demonstrated the relation between the size and diffusion coefficient. We have also evaluated the presence of ONBs in the nucleus with respect to an increase in incubation time and have quantified the uptake in single cells in ex vivo tumor tissues. Our results demonstrate that HSDFM can be a versatile platform to detect and measure cellulosic nanoparticles at the single-cell level and to assess the dynamics and trajectories of this delivery system.

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.

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

Analysis of MCNP simulated gamma spectra of CdTe detectors for boron neutron capture therapy.

PubMed

Winkler, Alexander; Koivunoro, Hanna; Savolainen, Sauli

2017-06-01

The next step in the boron neutron capture therapy (BNCT) is the real time imaging of the boron concentration in healthy and tumor tissue. Monte Carlo simulations are employed to predict the detector response required to realize single-photon emission computed tomography in BNCT, but have failed to correctly resemble measured data for cadmium telluride detectors. In this study we have tested the gamma production cross-section data tables of commonly used libraries in the Monte Carlo code MCNP in comparison to measurements. The cross section data table TENDL-2008-ACE is reproducing measured data best, whilst the commonly used ENDL92 and other studied libraries do not include correct tables for the gamma production from the cadmium neutron capture reaction that is occurring inside the detector. Furthermore, we have discussed the size of the annihilation peaks of spectra obtained by cadmium telluride and germanium detectors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Size and metabolic properties of fibers in rat fast-twitch muscles after hindlimb suspension

NASA Technical Reports Server (NTRS)

Roy, Roland R.; Bello, Maureen A.; Bouissou, Phillip; Edgerton, V. Reggie

1987-01-01

The effect of hind-limb suspension (HS) on single fibers of the medial gastrocnemius (MG) and the tibialis anterior (TA) muscles were studied in rats. Fiber area and the activities of succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) were determined in tissue sections using an image analysis system. After 28 days of HS, the MG atrophied 28 percent, whereas the TA weight was maintained. Both dark- and light-ATPase fibers in the deep region of the MG had decreased cross-sectional areas following HS, with the atrophic response being twice as great in the light-ATPase fibers than in the dark-ATPase fibers. Following HS, mean SDH activities of both fiber types were significantly lower in the MG and TA than in the CON; by contrast, mean GPD activities were either maintained at the CON level or were higher in both MG and TA muscles. The data suggest an independence of the mechanisms determining the muscle fiber size and the metabolic adaptations associated with HS.

Population transcriptomics with single-cell resolution: a new field made possible by microfluidics: a technology for high throughput transcript counting and data-driven definition of cell types.

PubMed

Plessy, Charles; Desbois, Linda; Fujii, Teruo; Carninci, Piero

2013-02-01

Tissues contain complex populations of cells. Like countries, which are comprised of mixed populations of people, tissues are not homogeneous. Gene expression studies that analyze entire populations of cells from tissues as a mixture are blind to this diversity. Thus, critical information is lost when studying samples rich in specialized but diverse cells such as tumors, iPS colonies, or brain tissue. High throughput methods are needed to address, model and understand the constitutive and stochastic differences between individual cells. Here, we describe microfluidics technologies that utilize a combination of molecular biology and miniaturized labs on chips to study gene expression at the single cell level. We discuss how the characterization of the transcriptome of each cell in a sample will open a new field in gene expression analysis, population transcriptomics, that will change the academic and biomedical analysis of complex samples by defining them as quantified populations of single cells. Copyright © 2013 WILEY Periodicals, Inc.

Soft tissue modelling with conical springs.

PubMed

Omar, Nadzeri; Zhong, Yongmin; Jazar, Reza N; Subic, Aleksandar; Smith, Julian; Shirinzadeh, Bijan

2015-01-01

This paper presents a new method for real-time modelling soft tissue deformation. It improves the traditional mass-spring model with conical springs to deal with nonlinear mechanical behaviours of soft tissues. A conical spring model is developed to predict soft tissue deformation with reference to deformation patterns. The model parameters are formulated according to tissue deformation patterns and the nonlinear behaviours of soft tissues are modelled with the stiffness variation of conical spring. Experimental results show that the proposed method can describe different tissue deformation patterns using one single equation and also exhibit the typical mechanical behaviours of soft tissues.

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.

Alveolar Ridge Contouring with Free Connective Tissue Graft at Implant Placement: A 5-Year Consecutive Clinical Study.

PubMed

Hanser, Thomas; Khoury, Fouad

2016-01-01

This study evaluated volume stability after alveolar ridge contouring with free connective tissue grafts at implant placement in single-tooth gaps. A total of 52 single-tooth gaps with labial volume deficiencies in the maxilla (incisors, canines, and premolars) were consecutively treated with implants and concomitant free palatal connective tissue grafts in 46 patients between 2006 and 2009. Implants had to be covered with at least 2 mm peri-implant local bone after insertion. At implant placement, a free connective tissue graft from the palate was fixed inside a labial split-thickness flap to form an existing concave buccal alveolar ridge contour due to tissue volume deficiency into a convex shape. Standardized volumetric measurements of the labial alveolar contour using a template were evaluated before connective tissue grafting and at 2 weeks, 1 year, and 5 years after implantprosthetic incorporation. Tissue volume had increased significantly (P < .05) in all six reference points representing the outer alveolar soft tissue contour of the implant before connective tissue grafting to baseline (2 weeks after implant-prosthetic incorporation). Statistically, 50% of the reference points (P > .05) kept their volume from baseline to 1 year after prosthetic incorporation and from baseline to 5 years after prosthetic incorporation, respectively, whereas reference points located within the area of the implant sulcus showed a significant (P < .05) decrease in volume. Clinically, 5 years after prosthetic incorporation the originally concave buccal alveolar contour was still convex in all implants, leading to a continuous favorable anatomical shape and improved esthetic situation. Intraoral radiographs confirmed osseointegration and stable peri-implant parameters with a survival rate of 100% after a follow-up of approximately 5 years. Implant placement with concomitant free connective tissue grafting appears to be an appropriate long-term means to contour preexisting buccal alveolar volume deficiencies in single implants.

Jamming and liquidity in 3D cancer cell aggregates

NASA Astrophysics Data System (ADS)

Oswald, Linda; Grosser, Steffen; Lippoldt, Jürgen; Pawlizak, Steve; Fritsch, Anatol; KäS, Josef A.

Traditionally, tissues are treated as simple liquids, which holds for example for embryonic tissue. However, recent experiments have shown that this picture is insufficient for other tissue types, suggesting possible transitions to solid-like behavior induced by cellular jamming. The coarse-grained self-propelled Voronoi (SPV) model predicts such a transition depending on cell shape which is thought to arise from an interplay of cell-cell adhesion and cortical tension. We observe non-liquid behavior in 3D breast cancer spheroids of varying metastatic potential and correlate single cell shapes, single cell dynamics and collective dynamic behavior of fusion and segregation experiments via the SPV model.

Systematic Review of Soft Tissue Alterations and Esthetic Outcomes Following Immediate Implant Placement and Restoration of Single Implants in the Anterior Maxilla.

PubMed

Khzam, Nabil; Arora, Himanshu; Kim, Paul; Fisher, Anthony; Mattheos, Nikos; Ivanovski, Saso

2015-12-01

The aim of this review is to assess the outcome of single-tooth immediate implant placement and restoration (IPR) in the maxillary anterior region, with a particular emphasis on soft tissue and esthetic outcomes. An electronic search in Medline, EBSCOhost, and Ovid (PubMed) was performed to identify studies that reported on soft tissue outcomes following immediate placement and restoration of implants in the maxillary esthetic region with a mean follow-up of ≥1 year. Nineteen studies on single implants inserted immediately into fresh extraction sockets and provisionally restored in the maxillary esthetic region were included. Soft tissue changes were found to be acceptable, with most studies reporting mean gingival recession of 0.27 ± 0.38 mm and mean papillary height loss of 0.23 ± 0.27 mm after follow-up of ≥1 year. Advanced buccal recession (>1 mm) occurred in 11% of cases. Long-term follow-up studies (>2 years) reported that the interdental papillae, in particular, showed a tendency to rebound over time. The few studies that reported on patient-centered outcomes showed a high level of patient satisfaction with the outcomes of IPR treatment. The IPR protocol resulted in generally acceptable soft tissue and esthetic outcomes, with suboptimal results reported in ≈11% of low-risk cases. Factors such as preoperative tissue biotype or use of a flap or connective tissue graft did not significantly influence soft tissue and esthetic outcomes. Long-term prospective controlled clinical trials are necessary to identify factors that may influence the esthetic outcomes associated with IPR.

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.

Preliminary results of laser tissue welding in extravesical reimplantation of the ureters.

PubMed

Kirsch, A J; Dean, G E; Oz, M C; Libutti, S K; Treat, M R; Nowygrod, R; Hensle, T W

1994-02-01

One exciting potential use of laparoscopic technology is the extravesical reimplantation of the ureters. We have assessed the efficacy of laser-activated fibrinogen solder to close vesical muscle flaps over submucosal ureters (Lich-Gregoir technique) in a canine model. Four dogs were subjected to unilateral flap closures via a protein solder (indocyanine green and fibrinogen) applied to the bladder serosa and exposed to 808 nm. continuous wave diode laser energy. Contralateral reimplantation was performed using 4-zero vicryl muscle flap closures (controls). At 7, 14 and 28 days postoperatively, intravenous pyelograms confirmed bilateral ureteral patency. At intravesical pressures above 100 cm. H2O, there was no evidence of wound disruption in either group. Nondisrupted wound closures were sectioned and strained until ultimate breakage to determine tensile strength. At each study interval the laser-welded closures withstood greater stress than the controls. Although these data represent single tissue samples and are not amenable to statistical analysis, laser-welded closures appeared to be stronger at each study interval. In conclusion, laser-welded vesical wound closures appear at least as strong as suture closures in the canine model.

[Concentration of glutathione (GSH), ascorbic acid (vitamin C) and substances reacting with thiobarbituric acid (TBA-rs) in single human brain metastases].

PubMed

Dudek, Henryk; Farbiszewski, Ryszard; Rydzewska, Maria; Michno, Tadeusz; Kozłowski, Andrzej

2005-01-01

The aim of the study was to estimate the concentration of glutathione (GSH), ascorbic acid (vitamin C) and thiobarbituric acid (TBA-rs) in single human brain metastases and histologically unchanged nerve tissue. The research was conducted on fragments of neoplasmatic tissue collected from 45 patients undergoing surgery in the Department of Neurosurgery, Medical University of Białystok in years 1996-2002. Concentration of GSH was evaluated using the GSH-400 method, vitamin C using the method of Kyaw and TBA-rs using the method of Salaris and Babs. It has been found that there is a decrease of concentration of GSH and vitamin C and a considerable increase (p < 0.05) of concentration of TBA-rs in investigated single brain human metastasis in correlation to the concentration of the mentioned above substances in unchanged nerve tissue.

Comparison of dual and single exposure techniques in dual-energy chest radiography.

PubMed

Ho, J T; Kruger, R A; Sorenson, J A

1989-01-01

Conventional chest radiography is the most effective tool for lung cancer detection and diagnosis; nevertheless, a high percentage of lung cancer tumors are missed because of the overlap of lung nodule image contrast with bone image contrast in a chest radiograph. Two different energy subtraction strategies, dual exposure and single exposure techniques, were studied for decomposing a radiograph into bone-free and soft tissue-free images to address this problem. For comparing the efficiency of these two techniques in lung nodule detection, the performances of the techniques were evaluated on the basis of residual tissue contrast, energy separation, and signal-to-noise ratio. The evaluation was based on both computer simulation and experimental verification. The dual exposure technique was found to be better than the single exposure technique because of its higher signal-to-noise ratio and greater residual tissue contrast. However, x-ray tube loading and patient motion are problems.

Effect of single and binary combinations of plant-derived molluscicides on different enzyme activities in the nervous tissue of Achatina fulica.

