Epoxy Embedding Research Articles

MORPHOMETRIC FEATURES OF THE STRUCTURAL COMPONENTS OF THE HEMOMICROCIRCULATORY BED IN THE PERIVULNAR REGION OF THE CAECUM IN WOUND DEFECT SUTURED WITH POLYFILAMENT SUTURE MATERIAL

The aim: The study focuses on the morphometric features specification concerning the structural components of the hemomicrocirculatory bed of the perivulnar region of the cecum in rabbits when suturing a wound defect with synthetic polyfilament surgical suture Vicryl. Materials and methods: The research was carried out on rabbits (n = 30: 5 animals made up the control group and 25 animals made up the experimental group) and included surgery based on the method of surgical intervention on the large intestine patented by the authors, which was performed in the conditions of the operating room at the Department of Clinical Anatomy and Operative Surgery. When determining the main morphometric parameters, we took biopsy specimens of the cecum and used paraffin and epoxy resin embedding considering the generally accepted methods. Results: The study determined that the implantation of Vicryl surgical suture material in the tissues of the cecum became a trigger for the formation of active inflammatory focus and occurrence of leukocyte infiltrate in the perivulnar region. Conclusions: Arterioles, as a resistive link, capillaries, as an exchange link and venules, as a capacitive link in the hemomicrocirculatory bed, actively participate in response to the inflammatory process and formation of a connective tissue scar. The process of structural organization of microvessels begins in the formed granulation tissue in the perivulnar region of the cecum starting from the 14th day of the experiment.

Evaluation of the Effect of the Microscopic Glass Surface Protonation on the Hard Tissue Thin Section Preparation

In this study, a new procedure for mounting tissue blocks was described while cutting and grinding the section remains tightly bound to the inert glass surface both chemically and micro mechanically allowing good quality specimens for staining and microscopic analysis. The micromechanical interlocking was achieved by using of frosted glass, the chemical binding was made with 10-methacryloyloxydecyl dihydrogen phosphate monomer (10-MDP) containing bond material. The glass surface activation was achieved by nitric acid etching and the surface was characterized by zeta potential, X-ray photoelectron spectroscopy (XPS), and contact angle measurements. Cylindrical samples were prepared from epoxy embedding materials, cortical bovine bone, and dental titanium to investigate the shear bond strengths (SBS) to microscopic glass slide compared to a routinely used thermoplastic adhesive. Based on the experiments it was found that the micromechanical retention combined with MDP containing bond material improved the SBS data compared to the thermoplastic adhesive. The acid etched glass became positively charged that significantly increased the SBS data of bone and titanium compared with the uncharged version. Therefore, the thickness of the undecalcified bone section with metal can safely reduce to improve histological microscopic analysis.

A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes

This manuscript reports a novel procedure to imaging growth plate chondrocytes by using confocal microscopy. The method is based on fixed undecalcified bone samples, in-block staining with eosin, epoxy resin embedding and grinding to obtain thick sections. It is simple, inexpensive and provides three-dimensional images of entire chondrocytes inside their native lacunae. Quantitative analysis of volume, shape and cytoplasm density of chondrocytes at different strata of the growth plate allowed to objectively grade chondrocytes of the growth plate in seven different clusters. These seven categories of chondrocytes were subsequently evaluated by immunohistochemistry of some well-defined molecular landmarks of chondrocyte differentiation. Furthermore, immunohistochemical analysis of proteins responsible for ionic changes and water transport allowing chondrocyte swelling during hypertrophy was also performed. Results obtained indicate that four subphases can be defined in the pre-hypertrophic zone and three subphases in the hypertrophic zone, a fact that raises that chondrocytes of the growth plate are less homogeneous than usually considered when different zones are defined according to subjective cell morphological criteria. Results in the present study provide a technological innovation and gives new insights into the complexity of the process of chondrocyte differentiation in the growth plate.