PubMed

Rao, I G; Singh, Amrita; Singh, V K; Singh, D K

2003-01-01

Effect of single and binary treatments of plant-derived molluscicides on different enzymes--acetylcholinesterase (AChE), lactic dehydrogenase (LDH) and acid/alkaline phosphatase (ACP/ALP)--in the nervous tissue of the harmful terrestrial snail Achatina fulica were studied. Sublethal in vivo 24-h exposure to 40% and 80% LC(50) of Azadirachta indica oil, Cedrus deodara oil, Allium sativum bulb powder, Nerium indicum bark powder and binary combinations of A. sativum (AS) + C. deodara (CD) and CD + A. indica (AI) oils significantly altered the activity of these enzymes in the nervous tissue of Achatina fulica. The binary treatment of AS + CD was more effective against AChE, LDH, and ALP than the single ones. However, binary treatment of AI + CD was more effective against ALP. Copyright 2003 John Wiley & Sons, Ltd.

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Minced Skin for Tissue Engineering of Epithelialized Subcutaneous Tunnels

PubMed Central

Fossum, Magdalena; Zuhaili, Baraa; Hirsch, Tobias; Spielmann, Malte; Reish, Richard G.; Mehta, Priyesh

2009-01-01

We used minced, autologous skin for neoepithelialization of surgically created subcutaneous tunnels in a large animal model. Partial-thickness skin grafts were harvested from the back region of five 50–60 kg Yorkshire pigs. The skin was minced to 0.8 × 0.8 × 0.3 mm particles. Silicone-latex tubes were covered with fibrin, rolled in minced skin, and placed in subcutaneous tunnels created in the abdominal area. For comparison, single cell suspensions of keratinocytes and fibroblasts in fibrin or fibrin only were transplanted on tubes. Tunnels were extracted after 14, 21, and 28 days for microscopic evaluation. All tubes transplanted with minced skin particles showed neoepithelialization. The epithelium was stratified and differentiated after 2 weeks in vivo, and the stratum corneum was directed toward the implanted tube. No epithelium formed from tubes transplanted with single cell suspensions, and only sparse keratinocytes could be detected by serial sectioning and immunostaining on day 14, but not later. No epithelial lining was found in tunnels with fibrin-only-coated tubes. Epithelial cysts could be found the first 2 weeks after transplantation in the minced skin group but not later. In conclusion, a minced skin technique could serve as a potential source for tissue engineering of tubular conduits for reconstructive purposes of the urethra and for cutaneous stomas for bladder catheterization, or intestinal irrigations. The method would have the advantage of being simple and expeditious and not requiring in vitro culturing. PMID:19292681

Inflammatory myofibroblastic tumor of the lung with unique histological pattern and association with Sjögren's disease and systemic lupus erythematosus.

PubMed

Shlopov, Boris V; French, Samuel W

2011-10-01

Inflammatory myofibroblastic tumor (IMT) of the lung is a rare condition. Radiological properties and clinical presentation of this disease can mimic malignant process. We present a case of IMT of the lung in a 58 year old female patient with a single lung nodule. Tumor was unencapsulated, firm, and well circumscribed. Microscopically tumor had multinodular structure with single or multiple small blood vessels in the center of each nodule surrounded in circular pattern by connective tissue containing spindle cells embedded into the thick layers of extracellular matrix. Extracellular matrix was identified as type I and type III collagen fibrils embedded into type IV collagen and laminin. The tumor was surrounded by T-, B-lymphocytes and polyclonal plasma cells. Histological organization of this lesion's stromal component was unique, but cell composition was similar to inflammatory pseudotumor of the lung. In addition, tumor tissue sections exhibited strong positivity for IgG, weak positivity for IgA, 1Cq, but were negative for IgM, and C3. Mutational analysis of the EGFR, KRAS genes and ALK locus rearrangement were performed and did not reveal any mutations. This is the first report of an IMT associated with Sjögren's disease, systemic lupus erythematosus and Non-Hodgkin lymphoma developing in the lungs. Patient was clinically followed up for 18 months and no recurrence of the tumor observed. Copyright © 2011 Elsevier Inc. All rights reserved.

Reproducibility of the pink esthetic score--rating soft tissue esthetics around single-implant restorations with regard to dental observer specialization.

PubMed

Gehrke, Peter; Lobert, Markus; Dhom, Günter

2008-01-01

The pink esthetic score (PES) evaluates the esthetic outcome of soft tissue around implant-supported single crowns in the anterior zone by awarding seven points for the mesial and distal papilla, soft-tissue level, soft-tissue contour, soft-tissue color, soft-tissue texture, and alveolar process deficiency. The aim of this study was to measure the reproducibility of the PES and assess the influence exerted by the examiner's degree of dental specialization. Fifteen examiners (three general dentists, three oral maxillofacial surgeons, three orthodontists, three postgraduate students in implant dentistry, and three lay people) applied the PES to 30 implant-supported single restorations twice at an interval of 4 weeks. Using a 0-1-2 scoring system, 0 being the lowest, 2 being the highest value, the maximum achievable PES was 14. At the second assessment, the photographs were scored in reverse order. Differences between the two assessments were evaluated with the Spearman's rank correlation coefficient (R). The Wilcoxon signed-rank test was used for comparisons of differences between the ratings. A significance level of p < 0.05 was chosen for both tests. Observer results indicated that the agreement between the first and second rating for all occupational groups was 70.5%, with a broad correlation between the two ratings and a high statistical significance (Spearman's R = 0.58, p = 0; Wilcoxon T = 163,182, Z = 3.383599, p = 0.000716). The most agreement between the first and second rating was obtained by orthodontists with 73.5% (R = 0.67), and the least by lay people 65.9% (R = 0.50). Very poor and very esthetic restorations showed the smallest deviations. Orthodontists were found to have assigned significantly poorer ratings than any other group. The assessment of postgraduate students and laypersons were the most favorable. The PES allows for a more objective appraisal of the esthetic short- and long-term results of various surgical and prosthetic implant procedures. It reproducibly evaluates the peri-implant soft tissue around single-implant restorations and results in good intra-examiner agreement. However, an effect of observer specialization on rating soft-tissue esthetics can be shown.

Esthetic outcome and alterations of soft tissue around single implant crowns: a 2-year prospective study.

PubMed

Gu, Ying-Xin; Shi, Jun-Yu; Zhuang, Long-Fei; Qiao, Shi-Chong; Xu, You-You; Lai, Hong-Chang

2015-08-01

The aim of this prospective study was to assess the esthetic outcome and alterations of peri-implant soft tissue using tissue-level implants. Furthermore, the influencing factors, including grafting and gingival biotype, of esthetic outcome of peri-implant soft tissue were also evaluated. Of 38 patients with single missing anterior tooth in maxilla were treated with a Straumann (®) Standard Plus SLA implant. Bone augmentation was performed in 24 patients. Follow-up was conducted at 12 and 24 months after definitive crowns placement. Esthetic outcome using the pink esthetic score/white esthetic score (PES/WES) and clinical parameters were evaluated. The mean PES/WES value at baseline, 1-year, and 2-year examination was 13.79, 14.87, and 14.96. Significant improvement was found between baseline and 1-year examination (P < 0.01). And the improvement between 1-year and 2-year examination was not significant (P = 0.40). The mean PES changing value in patients with thick biotype was significantly higher than those with thin biotype at 2-year after definitive crowns placement (P = 0.03). Graft procedure had an unfavorable effect on mean PES value both at baseline and at follow-up (P < 0.01). No implants were lost at 2-year examination. Three patients experienced peri-implant infection. No significant difference was found with the passage of time in modified plaque index (mPI), probing pocket depth (PPD), and modified bleeding index (mBI). According to the present prospective clinical study, it can be concluded that it is feasible to use tissue-level implant to support single crowns in esthetic area. Favorable short-term esthetic outcome and stability of soft tissue around single implant crowns can be expected in patients with or without graft. However, graft procedures might have an unfavorable effect on the esthetic outcome. Gingival biotype can be considered as prognostic factor for esthetic outcome. RCTs with long-term follow-up are needed to provide evidence for the long-term stability of peri-implant soft tissue using tissue-level implant systems. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A microprobe for parallel optical and electrical recordings from single neurons in vivo.

PubMed

LeChasseur, Yoan; Dufour, Suzie; Lavertu, Guillaume; Bories, Cyril; Deschênes, Martin; Vallée, Réal; De Koninck, Yves

2011-04-01

Recording electrical activity from identified neurons in intact tissue is key to understanding their role in information processing. Recent fluorescence labeling techniques have opened new possibilities to combine electrophysiological recording with optical detection of individual neurons deep in brain tissue. For this purpose we developed dual-core fiberoptics-based microprobes, with an optical core to locally excite and collect fluorescence, and an electrolyte-filled hollow core for extracellular single unit electrophysiology. This design provides microprobes with tips < 10 μm, enabling analyses with single-cell optical resolution. We demonstrate combined electrical and optical detection of single fluorescent neurons in rats and mice. We combined electrical recordings and optical Ca²(+) measurements from single thalamic relay neurons in rats, and achieved detection and activation of single channelrhodopsin-expressing neurons in Thy1::ChR2-YFP transgenic mice. The microprobe expands possibilities for in vivo electrophysiological recording, providing parallel access to single-cell optical monitoring and control.

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

PubMed

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

2017-09-20

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

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...

In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups

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

Burbach, J.P.; Voorhuis, T.A.; van Tol, H.H.

1987-05-29

Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded /sup 35/S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at thesemore » various sites.« less

Azimuthally invariant Mueller-matrix mapping of biological optically anisotropic network

NASA Astrophysics Data System (ADS)

Ushenko, Yu. O.; Vanchuliak, O.; Bodnar, G. B.; Ushenko, V. O.; Grytsyuk, M.; Pavlyukovich, N.; Pavlyukovich, O. V.; Antonyuk, O.

2017-09-01

A new technique of Mueller-matrix mapping of polycrystalline structure of histological sections of biological tissues is suggested. The algorithms of reconstruction of distribution of parameters of linear and circular dichroism of histological sections liver tissue of mice with different degrees of severity of diabetes are found. The interconnections between such distributions and parameters of linear and circular dichroism of liver of mice tissue histological sections are defined. The comparative investigations of coordinate distributions of parameters of amplitude anisotropy formed by Liver tissue with varying severity of diabetes (10 days and 24 days) are performed. The values and ranges of change of the statistical (moments of the 1st - 4th order) parameters of coordinate distributions of the value of linear and circular dichroism are defined. The objective criteria of cause of the degree of severity of the diabetes differentiation are determined.

CdSe/CdS semiconductor quantum rods as robust fluorescent probes for paraffin-embedded tissue imaging.

PubMed

Zacheo, Antonella; Quarta, Alessandra; Mangoni, Antonella; Pompa, Pier Paolo; Mastria, Rosanna; Capogrossi, Maurizio C; Rinaldi, Ross; Pellegrino, Teresa

2011-09-01

Immunofluorescence techniques on formalin fixed paraffin-embedded sections allow for the evaluation of the expression and spatial distribution of specific markers in patient tissue specimens or for monitoring the fate of labeled cells after in vivo injection. This technique suffers however from the auto-fluorescence background signal of the embedded tissue that eventually confounds the analysis. Here we show that rod-like semiconductor nanocrystals (QRs), intramuscularly injected in living mice, could be clearly detected by confocal microscopy in formalin fixed paraffin-embedded tissue sections. Despite the low amount of QRs amount injected (25 picomoles), these were clearly visible after 24 h in the muscle sections and their fluorescence signal was stronger than that of CdSe/ZnS quantum dots (QDs) similarly functionalized and in the case of QRs only, the signal lasted even after 21 days after the injection. © 2011 IEEE

Ex vivo applications of multiphoton microscopy in urology

NASA Astrophysics Data System (ADS)

Jain, Manu; Mukherjee, Sushmita

2016-03-01

Background: Routine urological surgery frequently requires rapid on-site histopathological tissue evaluation either during biopsy or intra-operative procedure. However, resected tissue needs to undergo processing, which is not only time consuming but may also create artifacts hindering real-time tissue assessment. Likewise, pathologist often relies on several ancillary methods, in addition to H&E to arrive at a definitive diagnosis. Although, helpful these techniques are tedious and time consuming and often show overlapping results. Therefore, there is a need for an imaging tool that can rapidly assess tissue in real-time at cellular level. Multiphoton microscopy (MPM) is one such technique that can generate histology-quality images from fresh and fixed tissue solely based on their intrinsic autofluorescence emission, without the need for tissue processing or staining. Design: Fresh tissue sections (neoplastic and non-neoplastic) from biopsy and surgical specimens of bladder and kidney were obtained. Unstained deparaffinized slides from biopsy of medical kidney disease and oncocytic renal neoplasms were also obtained. MPM images were acquired using with an Olympus FluoView FV1000MPE system. After imaging, fresh tissues were submitted for routine histopathology. Results: Based on the architectural and cellular details of the tissue, MPM could characterize normal components of bladder and kidney. Neoplastic tissue could be differentiated from non-neoplastic tissue and could be further classified as per histopathological convention. Some of the tumors had unique MPM signatures not otherwise seen on H&E sections. Various subtypes of glomerular lesions were identified as well as renal oncocytic neoplasms were differentiated on unstained deparaffinized slides. Conclusions: We envision MPM to become an integral part of regular diagnostic workflow for rapid assessment of tissue. MPM can be used to evaluate the adequacy of biopsies and triage tissues for ancillary studies. It can also be used as an adjunct to frozen section analysis for intra-operative margin assessment. Further, it can play an important role for pathologist for guiding specimen grossing, selecting tissue for tumor banking and as a rapid ancillary diagnostic tool.

Improved tumor identification using dual tracer molecular imaging in fluorescence guided brain surgery

NASA Astrophysics Data System (ADS)

Xu, Xiaochun; Torres, Veronica; Straus, David; Brey, Eric M.; Byrne, Richard W.; Tichauer, Kenneth M.

2015-03-01

Brain tumors represent a leading cause of cancer death for people under the age of 40 and the probability complete surgical resection of brain tumors remains low owing to the invasive nature of these tumors and the consequences of damaging healthy brain tissue. Molecular imaging is an emerging approach that has the potential to improve the ability for surgeons to correctly discriminate between healthy and cancerous tissue; however, conventional molecular imaging approaches in brain suffer from significant background signal in healthy tissue or an inability target more invasive sections of the tumor. This work presents initial studies investigating the ability of novel dual-tracer molecular imaging strategies to be used to overcome the major limitations of conventional "single-tracer" molecular imaging. The approach is evaluated in simulations and in an in vivo mice study with animals inoculated orthotopically using fluorescent human glioma cells. An epidermal growth factor receptor (EGFR) targeted Affibody-fluorescent marker was employed as a targeted imaging agent, and the suitability of various FDA approved untargeted fluorescent tracers (e.g. fluorescein & indocyanine green) were evaluated in terms of their ability to account for nonspecific uptake and retention of the targeted imaging agent. Signal-to-background ratio was used to measure and compare the amount of reporter in the tissue between targeted and untargeted tracer. The initial findings suggest that FDA-approved fluorescent imaging agents are ill-suited to act as untargeted imaging agents for dual-tracer fluorescent guided brain surgery as they suffer from poor delivery to the healthy brain tissue and therefore cannot be used to identify nonspecific vs. specific uptake of the targeted imaging agent where current surgery is most limited.

Multiple high-intensity focused ultrasound probes for kidney-tissue ablation.

PubMed

Häcker, Axel; Chauhan, Sunita; Peters, Kristina; Hildenbrand, Ralf; Marlinghaus, Ernst; Alken, Peter; Michel, Maurice Stephan

2005-10-01

To investigate kidney-tissue ablation by high-intensity focused ultrasound (HIFU) using multiple and single probes. Ultrasound beams (1.75 MHz) produced by a piezoceramic element (focal distance 80 mm) were focused at the center of renal parenchyma. One of the three probes (mounted on a jig) could also be used for comparison with a single probe at comparable power ratings. Lesion dimensions were examined in perfused and unperfused ex vivo porcine kidneys at different power levels (40, 60, and 80 W) and treatment times (4, 6, and 8 seconds). At identical power levels, the lesions induced by multiple probes were larger than those induced by a single probe. Lesion size increased with increasing pulse duration and generator power. The sizes and shapes of the lesions were predictably repeatable in all samples. Lesions in perfused kidneys were smaller than those in unperfused kidneys. Ex vivo, kidney-tissue ablation by means of multiple HIFU probes offers significant advantages over single HIFU probes in respect of lesion size and formation. These advantages need to be confirmed by tests in vivo at higher energy levels.

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.

Detecting skin malignancy using elastic light scattering spectroscopy

NASA Astrophysics Data System (ADS)

Canpolat, Murat; Akman, Ayşe; Çiftçioğlu, M. Akif; Alpsoy, Erkan

2007-07-01

We have used elastic light scattering spectroscopy to differentiate between malign and benign skin lesions. The system consists of a UV spectrometer, a single optical fiber probe and a laptop. The single optical fiber probe was used for both delivery and detection of white light to tissue and from the tissue. The single optical fiber probe received singly scattered photons rather than diffused photons in tissue. Therefore, the spectra are correlated with morphological differences of the cells. It has been shown that spectra of malign skin lesions are different than spectra of benign skin lesions. While slopes of the spectra taken on benign lesions or normal skin tissues were positive, slopes of the spectra taken on malign skin lesions tissues were negative. In vivo experiments were conducted on 20 lesions from 18 patients (11 men with mean age of 68 +/- 9 years and 7 women with mean age of 52 +/- 20 years) applied to the Department of Dermatology and Venerology. Before the biopsy, spectra were taken on the lesion and adjacent (approximately 1 cm distant) normal-appearing skin. Spectra of the normal skin were used as a control group. The spectra were correlated to the pathology results with sensitivity and specificity of 82% and 89%, respectively. Due to small diameter of fiber probe and limited number of sampling (15), some positive cases are missed, which is lowered the sensitivity of the system. The results are promising and could suggest that the system may be able to detect malignant skin lesion non-invasively and in real time.

Probability of cavitation for single ultrasound pulses applied to tissues and tissue-mimicking materials.

PubMed

Maxwell, Adam D; Cain, Charles A; Hall, Timothy L; Fowlkes, J Brian; Xu, Zhen

2013-03-01

In this study, the negative pressure values at which inertial cavitation consistently occurs in response to a single, two-cycle, focused ultrasound pulse were measured in several media relevant to cavitation-based ultrasound therapy. The pulse was focused into a chamber containing one of the media, which included liquids, tissue-mimicking materials, and ex vivo canine tissue. Focal waveforms were measured by two separate techniques using a fiber-optic hydrophone. Inertial cavitation was identified by high-speed photography in optically transparent media and an acoustic passive cavitation detector. The probability of cavitation (P(cav)) for a single pulse as a function of peak negative pressure (p(-)) followed a sigmoid curve, with the probability approaching one when the pressure amplitude was sufficient. The statistical threshold (defined as P(cav) = 0.5) was between p(-) = 26 and 30 MPa in all samples with high water content but varied between p(-) = 13.7 and >36 MPa in other media. A model for radial cavitation bubble dynamics was employed to evaluate the behavior of cavitation nuclei at these pressure levels. A single bubble nucleus with an inertial cavitation threshold of p(-) = 28.2 megapascals was estimated to have a 2.5 nm radius in distilled water. These data may be valuable for cavitation-based ultrasound therapy to predict the likelihood of cavitation at various pressure levels and dimensions of cavitation-induced lesions in tissue. Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

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.

Contact lens-induced peripheral ulcers with extended wear of disposable hydrogel lenses: histopathologic observations on the nature and type of corneal infiltrate.

PubMed

Holden, B A; Reddy, M K; Sankaridurg, P R; Buddi, R; Sharma, S; Willcox, M D; Sweeney, D F; Rao, G N

1999-09-01

Contact lens-induced peripheral ulcer (CLPU), a sudden-onset adverse event observed with extended wear of hydrogel lenses, is characterized by a single, small, circular, focal anterior stromal infiltrate in the corneal periphery or midperiphery. The condition is always associated with a significant overlying epithelial loss and resolves in a scar. The aim was to determine, by using histopathologic techniques, the nature and type of the corneal infiltrate of these events. Three CLPUs observed in three patients using disposable hydrogel lenses on an extended-wear schedule were examined. The eye was topically anesthetized, and a corneal section including all of the infiltrate was taken. A small triangular piece of conjunctiva immediately adjacent to the infiltrate was sectioned. The tissue was immediately fixed, processed, stained using hematoxylin and eosin and periodic acid-Schiff stains, and examined by using light microscopy. The diameter of these three corneal infiltrates varied from 0.3 to 0.6 mm. Histopathology of the corneal sections revealed a focal epithelial loss corresponding to the infiltrated stroma in all three patients. The adjacent epithelium was thinned. Bowman's layer was intact in two patients and had a localized area of loss in the remaining patient. The anterior stroma was densely infiltrated with polymorphonuclear leukocytes and had focal areas of necrosis. The infiltration was most dense in the region immediately underlying Bowman's layer. No other infiltrative cell type was seen in any of the sections. Histopathology of the conjunctiva revealed features consistent with normal conjunctival tissue. On histopathology of CLPU, distinctive features (i.e., focal corneal epithelial loss, an intact Bowman's membrane, and a localized infiltration of the anterior stroma with polymorphonuclear leukocytes) were seen. These features suggest that the event is an acute inflammatory process and probably noninfective in nature.

Ultraviolet radiation exposure triggers neurokinin-1 receptor upregulation in ocular tissues in vivo.

PubMed

Gross, Janine; Wegener, Alfred R; Kronschlaeger, Martin; Holz, Frank G; Schönfeld, Carl-Ludwig; Meyer, Linda M

2018-04-26

The purpose of this study was to investigate the neurokinin receptor-1 (NKR-1) protein expression in ocular tissues before and after supra-cataract threshold ultraviolet radiation (UVR-B peak at 312 nm) exposure in vivo in a mouse model. Six-week-old C57Bl/6 mice were unilaterally exposed to a single (2.9 kJ/m 2 ) and an above 3-fold UVR-B cataract threshold dose (9.4 kJ/m 2 ) of UVR. UVR-exposure (λpeak = 312 nm) was performed in mydriasis using a Bio-Spectra exposure system. After latency periods of 3 and 7 days, eyes were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and stained with fluorescence coupled antibody for NKR-1 and DAPI for cell nuclei staining. Control animals received only anesthesia but no UVR-exposure. Cataract development was documented with a Leica dark-field microscope and quantified as integrated optical density (IOD). NKR-1 is ubiquitously present in ocular tissues. An above 3-fold cataract threshold dose of UV-radiation induced NKR-1 upregulation after days 3 and 7 in the epithelium and endothelium of the cornea, the endothelial cells of the iris vessels, the pigmented epithelium/stroma of the ciliary body, the lens epithelium, pronounced in the nuclear bow region and the inner plexiform layer of the retina. A significant upregulation of NKR-1 could not be provoked with a single cataract threshold dose (2.9 kJ/m 2 UVR-B) ultraviolet irradiation. All exposed eyes developed anterior subcapsular cataracts. Neurokinin-1 receptor is present ubiquitously in ocular tissues including the lens epithelium and the nuclear bow region of the lens. UV-radiation exposure to an above 3-fold UVR-B cataract threshold dose triggers NKR-1 upregulation in the eye in vivo. The involvement of inflammation in ultraviolet radiation induced cataract and the role of neuroinflammatory peptides such as substance P and its receptor, NKR-1, might have been underestimated to date. Copyright © 2018. Published by Elsevier Ltd.