Influence of unmodified and modified sutures on experimental abdominal adhesive process

Experimental assessment of the effect of modified and unmodified surgical suture material on abdominal adhesive process. The study was performed on male rats of the Wistar subpopulation. There were 5 animals in each group. In all animals, midline abdominal incision was followed by suturing the parietal peritoneum with modified and unmodified suture material. All animals were euthanized with carbon dioxide vapors in 14 days after surgery. Macro- and microscopic assessment of severity of abdominal adhesive process was carried out. Two types of preparation of excised complexes 'peritoneum-suture material-adhesion' were applied for histological examination: paraffin sections and embedding in epoxy resin. Specimens were stained by Van Gieson and with methylene blue solution. Histological specimens were examined using Axio Imager A1 light microscope (Zeiss, Germany). Polypropylene filaments result extensive adhesions occupying about 75% of the area. Adhesions have a dense structure with signs of vascularization. Modification of suture material with solution of polyhydroxybutyrate/hydroxyvalerate and heparin reduce severity of adhesions. The use of modified suture material was followed by adhesions with more loose structure, no signs of vascularization. Adhesions occupied less than 25% of the area. Histological examination of excised complexes 'peritoneum-suture material-adhesion' revealed accumulation of inflammatory cells around the unmodified suture material, while there were no signs of tissue inflammatory process around the modified sutures. Application of polyhydroxybutyrate/hydroxyvalerate and heparin on the surface of surgical sutures is an effective method for prevention of abdominal adhesions.

STRUCTURE OF CALCIFICATES IN HUMAN CAROTID ARTERY ATHEROSCLEROTIC PLAQUES BY MEANS OF BACKSCATTERED SCANNING ELECTRON MICROSCOPY

Aim of the study was to investigate the atherosclerotic calcification employing our original technique of tissue staining, embedding, and backscattered scanning electron microscopy. Materials and methods . Atherosclerotic plaques excised during the carotid endarterectomy have been fixed in 10 % neutral phosphate buffered saline formalin for 24 hours, stained in 2 % osmium tetroxide for 60 hours and in alcoholic uranyl acetate for 5 hours with the subsequent epoxy resin embedding, grinding, polishing, lead citrate counterstaining for 7 minutes, sputter coating with carbon and backscattered scanning electron microscopy. We then analysed localisation, structure, and microenvironment of the calcium deposits. Results . Within the atherosclerotic plaques, we identified 3 distinct calcification morphologies: compact homogenous macrocalcifications, sheet-like heterogeneous macrocalcifications along the destructed collagen and elastin fibers, and microcalcifications around large calcium deposits with the sharp margins. However, even relatively homogenous compact macrocalcifications had uneven distribution of electron density. In conjunction with groups of mineral deposits of distinct calcium phosphate phases including nascent calcified loci, this testified to ongoing calcium phosphate maturation while multiple microcalcifications merging into the single macrocalcification indicated ossification. Compact ossifying macrocalcifications with smooth margins frequently had a dense connective tissue capsule. The microenvironment of calcium deposits was often characterised by leaky neovessels. Conclusion . Diversity of calcification morphologies and ossification in atherosclerotic plaques is similar to those observed in calcific aortic valve disease and bioprosthetic heart valve failure. Combined with atherosclerotic plaque disaggregation followed by flow cytometry and singlecell RNA sequencing, our technique may improve our understanding of atherosclerotic calcification and lead to the identification of appropriate therapeutic targets to prevent or retard this process.

High-Throughput Immunofluorescence and Electron Tomography to Characterize Centrosomal Aberrations in Plasma Cell Neoplasia

High-Throughput Immunofluorescence and Electron Tomography to Characterize Centrosomal Aberrations in Plasma Cell Neoplasia

MEosEM withstands osmium staining and Epon embedding for super-resolution CLEM.

Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO4) treatment for improved SR-CLEM.

Optimization of ultrastructural preservation of human brain for transmission electron microscopy after long post-mortem intervals

Electron microscopy (EM) provides the necessary resolution to visualize the finer structures of nervous tissue morphology, which is important to understand healthy and pathological conditions in the brain. However, for the interpretation of the micrographs the tissue preservation is crucial. The quality of the tissue structure is mostly influenced by the post mortem interval (PMI), the time of death until the preservation of the tissue. Therefore, the aim of this study was to optimize the preparation-procedure for the human frontal lobe to preserve the ultrastructure as well as possible despite the long PMIs. Combining chemical pre- and post-fixation with cryo-fixation and cryo-substitution (“hybrid freezing”), it was possible to improve the preservation of the neuronal profiles of human brain samples compared to the “standard” epoxy resin embedding method. In conclusion short PMIs are generally desirable but up to a PMI of 16 h the ultrastructure can be preserved on an acceptable level with a high contrast using the “hybrid freezing” protocol described here.

Correlations between As in Earthworms' Coelomic Fluid and As Bioavailability in Highly Polluted Soils as Revealed by Combined Laboratory X-ray Techniques.