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

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.

Finite element modelling of stapled colorectal end-to-end anastomosis: advantages of variable height stapler design.

PubMed

Nováček, V; Tran, T N; Klinge, U; Tolba, R H; Staat, M; Bronson, D G; Miesse, A M; Whiffen, J; Turquier, F

2012-10-11

The impact of surgical staplers on tissues has been studied mostly in an empirical manner. In this paper, finite element method was used to clarify the mechanics of tissue stapling and associated phenomena. Various stapling modalities and several designs of circular staplers were investigated to evaluate the impact of the device on tissues and mechanical performance of the end-to-end colorectal anastomosis. Numerical simulations demonstrated that a single row of staples is not adequate to resist leakage due to non-linear buckling and opening of the tissue layers between two adjacent staples. Compared to the single staple row configuration, significant increase in stress experienced by the tissue at the inner staple rows was observed in two and three rows designs. On the other hand, adding second and/or third staple row had no effect on strain in the tissue inside the staples. Variable height design with higher staples in outer rows significantly reduced the stresses and strains in outer rows when compared to the same configuration with flat cartridge. Copyright © 2012 Elsevier Ltd. All rights reserved.

Single-Pass Microkeratome System for Eye Bank DSAEK Tissue Preparation: Is Stromal Bed Thickness Predictable and Reproducible?

PubMed

Choulakian, Mazen Y; Li, Jennifer Y; Ramos, Samuel; Mannis, Mark J

2016-01-01

To evaluate the predictability and reproducibility of stromal bed thickness for single-pass donor Descemet stripping automated endothelial keratoplasty (DSAEK) tissue preparation, using the ML7 Microkeratome Donor Cornea System (Med-logics Inc, Athens, TX). In this retrospective chart review of 256 consecutive corneal tissue preparations for DSAEK surgery, from June 2013 to August 2014, tissue thicknesses were divided into 3 groups, depending on surgeon preference: <91 μm (group A), 90 to 120 μm (group B), and 120 to 160 μm (group C). Precut and postcut data were recorded. Average postcut donor corneal thickness was 114 ± 30 μm (range 60-183 μm), whereas the average in group A was 97 ± 23 μm (range 60-128), in group B was 113 ± 21 μm (range 77-179), and in group C was 134 ± 43 (range 89-183). Average postcut endothelial cell density was very adequate at 3013 ± 250 cells per square millimeter. There were a total of 7 failed procedures from 256 attempts, which represents a rate of 2.7%. This rate decreases to 1.5% when analyzing the last 200 cuts. The ML7 Microkeratome Donor Cornea System allows for reliable and reproducible DSAEK tissue preparation. Ultrathin DSAEK tissues can be prepared with a single-pass. Aiming for a graft thickness between 90 and 120 μm seems to be most reliable.

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.

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.

Frozen section analysis of margins for head and neck tumor resections: reduction of sampling errors with a third histologic level.

PubMed

Olson, Stephen M; Hussaini, Mohammad; Lewis, James S

2011-05-01

Frozen section analysis is an essential tool for assessing margins intra-operatively to assure complete resection. Many institutions evaluate surgical defect edge tissue provided by the surgeon after the main lesion has been removed. With the increasing use of transoral laser microsurgery, this method is becoming even more prevalent. We sought to evaluate error rates at our large academic institution and to see if sampling errors could be reduced by the simple method change of taking an additional third section on these specimens. All head and neck tumor resection cases from January 2005 through August 2008 with margins evaluated by frozen section were identified by database search. These cases were analyzed by cutting two levels during frozen section and a third permanent section later. All resection cases from August 2008 through July 2009 were identified as well. These were analyzed by cutting three levels during frozen section (the third a 'much deeper' level) and a fourth permanent section later. Error rates for both of these periods were determined. Errors were separated into sampling and interpretation types. There were 4976 total frozen section specimens from 848 patients. The overall error rate was 2.4% for all frozen sections where just two levels were evaluated and was 2.5% when three levels were evaluated (P=0.67). The sampling error rate was 1.6% for two-level sectioning and 1.2% for three-level sectioning (P=0.42). However, when considering only the frozen section cases where tumor was ultimately identified (either at the time of frozen section or on permanent sections) the sampling error rate for two-level sectioning was 15.3 versus 7.4% for three-level sectioning. This difference was statistically significant (P=0.006). Cutting a single additional 'deeper' level at the time of frozen section identifies more tumor-bearing specimens and may reduce the number of sampling errors.

Virtual taphonomy using synchrotron tomographic microscopy reveals cryptic features and internal structure of modern and fossil plants

PubMed Central

Smith, Selena Y.; Collinson, Margaret E.; Rudall, Paula J.; Simpson, David A.; Marone, Federica; Stampanoni, Marco

2009-01-01

While more commonly applied in zoology, synchrotron radiation X-ray tomographic microscopy (SRXTM) is well-suited to nondestructive study of the morphology and anatomy of both fossil and modern plants. SRXTM uses hard X-rays and a monochromatic light source to provide high-resolution data with little beam-hardening, resulting in slice data with clear boundaries between materials. Anatomy is readily visualized, including various planes of section from a single specimen, as clear as in traditional histological sectioning at low magnifications. Thus, digital sectioning of rare or difficult material is possible. Differential X-ray attenuation allows visualization of different layers or chemistries to enable virtual 3-dimensional (3D) dissections of material. Virtual potential fossils can be visualized and digital tissue removal reveals cryptic underlying morphology. This is essential for fossil identification and for comparisons between assemblages where fossils are preserved by different means. SRXTM is a powerful approach for botanical studies using morphology and anatomy. The ability to gain search images in both 2D and 3D for potential fossils gives paleobotanists a tool—virtual taphonomy—to improve our understanding of plant evolution and paleobiogeography. PMID:19574457

Single-cell epigenomics: techniques and emerging applications.

PubMed

Schwartzman, Omer; Tanay, Amos

2015-12-01

Epigenomics is the study of the physical modifications, associations and conformations of genomic DNA sequences, with the aim of linking these with epigenetic memory, cellular identity and tissue-specific functions. While current techniques in the field are characterizing the average epigenomic features across large cell ensembles, the increasing interest in the epigenetics within complex and heterogeneous tissues is driving the development of single-cell epigenomics. We review emerging single-cell methods for capturing DNA methylation, chromatin accessibility, histone modifications, chromosome conformation and replication dynamics. Together, these techniques are rapidly becoming a powerful tool in studies of cellular plasticity and diversity, as seen in stem cells and cancer.

Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.

PubMed

Soeller, Christian; Hou, Yufeng; Jayasinghe, Isuru D; Baddeley, David; Crossman, David

2017-01-01

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

MRI of articular cartilage at microscopic resolution

PubMed Central

Xia, Y.

2013-01-01

This review briefly summarises some of the definitive studies of articular cartilage by microscopic MRI (µMRI) that were conducted with the highest spatial resolutions. The article has four major sections. The first section introduces the cartilage tissue, MRI and µMRI, and the concept of image contrast in MRI. The second section describes the characteristic profiles of three relaxation times (T1, T2 and T1ρ) and self-diffusion in healthy articular cartilage. The third section discusses several factors that can influence the visualisation of articular cartilage and the detection of cartilage lesion by MRI and µMRI. These factors include image resolution, image analysis strategies, visualisation of the total tissue, topographical variations of the tissue properties, surface fibril ambiguity, deformation of the articular cartilage, and cartilage lesion. The final section justifies the values of multidisciplinary imaging that correlates MRI with other technical modalities, such as optical imaging. Rather than an exhaustive review to capture all activities in the literature, the studies cited in this review are merely illustrative. PMID:23610697

Aesthetic outcome of single-tooth implant restorations following early implant placement and guided bone regeneration: crown and soft tissue dimensions compared with contralateral teeth.

PubMed

Cosyn, Jan; De Rouck, Tim

2009-10-01

The aim of this study was to compare crown and soft tissue dimensions of single-tooth implant restorations following early implant placement and guided bone regeneration (GBR) with contralateral non-restored teeth. Twenty-seven patients treated by one and the same surgeon and prosthodontist to restore a single-tooth gap with a class I bone defect in the premaxilla by means of an implant-supported restoration were reviewed. Patients were examined at least 6 months following placement of the crown. All implants had been inserted 6-8 weeks following tooth extraction in conjunction with GBR. At evaluation, crown dimensions, soft tissue dimensions, clinical conditions and patients' aesthetic satisfaction were assessed by one clinician who had not been involved in the treatment. Implant-supported crowns were not significantly longer than contralateral teeth and midfacial soft tissues showed comparable levels after on average 21 months of function. Our data also indicated significant papilla loss especially at the distal aspect of the implants. As the patient's aesthetic appreciation was favourable in 88% of the cases, this appeared to be of trivial importance. Favourable aesthetics may be achieved for single-tooth implant restorations following early implant placement and GBR. The impact of the latter on papilla levels, however, remains to be determined in longitudinal studies.

Long-term Stability of Soft Tissue Esthetic Outcomes Following Conventional Single Implant Treatment in the Anterior Maxilla: 10-12 Year Results.

PubMed

Rokn, A; Bassir, S H; Rasouli Ghahroudi, A A; Kharazifard, M J; Manesheof, R

2016-01-01

The present study aimed to evaluate the long-term stability of esthetic outcomes of soft tissue around maxillary anterior single-tooth implants after 10-to-12 years of loading. Patients who had been treated for single-tooth implants in the anterior maxilla between February 2000 and July 2002 were invited to participate in the study. All implants had been placed according to delayed implant placement and conventional loading protocols without any connective tissue graft or papilla preservation flaps. Pink Esthetic Score (PES) was rated using standardized clinical photographs to assess the esthetic outcomes of the implant treatment at the time of crown placement and at time of follow-up examination which was at least 10 years after the crown placement. A total of 19 patients were included. The mean score of PES was 11.63 (SD 1.61; range 7-14) at baseline. After 10-to-12 years of function, a mean PES score of 11.05 (SD 2.09; range 6-14) was recorded. No significant differences were found in the esthetic outcomes, categorized based on clinically relevant levels, between the baseline and follow-up session (p>0.05). Within limitation of the present study, it can be concluded that the esthetic outcomes of soft tissue around the maxillary anterior single-tooth implants placed using conventional implant placement technique remained stable in the long-term.

Pharmacodynamic Activity of Dapivirine and Maraviroc Single Entity and Combination Topical Gels for HIV-1 Prevention.

PubMed

Dezzutti, Charlene S; Yandura, Sarah; Wang, Lin; Moncla, Bernard; Teeple, Elizabeth A; Devlin, Brid; Nuttall, Jeremy; Brown, Elizabeth R; Rohan, Lisa C

2015-11-01

Dapivirine (DPV), a non-nucleoside reverse transcriptase inhibitor, and maraviroc (MVC), a CCR5 antagonist, were formulated into aqueous gels designed to prevent mucosal HIV transmission. 0.05% DPV, 0.1% MVC, 0.05% DPV/0.1% MVC and placebo gels were evaluated for pH, viscosity, osmolality, and in vitro release. In vitro assays and mucosal tissues were used to evaluate anti-HIV activity. Viability (Lactobacilli only) and epithelial integrity in cell lines and mucosal tissues defined safety. The gels were acidic and viscous. DPV gel had an osmolality of 893 mOsm/kg while the other gels had an osmolality of <100 mOsm/kg. MVC release was similar from the single and combination gels (~5 μg/cm(2)/min(1/2)), while DPV release was 10-fold less from the single as compared to the combination gel (0.4331 μg/cm(2)/min(1/2)). Titrations of the gels showed 10-fold more drug was needed to protect ectocervical than colonic tissue. The combination gel showed ~10- and 100-fold improved activity as compared to DPV and MVC gel, respectively. All gels were safe. The DPV/MVC gel showed a benefit blocking HIV infection of mucosal tissue compared to the single entity gels. Combination products with drugs affecting unique steps in the viral replication cycle would be advantageous for HIV prevention.

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

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.

A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

PubMed Central

2013-01-01

Background In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples. Results We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with small geometric deviations occurring only in the peripheral areas of the specimen. Based on co-registered datasets the excretory organs, which were chosen as ROI for this study, could be investigated regarding both their ultrastructure as well as their position in the organism and their spatial relationship to adjacent tissues. We found structures typical for mollusc excretory systems, including ultrafiltration sites at the pericardial wall, and ducts leading from the pericardium towards the kidneys, which exhibit a typical basal infolding system. Conclusions The presented approach allows a comprehensive analysis and presentation of small objects regarding both the overall organization as well as cellular and subcellular details. Although our protocol involves a variety of different equipment and procedures, we maintain that it offers savings in both effort and cost. Co-registration of datasets from different imaging modalities can be accomplished with high-end desktop computers and offers new opportunities for understanding and communicating structural relationships within organisms and tissues. In general, the correlative use of different microscopic imaging techniques will continue to become more widespread in morphological and structural research in zoology. Classical TEM serial section investigations are extremely time consuming, and modern methods for 3D analysis of ultrastructure such as SBF-SEM and FIB-SEM are limited to very small volumes for examination. Thus the re-sectioning of LM sections is suitable for speeding up TEM examination substantially, while microCT could become a key-method for complementing ultrastructural examinations. PMID:23915384

Proteomic analysis of formalin-fixed paraffin embedded tissue by MALDI imaging mass spectrometry

PubMed Central

Casadonte, Rita; Caprioli, Richard M

2012-01-01

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE -TMAs using matrix-assisted laser desorption/ionization (MAL DI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE -TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis. PMID:22011652

siRNA Nanoparticle Functionalization of Nanostructured Scaffolds Enables Controlled Multilineage Differentiation of Stem Cells

PubMed Central

Andersen, Morten Ø; Nygaard, Jens V; Burns, Jorge S; Raarup, Merete K; Nyengaard, Jens R; Bünger, Cody; Besenbacher, Flemming; Howard, Kenneth A; Kassem, Moustapha; Kjems, Jørgen

2010-01-01

The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas of an implant. As a consequence of this, we predict that complex tissues and organs can be engineered by the in situ development of multiple cell types guided by spatially restricted nanoparticles. PMID:20808289

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies

PubMed Central

Kirkton, Robert D.; Bursac, Nenad

2012-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair. PMID:21556054

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.

[Application of polyclonal break-apart probes in the diagnosis of Xp11.2 translocation renal cell carcinoma].

PubMed

Chen, Xiancheng; Gan, Weidong; Ye, Qing; Yang, Jun; Guo, Hongqian; Li, Dongmei

2014-12-16

To explore the value of self-designed fluorescent in situ hybridization (FISH) polyclonal break-apart probes specific for TFE3 gene in the diagnosis of Xp11.2 translocation renal cell carcinoma. All tissue samples were collected from 2006 to 2013, including Xp11.2 translocation renal cell carcinoma (n = 10), renal clear cell carcinoma (n = 10) and renal papillary cell carcinoma (n = 10). FISH was conducted for paraffin-embedded tumor tissue sections with probes. The types of fluorescence were observed by fluorescent microscopy to determine the existence or non-existence of translocated TFE3 gene. All sections were successfully probed. The split red and green signals within a single nucleus were detected simultaneously in 9 cases of Xp11.2 translocation renal cell carcinoma as diagnosed by traditional pathological and immunohistochemical methods. And it was consistent with the initial diagnosis. Detection of fusion signal in 1/10 and negative FISH result did not conform to the initial diagnosis. The fluorescent types of renal clear cell carcinoma and renal papillary cell carcinoma were all fusion signals. FISH tests were negative for renal clear and papillary cell carcinomas. Xp11.2 translocation renal cell carcinomas diagnosed by traditional pathological and immunohistochemical methods are sometimes misdiagnosed. Detecting the translocation of TFE3 gene with FISH polyclonal break-apart probes is both accurate and reliable for diagnosing Xp11.2 translocation renal cell carcinoma.

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

An integrated single- and two-photon non-diffracting light-sheet microscope

NASA Astrophysics Data System (ADS)

Lau, Sze Cheung; Chiu, Hoi Chun; Zhao, Luwei; Zhao, Teng; Loy, M. M. T.; Du, Shengwang

2018-04-01

We describe a fluorescence optical microscope with both single-photon and two-photon non-diffracting light-sheet excitations for large volume imaging. With a special design to accommodate two different wavelength ranges (visible: 400-700 nm and near infrared: 800-1200 nm), we combine the line-Bessel sheet (LBS, for single-photon excitation) and the scanning Bessel beam (SBB, for two-photon excitation) light sheet together in a single microscope setup. For a transparent thin sample where the scattering can be ignored, the LBS single-photon excitation is the optimal imaging solution. When the light scattering becomes significant for a deep-cell or deep-tissue imaging, we use SBB light-sheet two-photon excitation with a longer wavelength. We achieved nearly identical lateral/axial resolution of about 350/270 nm for both imagings. This integrated light-sheet microscope may have a wide application for live-cell and live-tissue three-dimensional high-speed imaging.

Single-band upconversion nanoprobes for multiplexed simultaneous in situ molecular mapping of cancer biomarkers.

PubMed

Zhou, Lei; Wang, Rui; Yao, Chi; Li, Xiaomin; Wang, Chengli; Zhang, Xiaoyan; Xu, Congjian; Zeng, Aijun; Zhao, Dongyuan; Zhang, Fan

2015-04-24

The identification of potential diagnostic markers and target molecules among the plethora of tumour oncoproteins for cancer diagnosis requires facile technology that is capable of quantitatively analysing multiple biomarkers in tumour cells and tissues. Diagnostic and prognostic classifications of human tumours are currently based on the western blotting and single-colour immunohistochemical methods that are not suitable for multiplexed detection. Herein, we report a general and novel method to prepare single-band upconversion nanoparticles with different colours. The expression levels of three biomarkers in breast cancer cells were determined using single-band upconversion nanoparticles, western blotting and immunohistochemical technologies with excellent correlation. Significantly, the application of antibody-conjugated single-band upconversion nanoparticle molecular profiling technology can achieve the multiplexed simultaneous in situ biodetection of biomarkers in breast cancer cells and tissue specimens and produce more accurate results for the simultaneous quantification of proteins present at low levels compared with classical immunohistochemical technology.

Dissecting Cell-Type Composition and Activity-Dependent Transcriptional State in Mammalian Brains by Massively Parallel Single-Nucleus RNA-Seq.

PubMed

Hu, Peng; Fabyanic, Emily; Kwon, Deborah Y; Tang, Sheng; Zhou, Zhaolan; Wu, Hao

2017-12-07

Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues such as adult mammalian brains is challenging. Here, we integrate sucrose-gradient-assisted purification of nuclei with droplet microfluidics to develop a highly scalable single-nucleus RNA-seq approach (sNucDrop-seq), which is free of enzymatic dissociation and nucleus sorting. By profiling ∼18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution, in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Production of sperm from porcine fetal testicular tissue after cryopreservation and grafting into nude mice.

PubMed

Kaneko, Hiroyuki; Kikuchi, Kazuhiro; Men, Nguyen Thi; Nakai, Michiko; Noguchi, Junko; Kashiwazaki, Naomi; Ito, Junya

2017-03-15

A major goal of testicular xenografting is to salvage germ cells from immature animals that cannot be used for reproduction and generate their offspring. In this study, we investigated whether porcine fetal testicular tissue would acquire the ability to produce sperm with full developmental competence after they had been cryopreserved and grafted into nude mice. Testicular fragments from fetuses at 35, 55 and 90 days postartificial insemination (dpi) were vitrified and stored in liquid nitrogen. Immediately after warming, testicular fragments at each fetal stage were transplanted under the back skin of castrated nude mice (Crlj:CD1-Foxn1 nu ) (35-, 55- and 90-dpi groups, respectively) (day 0 = grafting). Before grafting, the testicular fragments contained seminiferous cords consisting of only gonocytes and Sertoli cells. Histological analyses of the testicular grafts revealed that the differentiation of seminiferous tubules was largely dependent on the time after grafting, and not on donor age. On day 180 in each group, 10-20% of the total number of tubule/cord cross-sections examined had germ cells that had progressed beyond the spermatogonial stage. Fewer than 5% of tubule cross-sections contained elongated spermatids or sperm. Between days 360 and 420, tubule differentiation advanced further, until more than 45% of the tubule cross-sections contained elongated spermatids or sperm. Sperm were recovered for the first time from a single mouse in the 55-dpi group on day 180, although on days 360-420 sperm were recovered from most mice in all of the groups. Serum concentrations of inhibin and testosterone in host mice in all of the groups were higher than those in castrated mice that had received no testicular grafts. Single sperm collected from mice in each group on day 300 or later were injected into individual in vitro-matured oocytes, and these sperm-injected oocytes were transferred to the oviducts of 2 or 3 estrus-synchronized recipient gilts. None of the recipients in any of the groups produced piglets. The present results clearly indicate that porcine fetal testes during the gestational period acquire endocrine and exocrine functions after being cryopreserved and grafted into nude mice. However, the ability of xenogeneic sperm derived from fetal testis to generate piglets was not confirmed in the present study. Copyright © 2017 Elsevier Inc. All rights reserved.

The tissue microarray data exchange specification: A community-based, open source tool for sharing tissue microarray data

PubMed Central

Berman, Jules J; Edgerton, Mary E; Friedman, Bruce A

2003-01-01

Background Tissue Microarrays (TMAs) allow researchers to examine hundreds of small tissue samples on a single glass slide. The information held in a single TMA slide may easily involve Gigabytes of data. To benefit from TMA technology, the scientific community needs an open source TMA data exchange specification that will convey all of the data in a TMA experiment in a format that is understandable to both humans and computers. A data exchange specification for TMAs allows researchers to submit their data to journals and to public data repositories and to share or merge data from different laboratories. In May 2001, the Association of Pathology Informatics (API) hosted the first in a series of four workshops, co-sponsored by the National Cancer Institute, to develop an open, community-supported TMA data exchange specification. Methods A draft tissue microarray data exchange specification was developed through workshop meetings. The first workshop confirmed community support for the effort and urged the creation of an open XML-based specification. This was to evolve in steps with approval for each step coming from the stakeholders in the user community during open workshops. By the fourth workshop, held October, 2002, a set of Common Data Elements (CDEs) was established as well as a basic strategy for organizing TMA data in self-describing XML documents. Results The TMA data exchange specification is a well-formed XML document with four required sections: 1) Header, containing the specification Dublin Core identifiers, 2) Block, describing the paraffin-embedded array of tissues, 3)Slide, describing the glass slides produced from the Block, and 4) Core, containing all data related to the individual tissue samples contained in the array. Eighty CDEs, conforming to the ISO-11179 specification for data elements constitute XML tags used in the TMA data exchange specification. A set of six simple semantic rules describe the complete data exchange specification. Anyone using the data exchange specification can validate their TMA files using a software implementation written in Perl and distributed as a supplemental file with this publication. Conclusion The TMA data exchange specification is now available in a draft form with community-approved Common Data Elements and a community-approved general file format and data structure. The specification can be freely used by the scientific community. Efforts sponsored by the Association for Pathology Informatics to refine the draft TMA data exchange specification are expected to continue for at least two more years. The interested public is invited to participate in these open efforts. Information on future workshops will be posted at (API we site). PMID:12769826

Plant Tissue Culture in a Bag.