Combined X-ray-based spectroscopy techniques were applied to investigate arsenic (As) bioaccumulation in earthworms (Eisenia andrei) exposed to six field-collected polluted soils (58-13 330 mg As kg-1). After 14 days of exposure to the arsenious soils, the As distribution in earthworms was examined by micro-X-ray fluorescence spectroscopy (μXRF), after epoxy resin embedding and preparing thin sections. Similar to μXRF data, XRF-computed tomography (XRF-CT) confirmed As accumulation in the coelom of intact earthworms. Therefore, total-reflection XRF was used to determine total As within both the whole earthworm's body (AsE) and coelomic fluid extracts (AsF). Bioaccumulation data (AsE and AsF) were thereafter evaluated in relation to total As concentration in soils (AsT) and to As mobile fraction in soils. A significant linear correlation (R2 = 0.97) was found between AsE and AsF, indicating that the As sequestrated into the coelomic fluid may reflect the total body concentration. Therefore, we may conclude that the As concentration in the coelomic fluid can be used as an index of As availability. This paper demonstrates that by combining different laboratory X-ray analytical techniques, compartmentalization and bioavailability of potentially toxic elements can be visualized and quantified within indicator-living organisms, thus contributing to an improved risk assessment for contaminated soils.

Hypertensive Neuropathy: A New Clinical Entity?

Hypertension is a main risk factor for stroke and vascular dementia and may cause important changes to the cerebrovascular tree, turning the brain more susceptible to infarcts, microaneurysms and ischemia. In spite of the well documented influence of hypertension on the brain, data on the sensitivity of peripheral nerves in hypertension is scarce. Our laboratory has been working for decades to help understanding how hypertension affects peripheral nerve morphology and function, using the spontaneously hypertensive rats (SHR) model. Spontaneously hypertensive rats (SHR), first inbred from Wistar Kyoto rats (WKY), are considered a good experimental model of human essential hypertension. The purpose of this study was to investigate the alterations on the morphology and morphometric data on sural, phrenic, vagus and recurrent laryngeal nerves of SHR, previous to the development of hypertension and with well‐established hypertension. Male and female SHR and normotensive WKY rats aged 5, 8 and 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural, vagus, phrenic and recurrent laryngeal nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter. Nerves from young animals did not show differences between sides and genders. Significant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR in all nerves. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified in nerves from adult hypertensive animals. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders. These results indicate that hypertension altered important morphometric parameters related to nerve conduction in hypertensive animals. Moreover the comparison between males and females of WKY and SHR showed that the morphological and morphometric alterations due to hypertension are not gender related. The presence of a so called “hypertensive neuropathy” is documented with endoneural vascular lesions, axonal atrophy and loss of small myelinated fibers.Support or Funding InformationSupport: CNPq, FAPESP and FAEPAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FIB-SEM imaging properties of Drosophila melanogaster tissues embedded in Lowicryl HM20.

Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high-pressure freezing and freeze-substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze-substitution with per-mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time-efficient FIB-SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM-TEM workflows to comprehensively analyse biological structures. LAY DESCRIPTION: Focused ion beam scanning electron microscopy is becoming a widely used technique for the three-dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy-based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub-cellular and sub-organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl-embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures.

Amorphous Calcium Carbonate Granules Form Within an Intracellular Compartment in Calcifying Cyanobacteria.

The recent discovery of cyanobacteria forming intracellular amorphous calcium carbonate (ACC) has challenged the former paradigm suggesting that cyanobacteria-mediated carbonatogenesis was exclusively extracellular. Yet, the mechanisms of intracellular biomineralization in cyanobacteria and in particular whether this takes place within an intracellular microcompartment, remain poorly understood. Here, we analyzed six cyanobacterial strains forming intracellular ACC by transmission electron microscopy. We tested two different approaches to preserve as well as possible the intracellular ACC inclusions: (i) freeze-substitution followed by epoxy embedding and room-temperature ultramicrotomy and (ii) high-pressure freezing followed by cryo-ultramicrotomy, usually referred to as cryo-electron microscopy of vitreous sections (CEMOVIS). We observed that the first method preserved ACC well in 500-nm-thick sections but not in 70-nm-thick sections. However, cell ultrastructures were difficult to clearly observe in the 500-nm-thick sections. In contrast, CEMOVIS provided a high preservation quality of bacterial ultrastructures, including the intracellular ACC inclusions in 50-nm-thick sections. ACC inclusions displayed different textures, suggesting varying brittleness, possibly resulting from different hydration levels. Moreover, an electron dense envelope of ∼2.5 nm was systematically observed around ACC granules in all studied cyanobacterial strains. This envelope may be composed of a protein shell or a lipid monolayer, but not a lipid bilayer as usually observed in other bacteria forming intracellular minerals. Overall, this study evidenced that ACC inclusions formed and were stabilized within a previously unidentified bacterial microcompartment in some species of cyanobacteria.