ERIC Educational Resources Information Center

Beck, Mike

2000-01-01

Describes the use of an oven bag as a sterile chamber for culture initiation and tissue transfer. Plant tissue culture is an ideal tool for introducing students to plants, cloning, and experimental design. Includes materials, methods, discussion, and conclusion sections. (SAH)

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.

3D Printer Generated Tissue iMolds for Cleared Tissue Using Single- and Multi-Photon Microscopy for Deep Tissue Evaluation.

PubMed

Miller, Sean J; Rothstein, Jeffrey D

2017-01-01

Pathological analyses and methodology has recently undergone a dramatic revolution. With the creation of tissue clearing methods such as CLARITY and CUBIC, groups can now achieve complete transparency in tissue samples in nano-porous hydrogels. Cleared tissue is then imagined in a semi-aqueous medium that matches the refractive index of the objective being used. However, one major challenge is the ability to control tissue movement during imaging and to relocate precise locations post sequential clearing and re-staining. Using 3D printers, we designed tissue molds that fit precisely around the specimen being imaged. First, images are taken of the specimen, followed by importing and design of a structural mold, then printed with affordable plastics by a 3D printer. With our novel design, we have innovated tissue molds called innovative molds (iMolds) that can be generated in any laboratory and are customized for any organ, tissue, or bone matter being imaged. Furthermore, the inexpensive and reusable tissue molds are made compatible for any microscope such as single and multi-photon confocal with varying stage dimensions. Excitingly, iMolds can also be generated to hold multiple organs in one mold, making reconstruction and imaging much easier. Taken together, with iMolds it is now possible to image cleared tissue in clearing medium while limiting movement and being able to relocate precise anatomical and cellular locations on sequential imaging events in any basic laboratory. This system provides great potential for screening widespread effects of therapeutics and disease across entire organ systems.

Raman molecular imaging of brain frozen tissue sections.

PubMed

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

2014-10-01

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

SU-E-T-480: Radiobiological Dose Comparison of Single Fraction SRS, Multi-Fraction SRT and Multi-Stage SRS of Large Target Volumes Using the Linear-Quadratic Formula

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

Ding, C; Hrycushko, B; Jiang, S

2014-06-01

Purpose: To compare the radiobiological effect on large tumors and surrounding normal tissues from single fraction SRS, multi-fractionated SRT, and multi-staged SRS treatment. Methods: An anthropomorphic head phantom with a centrally located large volume target (18.2 cm{sup 3}) was scanned using a 16 slice large bore CT simulator. Scans were imported to the Multiplan treatment planning system where a total prescription dose of 20Gy was used for a single, three staged and three fractionated treatment. Cyber Knife treatment plans were inversely optimized for the target volume to achieve at least 95% coverage of the prescription dose. For the multistage plan,more » the target was segmented into three subtargets having similar volume and shape. Staged plans for individual subtargets were generated based on a planning technique where the beam MUs of the original plan on the total target volume are changed by weighting the MUs based on projected beam lengths within each subtarget. Dose matrices for each plan were export in DICOM format and used to calculate equivalent dose distributions in 2Gy fractions using an alpha beta ratio of 10 for the target and 3 for normal tissue. Results: Singe fraction SRS, multi-stage plan and multi-fractionated SRT plans had an average 2Gy dose equivalent to the target of 62.89Gy, 37.91Gy and 33.68Gy, respectively. The normal tissue within 12Gy physical dose region had an average 2Gy dose equivalent of 29.55Gy, 16.08Gy and 13.93Gy, respectively. Conclusion: The single fraction SRS plan had the largest predicted biological effect for the target and the surrounding normal tissue. The multi-stage treatment provided for a more potent biologically effect on target compared to the multi-fraction SRT treatments with less biological normal tissue than single-fraction SRS treatment.« less

Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells

PubMed Central

Miragoli, Michele; Moshkov, Alexey; Novak, Pavel; Shevchuk, Andrew; Nikolaev, Viacheslav O.; El-Hamamsy, Ismail; Potter, Claire M. F.; Wright, Peter; Kadir, S.H. Sheikh Abdul; Lyon, Alexander R.; Mitchell, Jane A.; Chester, Adrian H.; Klenerman, David; Lab, Max J.; Korchev, Yuri E.; Harding, Sian E.; Gorelik, Julia

2011-01-01

Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies. PMID:21325316

Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Ckap4 as a New Modulator of Fibroblasts Activation.

PubMed

Gladka, Monika M; Molenaar, Bas; de Ruiter, Hesther; van der Elst, Stefan; Tsui, Hoyee; Versteeg, Danielle; Lacraz, Grègory P A; Huibers, Manon M H; van Oudenaarden, Alexander; van Rooij, Eva

2018-01-31

Background -Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. Methods -Here, we present a method to obtain high quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. Results -After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression we were also able to identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and the injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton associated protein 4 ( Ckap4 ) as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Ckap4 inhibition in activated fibroblasts treated with TGFβ triggered a greater increase in the expression of genes related to activated fibroblasts compared to control, suggesting a role of Ckap4 in modulating fibroblast activation in the injured heart. Conclusions -Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.

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.

SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle.

PubMed

Kettenbach, Arminja N; Sano, Hiroyuki; Keller, Susanna R; Lienhard, Gustav E; Gerber, Scott A

2015-01-30

The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues. Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results. Copyright © 2014 Elsevier B.V. All rights reserved.

Microvascular basis for growth of small infarcts following occlusion of single penetrating arterioles in mouse cortex

PubMed Central

Taylor, Zachary J; Hui, Edward S; Watson, Ashley N; Nie, Xingju; Deardorff, Rachael L; Jensen, Jens H; Helpern, Joseph A

2015-01-01

Small cerebral infarcts, i.e. microinfarcts, are common in the aging brain and linked to vascular cognitive impairment. However, little is known about the acute growth of these minute lesions and their effect on blood flow in surrounding tissues. We modeled microinfarcts in the mouse cortex by inducing photothrombotic clots in single penetrating arterioles. The resultant hemodynamic changes in tissues surrounding the occluded vessel were then studied using in vivo two-photon microscopy. We were able to generate a spectrum of infarct volumes by occluding arterioles that carried a range of blood fluxes. Those resulting from occlusion of high-flux penetrating arterioles (flux of 2 nL/s or higher) exhibited a radial outgrowth that encompassed unusually large tissue volumes. The gradual expansion of these infarcts was propagated by an evolving insufficiency in capillary flow that encroached on territories of neighboring penetrating arterioles, leading to the stagnation and recruitment of their perfusion domains into the final infarct volume. Our results suggest that local collapse of microvascular function contributes to tissue damage incurred by single penetrating arteriole occlusions in mice, and that a similar mechanism may add to pathophysiology induced by microinfarcts of the human brain. PMID:26661182

Half forehead reconstruction with a single rotational scalp flap for dermatofibrosarcoma protuberans treatment.

PubMed

Mori, Stefano; Di Monta, Gianluca; Marone, Ugo; Chiofalo, Maria Grazia; Caracò, Corrado

2012-05-06

Dermatofibrosarcoma protuberans (DFSP) is a soft tissue neoplasm of intermediate to low-grade malignancy. Although metastasis rarely occurs, DFSP has a locally aggressive behavior with a high recurrence rate. In the head and neck area, resection involving a wide margin of healthy tissue can be difficult because of functional and cosmetic considerations. We describe a novel reconstructive method for half forehead defects with an innovative single local wide scalp flap following excision of DFSP with a 3 cm margin of healthy tissue. Two patients underwent wide resection of forehead DFSP and reconstruction with a single rotational scalp flap. The scalp flap blood supply was provided from three main vessels: the superficial temporal artery, occipital artery and posterior auricular artery. No early or late complications were observed in either patient with no local recurrence after 18 months of follow-up. The donor area could be closed primarily in both cases and the flaps survived completely. This innovative technique allowed a radical excision of forehead DFSP with sufficient healthy margins, thus potentially decreasing tumor recurrence rate. Reconstruction was achieved avoiding microsurgery, skin expanders and large skin grafts. Moreover, all main reconstructive criteria, such as functional and cosmetic tissue characteristics, were completely fulfilled.

Assembly line plants take root

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

Comis, D.; Wood, M.

This paper discussed tissue-culture propagation of sugarcane, apple trees, peach trees, citrus, orchids, data palms, and carrots. Tissue-culture propagation is a term used for a variety of techniques used to grow or genetically modify, preserve, or study plant parts in laboratories, from tissue or even a single cell. The author examined the benefits and commercial applications of this propagation process.

Comparative experimental pharmacokinetics of benzimidazole derivatives.

PubMed

Sergeeva, S A; Gulyaeva, I L

2008-12-01

Comparative study of experimental kinetics of distribution of benzimidazole derivatives (bemithyl, etomerzole, and thietazole) in organs and tissues was carried out after single and course treatment. The drugs intensely passed into organs and tissues from the blood after treatment by all protocols. Specific features of drug distribution were detected; for example, splenic tissue selectively accumulated thietazole during course treatment.

A stochastic step model of replicative senescence explains ROS production rate in ageing cell populations.

PubMed

Lawless, Conor; Jurk, Diana; Gillespie, Colin S; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F

2012-01-01

Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells.One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence.

A Stochastic Step Model of Replicative Senescence Explains ROS Production Rate in Ageing Cell Populations

PubMed Central

Lawless, Conor; Jurk, Diana; Gillespie, Colin S.; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F.

2012-01-01

Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells. One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence. PMID:22359661

High-Resolution Ultrasonic Imaging of Dento-Periodontal Tissues Using a Multi-Element Phased Array System.

PubMed

Nguyen, Kim-Cuong T; Le, Lawrence H; Kaipatur, Neelambar R; Zheng, Rui; Lou, Edmond H; Major, Paul W

2016-10-01

Intraoral ultrasonography uses high-frequency mechanical waves to study dento-periodontium. Besides the advantages of portability and cost-effectiveness, ultrasound technique has no ionizing radiation. Previous studies employed a single transducer or an array of transducer elements, and focused on enamel thickness and distance measurement. This study used a phased array system with a 128-element array transducer to image dento-periodontal tissues. We studied two porcine lower incisors from a 6-month-old piglet using 20-MHz ultrasound. The high-resolution ultrasonographs clearly showed the cross-sectional morphological images of the hard and soft tissues. The investigation used an integration of waveform analysis, travel-time calculation, and wavefield simulation to reveal the nature of the ultrasound data, which makes the study novel. With the assistance of time-distance radio-frequency records, we robustly justified the enamel-dentin interface, dentin-pulp interface, and the cemento-enamel junction. The alveolar crest level, the location of cemento-enamel junction, and the thickness of alveolar crest were measured from the images and compared favorably with those from the cone beam computed tomography with less than 10% difference. This preliminary and fundamental study has reinforced the conclusions from previous studies, that ultrasonography has great potential to become a non-invasive diagnostic imaging tool for quantitative assessment of periodontal structures and better delivery of oral care.

Reproducibility of neuroimaging analyses across operating systems

PubMed Central

Glatard, Tristan; Lewis, Lindsay B.; Ferreira da Silva, Rafael; Adalat, Reza; Beck, Natacha; Lepage, Claude; Rioux, Pierre; Rousseau, Marc-Etienne; Sherif, Tarek; Deelman, Ewa; Khalili-Mahani, Najmeh; Evans, Alan C.

2015-01-01

Neuroimaging pipelines are known to generate different results depending on the computing platform where they are compiled and executed. We quantify these differences for brain tissue classification, fMRI analysis, and cortical thickness (CT) extraction, using three of the main neuroimaging packages (FSL, Freesurfer and CIVET) and different versions of GNU/Linux. We also identify some causes of these differences using library and system call interception. We find that these packages use mathematical functions based on single-precision floating-point arithmetic whose implementations in operating systems continue to evolve. While these differences have little or no impact on simple analysis pipelines such as brain extraction and cortical tissue classification, their accumulation creates important differences in longer pipelines such as subcortical tissue classification, fMRI analysis, and cortical thickness extraction. With FSL, most Dice coefficients between subcortical classifications obtained on different operating systems remain above 0.9, but values as low as 0.59 are observed. Independent component analyses (ICA) of fMRI data differ between operating systems in one third of the tested subjects, due to differences in motion correction. With Freesurfer and CIVET, in some brain regions we find an effect of build or operating system on cortical thickness. A first step to correct these reproducibility issues would be to use more precise representations of floating-point numbers in the critical sections of the pipelines. The numerical stability of pipelines should also be reviewed. PMID:25964757

Reproducibility of neuroimaging analyses across operating systems.

PubMed

Glatard, Tristan; Lewis, Lindsay B; Ferreira da Silva, Rafael; Adalat, Reza; Beck, Natacha; Lepage, Claude; Rioux, Pierre; Rousseau, Marc-Etienne; Sherif, Tarek; Deelman, Ewa; Khalili-Mahani, Najmeh; Evans, Alan C

2015-01-01

Neuroimaging pipelines are known to generate different results depending on the computing platform where they are compiled and executed. We quantify these differences for brain tissue classification, fMRI analysis, and cortical thickness (CT) extraction, using three of the main neuroimaging packages (FSL, Freesurfer and CIVET) and different versions of GNU/Linux. We also identify some causes of these differences using library and system call interception. We find that these packages use mathematical functions based on single-precision floating-point arithmetic whose implementations in operating systems continue to evolve. While these differences have little or no impact on simple analysis pipelines such as brain extraction and cortical tissue classification, their accumulation creates important differences in longer pipelines such as subcortical tissue classification, fMRI analysis, and cortical thickness extraction. With FSL, most Dice coefficients between subcortical classifications obtained on different operating systems remain above 0.9, but values as low as 0.59 are observed. Independent component analyses (ICA) of fMRI data differ between operating systems in one third of the tested subjects, due to differences in motion correction. With Freesurfer and CIVET, in some brain regions we find an effect of build or operating system on cortical thickness. A first step to correct these reproducibility issues would be to use more precise representations of floating-point numbers in the critical sections of the pipelines. The numerical stability of pipelines should also be reviewed.

Acceleration of hard and soft tissue healing in the oral cavity by a single transmucosal injection of fluvastatin-impregnated poly (lactic-co-glycolic acid) microspheres. An in vitro and rodent in vivo study.

PubMed

Yasunami, Noriyuki; Ayukawa, Yasunori; Furuhashi, Akihiro; Atsuta, Ikiru; Rakhmatia, Yunia Dwi; Moriyama, Yasuko; Masuzaki, Tomohiro; Koyano, Kiyoshi

2015-12-23

Antihyperlipidemic drug statins reportedly promote both bone formation and soft tissue healing. We examined the effect of sustained-release, fluvastatin-impregnated poly(lactic-co-glycolic acid) (PLGA) microspheres on the promotion of bone and gingival healing at an extraction socket in vivo, and the effect of fluvastatin on epithelial cells and fibroblasts in vitro. The maxillary right first molar was extracted in rats, then one of the following was immediately injected, as a single dose, into the gingivobuccal fold: control (no administration), PLGA microspheres without a statin (active control), or PLGA microspheres containing 20 or 40 μg kg(-1) of fluvastatin. At days 1, 3, 7, 14, and 28 after injection, bone and soft tissue healing were histologically evaluated. Cell proliferation was measured under the effect of fluvastatin at dosages of 0, 0.01, 0.1, 1.0, 10, and 50 μM. Cell migration and morphology were observed at dosages of 0 and 0.1 μM. Following tooth extraction, the statin significantly enhanced bone volume and density, connective tissue volume, and epithelial wound healing. In the in vitro study, it promoted significant proliferation and migration of epithelial cells and fibroblasts. A single dose of topically administered fluvastatin-impregnated PLGA microspheres promoted bone and soft tissue healing at the extraction site.

Toward optical guidance during endoscopic ultrasound-guided fine needle aspirations of pancreatic masses using single fiber reflectance spectroscopy: a feasibility study

NASA Astrophysics Data System (ADS)

Stegehuis, Paulien L.; Boogerd, Leonora S. F.; Inderson, Akin; Veenendaal, Roeland A.; van Gerven, P.; Bonsing, Bert A.; Sven Mieog, J.; Amelink, Arjen; Veselic, Maud; Morreau, Hans; van de Velde, Cornelis J. H.; Lelieveldt, Boudewijn P. F.; Dijkstra, Jouke; Robinson, Dominic J.; Vahrmeijer, Alexander L.

2017-02-01

Endoscopic ultrasound-guided fine needle aspirations (EUS-FNA) of pancreatic masses suffer from sample errors and low-negative predictive values. Fiber-optic spectroscopy in the visible to near-infrared wavelength spectrum can noninvasively extract physiological parameters from tissue and has the potential to guide the sampling process and reduce sample errors. We assessed the feasibility of single fiber (SF) reflectance spectroscopy measurements during EUS-FNA of pancreatic masses and its ability to distinguish benign from malignant pancreatic tissue. A single optical fiber was placed inside a 19-gauge biopsy needle during EUS-FNA and at least three reflectance measurements were taken prior to FNA. Spectroscopy measurements did not cause any related adverse events and prolonged procedure time with ˜5 min. An accurate correlation between spectroscopy measurements and cytology could be made in nine patients (three benign and six malignant). The oxygen saturation and bilirubin concentration were significantly higher in benign tissue compared with malignant tissue (55% versus 21%, p=0.038; 166 μmol/L versus 17 μmol/L, p=0.039, respectively). To conclude, incorporation of SF spectroscopy during EUS-FNA was feasible, safe, and relatively quick to perform. The optical properties of benign and malignant pancreatic tissue are different, implying that SF spectroscopy can potentially guide the FNA sampling.

Estimating needle tip deflection in biological tissue from a single transverse ultrasound image: application to brachytherapy.

PubMed

Rossa, Carlos; Sloboda, Ron; Usmani, Nawaid; Tavakoli, Mahdi

2016-07-01

This paper proposes a method to predict the deflection of a flexible needle inserted into soft tissue based on the observation of deflection at a single point along the needle shaft. We model the needle-tissue as a discretized structure composed of several virtual, weightless, rigid links connected by virtual helical springs whose stiffness coefficient is found using a pattern search algorithm that only requires the force applied at the needle tip during insertion and the needle deflection measured at an arbitrary insertion depth. Needle tip deflections can then be predicted for different insertion depths. Verification of the proposed method in synthetic and biological tissue shows a deflection estimation error of [Formula: see text]2 mm for images acquired at 35 % or more of the maximum insertion depth, and decreases to 1 mm for images acquired closer to the final insertion depth. We also demonstrate the utility of the model for prostate brachytherapy, where in vivo needle deflection measurements obtained during early stages of insertion are used to predict the needle deflection further along the insertion process. The method can predict needle deflection based on the observation of deflection at a single point. The ultrasound probe can be maintained at the same position during insertion of the needle, which avoids complications of tissue deformation caused by the motion of the ultrasound probe.

The maximum entropy method of moments and Bayesian probability theory

NASA Astrophysics Data System (ADS)

Bretthorst, G. Larry

2013-08-01

The problem of density estimation occurs in many disciplines. For example, in MRI it is often necessary to classify the types of tissues in an image. To perform this classification one must first identify the characteristics of the tissues to be classified. These characteristics might be the intensity of a T1 weighted image and in MRI many other types of characteristic weightings (classifiers) may be generated. In a given tissue type there is no single intensity that characterizes the tissue, rather there is a distribution of intensities. Often this distributions can be characterized by a Gaussian, but just as often it is much more complicated. Either way, estimating the distribution of intensities is an inference problem. In the case of a Gaussian distribution, one must estimate the mean and standard deviation. However, in the Non-Gaussian case the shape of the density function itself must be inferred. Three common techniques for estimating density functions are binned histograms [1, 2], kernel density estimation [3, 4], and the maximum entropy method of moments [5, 6]. In the introduction, the maximum entropy method of moments will be reviewed. Some of its problems and conditions under which it fails will be discussed. Then in later sections, the functional form of the maximum entropy method of moments probability distribution will be incorporated into Bayesian probability theory. It will be shown that Bayesian probability theory solves all of the problems with the maximum entropy method of moments. One gets posterior probabilities for the Lagrange multipliers, and, finally, one can put error bars on the resulting estimated density function.

A new technique for the rapid screening and selection of large pieces of tissue for ultrastructural evaluation.

PubMed

Dalley, B K; Seliger, W G

1980-05-01

A simple and rapid technique is described for the screening of Epon embedded organ slices for the location, isolation, and removal of small specific sites for ultrastructural study with the transmission electron microscope. This procedure consists of perfusion fixation followed by making 1 to 21/2 mm thick slices of relatively large pieces of the organs, control of the degree and evenness of the osmium staining by addition of 3% sodium iodate, and infiltration with a fluorescent dye prior to embedment in Epon. Tissue slices are embedded in wafer-shaped blocks, generally with several slices in one "wafer", and are examined in a controlled manner using a rapid form of serial surface polishing. Each level of the polished wafer is examined using an epi-illuminated fluorescence microscope, and selected sites are chosen at each level for ultrastructural study. Methods are also described for marking each selected site using a conventional slide marker, and for the removal of the selected site in the form of a small disc of Epon, after which the Epon wafer can be further serially polished and the examination continued. Areas to be thin-sectioned are removed using a core drill mounted on a model-maker's drill press. The technique is simple, does not require the destruction of remaining tissues to evaluate more critically a single small site, allows for the easy maintenance of tissue orientation, and the most time-consuming portions of the technique can be quickly taught to a person with no previous histological training.

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).

Identification of tissue-embedded ascarid larvae by ribosomal DNA sequencing.

PubMed

Ishiwata, Kenji; Shinohara, Akio; Yagi, Kinpei; Horii, Yoichiro; Tsuchiya, Kimiyuki; Nawa, Yukifumi

2004-01-01

Polymerase chain reaction (PCR) was applied to identify tissue-embedded ascarid nematode larvae. Two sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA), ITS1 and ITS2, of the ascarid parasites were amplified and compared with those of ascarid-nematodes registered in a DNA database (GenBank). The ITS sequences of the PCR products obtained from the ascarid parasite specimen in our laboratory were compatible with those of registered adult Ascaris and Toxocara parasites. PCR amplification of the ITS regions was sensitive enough to detect a single larva of Ascaris suum mixed with porcine liver tissue. Using this method, ascarid larvae embedded in the liver of a naturally infected turkey were identified as Toxocara canis. These results suggest that even a single larva embedded in tissues from patients with larva migrans could be identified by sequencing the ITS regions.

M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors.

PubMed

Yi, Hyunjung; Ghosh, Debadyuti; Ham, Moon-Ho; Qi, Jifa; Barone, Paul W; Strano, Michael S; Belcher, Angela M

2012-03-14

Second near-infrared (NIR) window light (950-1400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to 4-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control nontargeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas. © 2012 American Chemical Society

M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors

PubMed Central

HAM, MOON-HO; QI, JIFA; BARONE, PAUL W.; STRANO, MICHAEL S.; BELCHER, ANGELA M.

2014-01-01

Second near-infrared (NIR) window light (950-1,400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to four-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control non-targeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas. PMID:22268625

Quantum Dot-Based Hybrid Nanostructures and Energy Transfer on the Nanoscale for Single- and Multi-Photon Imaging and Cancer Diagnostics

NASA Astrophysics Data System (ADS)

Nabiev, Igor

2017-01-01

An ideal single-photon (1P) or multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible 1P or two-photon (2P) absorption cross section and the smallest possible highly specific recognition molecules conjugated with the nanoparticle in an oriented manner. However, the conditions used for conjugation of typical recognition molecules (conventional antibodies, Abs) with nanoparticles often provoke their unfolding and/or yield nanoprobes with irregular orientation of Abs on the nanoparticle surface. Conjugation of smaller Ab fragments, such as single-domain antibodies (sdAbs), with quantum dots (QDs) in an oriented manner can be considered as an attractive approach to engineering of ultrasmall diagnostic nanoprobes. QDs conjugated to 13-kDa sdAbs derived from camelid IgG or streptavidin have been used as efficient 1P or 2P excitation probes for imaging of cancer markers. The 2P absorption cross sections (TPACSs) for some conjugates are higher than 49,000 GM (Goeppert-Mayer units), which is close to the theoretical value calculated for CdSe QDs and considerably exceeds that of organic dyes. A further step in advanced QD-based cancer diagnostics has been made through implementation of efficient FRET-based imaging with 2P excitation, which has been demonstrated for double immunostaining complexes formed on the surface of cancer cells from sdAb-QD conjugates (donor) and a combination of monoclonal Abs and secondary antibodies labeled with the AlexaFluor dye (acceptor). The proposed approach permits obtaining an exceptional contrast of 2P imaging of cancer biomarkers without any contribution of cell and tissue autofluorescence in the recorded images.

High-Resolution Photoacoustic Imaging of Ocular Tissues

PubMed Central

Silverman, Ronald H.; Kong, Fanting; Chen, Y.C.; Lloyd, Harriet O.; Kim, Hyung Ham; Cannata, Jonathan M.; Shung, K. Kirk; Coleman, D Jackson

2010-01-01

Optical coherence tomography (OCT) and ultrasound (US) are methods widely used for diagnostic imaging of the eye. These techniques detect discontinuities in optical refractive index and acoustic impedance respectively. Because these both relate to variations in tissue density or composition, OCT and US images share a qualitatively similar appearance. In photoacoustic imaging (PAI), short light pulses are directed at tissues, pressure is generated due to a rapid energy deposition in the tissue volume, and thermoelastic expansion results in generation of broadband US. PAI thus depicts optical absorption, which is independent of the tissue characteristics imaged by OCT or US. Our aim was to demonstrate the application of PAI in ocular tissues and to do so with lateral resolution comparable to OCT. We developed two PAI assemblies, both of which used single-element US transducers and lasers sharing a common focus. The first assembly had optical and 35-MHz US axes offset by a 30° angle. The second assembly consisted of a 20-MHz ring transducer with a coaxial optics. The laser emitted 5-ns pulses at either 532-nm or 1064-nm, with spot sizes at the focus of 35-μm for the angled probe and 20-μm for the coaxial probe. We compared lateral resolution by scanning 12.5-μm diameter wire targets with pulse/echo US and PAI at each wavelength. We then imaged the anterior segment in whole ex vivo pig eyes and the choroid and ciliary body region in sectioned eyes. PAI data obtained at 1064 nm in the near infrared had higher penetration but reduced signal amplitude compared to that obtained using the 532-nm green wavelength. Images were obtained of the iris, choroid and ciliary processes. The zonules and anterior cornea and lens surfaces were seen at 532 nm. Because the laser spot size was significantly smaller than the US beamwidth at the focus, PAI images had superior resolution than those obtained using conventional US. PMID:20420969

Amyloid structure exhibits polymorphism on multiple length scales in human brain tissue

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

Liu, Jiliang; Costantino, Isabel; Venugopalan, Nagarajan

Although aggregation of Aβ amyloid fibrils into plaques in the brain is a hallmark of Alzheimer's Disease (AD), the correlation between amyloid burden and severity of symptoms is weak. One possible reason is that amyloid fibrils are structurally polymorphic and different polymorphs may contribute differentially to disease. However, the occurrence and distribution of amyloid polymorphisms in human brain is poorly documented. Here we seek to fill this knowledge gap by using X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid within individual plaques; among proximal plaques and in subjects with distinctmore » clinical histories. A 5 µ x-ray beam was used to generate diffraction data with each pattern arising from a scattering volume of only ~ 450 µ3 , making possible collection of dozens to hundreds of diffraction patterns from a single amyloid plaque. X-ray scattering from these samples exhibited all the properties expected for scattering from amyloid. Amyloid distribution was mapped using the intensity of its signature 4.7 Å reflection which also provided information on the orientation of amyloid fibrils across plaques. Margins of plaques exhibited a greater degree of orientation than cores and orientation around blood vessels frequently appeared tangential. Variation in the structure of Aβ fibrils is reflected in the shape of the 4.7 Å peak which usually appears as a doublet. Variations in this peak correspond to differences between the structure of amyloid within cores of plaques and at their periphery. Examination of tissue from a mismatch case - an individual with high plaque burden but no overt signs of dementia at time of death - revealed a diversity of structure and spatial distribution of amyloid that is distinct from typical AD cases. We demonstrate the existence of structural polymorphisms among amyloid within and among plaques of a single individual and suggest the existence of distinct differences in the organization of amyloid in subjects with different clinical presentations.« less

Amyloid structure exhibits polymorphism on multiple length scales in human brain tissue

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

Liu, Jiliang; Costantino, Isabel; Venugopalan, Nagarajan

Although aggregation of Aβ amyloid fibrils into plaques in the brain is a hallmark of Alzheimer's Disease (AD), the correlation between amyloid burden and severity of symptoms is weak. One possible reason is that amyloid fibrils are structurally polymorphic and different polymorphs may contribute differentially to disease. However, the occurrence and distribution of amyloid polymorphisms in human brain is poorly documented. Here we seek to fill this knowledge gap by using X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid within individual plaques; among proximal plaques and in subjects with distinctmore » clinical histories. A 5 µ x-ray beam was used to generate diffraction data with each pattern arising from a scattering volume of only ~ 450 µ3 , making possible collection of dozens to hundreds of diffraction patterns from a single amyloid plaque. X-ray scattering from these samples exhibited all the properties expected for scattering from amyloid. Amyloid distribution was mapped using the intensity of its signature 4.7 Å reflection which also provided information on the orientation of amyloid fibrils across plaques. Margins of plaques exhibited a greater degree of orientation than cores and orientation around blood vessels frequently appeared tangential. Variation in the structure of Aβ fibrils is reflected in the shape of the 4.7 Å peak which usually appears as a doublet. Variations in this peak correspond to differences between the structure of amyloid within cores of plaques and at their periphery. Examination of tissue from a mismatch case - an individual with high plaque burden but no overt signs of dementia at time of death - revealed a diversity of structure and spatial distribution of amyloid that is distinct from typical AD cases. As a result, we demonstrate the existence of structural polymorphisms among amyloid within and among plaques of a single individual and suggest the existence of distinct differences in the organization of amyloid in subjects with different clinical presentations.« less

Evaluation of the persistence, integration, histopathology and environmental release of DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2.

PubMed

Song, Xiaokai; Zhang, Zeyang; Liu, Chang; Xu, Lixin; Yan, Ruofeng; Li, Xiangrui

2016-10-15

In a previous study, the construction of the Eimeria tenella DNA vaccine pVAX1.0-TA4-IL-2 which provides effective protection against coccidiosis was described and the immunization procedure was optimized. However, the persistence, integration, histopathology and environmental release of the DNA vaccine remain unknown. In this study, the persistence, integration and histopathology of the DNA vaccine pVAX1.0-TA4-IL-2 was evaluated in chickens in the following immunization studies: (1) single-dose immunization in one-day-old chickens; (2) repeat-dose immunization in chickens; and (3) single-high-dose immunization of three batches of plasmid in chickens. The persistence, integration, histopathology of the DNA vaccine was also evaluated in mice. At 1, 1.5, 2-4 months post immunization, blood, duodenum, heart, liver, spleen, kidneys and the immunized muscle tissue were collected from ten animals of each group. Persistence and integration were evaluated using PCR with a confirmed sensitivity of 30 plasmid copies. Hematoxylin and eosin stained sections were examined for the presence of inflammation or abnormalities that may result from vaccination. Water and fecal samples were also collected from the chicken enclosures to evaluate the potential for environmental release of the DNA vaccine. Testing various tissues by PCR confirmed that plasmid DNA persisted 1.5 months in blood, heart, liver and spleen, 2 months in kidneys and muscle of injected site. Furthermore, the vaccine did not integrate with the host genome. The histopathological examinations did not show obvious inflammation or pathological damage in any tissue of the immunized chickens. Similar results were observed in mice. Moreover, the DNA vaccine was not released into the surrounding environment. These results indicate that the DNA vaccine pVAX1.0-TA4-IL-2 has potential as safe vaccine against coccidiosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Amyloid structure exhibits polymorphism on multiple length scales in human brain tissue

DOE PAGES

Liu, Jiliang; Costantino, Isabel; Venugopalan, Nagarajan; ...

2016-09-15

Although aggregation of Aβ amyloid fibrils into plaques in the brain is a hallmark of Alzheimer's Disease (AD), the correlation between amyloid burden and severity of symptoms is weak. One possible reason is that amyloid fibrils are structurally polymorphic and different polymorphs may contribute differentially to disease. However, the occurrence and distribution of amyloid polymorphisms in human brain is poorly documented. Here we seek to fill this knowledge gap by using X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid within individual plaques; among proximal plaques and in subjects with distinctmore » clinical histories. A 5 µ x-ray beam was used to generate diffraction data with each pattern arising from a scattering volume of only ~ 450 µ3 , making possible collection of dozens to hundreds of diffraction patterns from a single amyloid plaque. X-ray scattering from these samples exhibited all the properties expected for scattering from amyloid. Amyloid distribution was mapped using the intensity of its signature 4.7 Å reflection which also provided information on the orientation of amyloid fibrils across plaques. Margins of plaques exhibited a greater degree of orientation than cores and orientation around blood vessels frequently appeared tangential. Variation in the structure of Aβ fibrils is reflected in the shape of the 4.7 Å peak which usually appears as a doublet. Variations in this peak correspond to differences between the structure of amyloid within cores of plaques and at their periphery. Examination of tissue from a mismatch case - an individual with high plaque burden but no overt signs of dementia at time of death - revealed a diversity of structure and spatial distribution of amyloid that is distinct from typical AD cases. As a result, we demonstrate the existence of structural polymorphisms among amyloid within and among plaques of a single individual and suggest the existence of distinct differences in the organization of amyloid in subjects with different clinical presentations.« less

Tissue Expansion Using Hyaluronic Acid Filler for Single-Stage Ear Reconstruction: A Novel Concept for Difficult Areas.

PubMed

Inbal, Amir; Lemelman, Benjamin T; Millet, Eran; Greensmith, Andrew

2017-10-16

Auricular reconstruction is one of the most challenging procedures in plastic surgery. An adequate skin envelope is essential for cartilage framework coverage, yet few good options exist without additional surgery. We propose a novel method for minimally invasive tissue expansion, using hyaluronic acid (HA) filler to allow for single-stage ear reconstruction. To introduce the novel concept of HA filler for tissue expansion in ear reconstruction, and as an alternative to traditional expansion techniques. Macrolane is a large particle HA gel developed for large volume restoration. Expansion of the non-hair-bearing mastoid skin was performed in our clinic weekly or every other week. Final expansion was completed one week prior to reconstructive surgery. Tissue from one patient's expanded pocket was sent for histological analysis. Ten patients underwent single-stage auricular reconstruction with preoperative expansion. Injection sessions ranged from 7 to 13 (mean, 9.7). Mean injected volume per session was 2.03 mL per patient, for an average total of 19.8 mL (range, 14.5-30 mL). There were no major complications. One minor complication required removal of exposed wire from the antihelix in the office. Hematoxylin and eosin stain revealed similar histology to that seen with traditional expanders. This novel expansion technique using serial HA injections allowed for optimized skin coverage in single-stage ear reconstruction. The concept of tissue expansion using HA filler is a new frontier for research that may be applicable to other arenas of reconstruction. 4. © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com