Immunohistochemestry for nerve morphometry?

Peripheral nerve biopsy is an invaluable aid to diagnosis carefully selected patients. The nerves histological evaluation gives clues for investigating diseases mechanisms and causes, as well as guides the therapeutic planning of inflammatory, infectious, demyelinating or degenerative lesions [1]. Nerves histological processing method that provides the best image quality is the epoxy resin embedding. Thus, nerve sections can be observed by light and/or transmission electron microscopy (TEM) and classes of nerve fibers can be morphometrically identified and studied. Epoxy resin embedding and TEM is considered the gold standard method for unmyelinated fibers identification and quantification. More recently, the immunohistochemistry technique (IHC) is being added to the inflammatory neuropathy investigation and the most important of the panel of antibodies employed are those needed to differentiate lymphocytes into T (CD3þve) and B cells (CD20þve). In addition, antibodies against macrophages, myelin basic protein, neurofilaments, and epithelial membrane antigen (EMA) (for perineurial cells) are included [1]. Neurotransmitters and their fiber “carrier” are much less explored. We suggest that it would be possible to use the IHC to identify nerve fiber classes as an alternative to TEM and morphometry, with the gain on functional information. To investigate this hypothesis we used IHQ and immunofluorescence (IF) to immunolabel small myelinated and unmyelinated fibers in sural nerves of rats. The nerves were surgically removed, immersed in paraformaldehyde 4% for 18 hours, and cryoprotected in increasing concentrations of sucrose solutions, before transversal cryosectioning (12μm). For IHC, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:5.000, 18h) followed by anti‐rabbit IgG (1:500; 2h) and Extravidin‐HRP (1:1500, 2h). A reaction with diaminobenzidine intensified with nickel revealed immunoreactivity, observed by light microscopy. For IF, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:1.000, 18h) followed by anti‐rabbit IgG (Alexa Fluor® 594) (1:200, 2h). The immunoreactivity was observed by fluorescence microscopy. Both protocols worked on transverse sections to identify substance P immunolabeled small myelinated and unmyelinated fibers. Results were compared with transverse sections prepared for TEM and the IHC results showed labeled small myelinated and unmyelinated axons intermingled with large unlabeled myelinated axons, with preservation of their myelin sheath. The IHC transverse sections were comparable to the epoxy resin embedded for small fiber localization in the endoneural space. Further studies on quantification of these fibers will be performed comparing both methods (IHC and IF) with the TEM images in order to show that IHC might also be a reliable method for quantifying axons.Support or Funding InformationFinancial support: FAPESP, CNPq, CAPES and FAEPAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanical Properties of Alumina Nanofilled Polymeric Composites Cured with DDSA and MNA

Reinforced concrete is widely used in structures. New materials to replace both the steel and the concrete have been studied in many research centres. One of the possibilities for the reinforcement is the partial or total replacement of the steel bars by new composite materials. Nano composites are very promising, and an investigation line was developed to this end by an interdisciplinary team. On this work, the mechanical properties of epoxy resin nanocomposites (EPNCs) filled with α-Al2O3 nanoparticles (NPs) with irregular shape and approximately 100 nm maximum diameter size was investigated. The variable study was the alumina NPs contents: 1, 3 and 5 wt.%. The NPs were previously pretreated with a silane agent (APTES). Two hardeners, 3-dodec-2-enyloxolane-2,5-dione (DDSA) and 8-methyl-3a,4,7,7a-tetrahydro-4,7-methano-2-benzofuran-1,3-dione (MNA), frequently used in epoxy resin embedding tissues, were used simultaneously for this study. Unlike other hardeners, DDSA does not need curing treatment, constituting a novel application and a saving time-energy during the manufacturing process. Considering the mechanical behaviour, it was observed that the EPNCs filled with 5 wt.% of alumina NPs showed the maximum improvement in flexural modulus, around 14 % when compared to the pristine EP sample. No relevant effect was observed on the flexural strength by adding alumina NPs. Additionally, the maximum increase observed for hardness, and Young’s modulus were about 13 % and 28 %, respectively (the maximum increase was observed at 3 wt.%).

Co-localized AFM-Raman: A powerful tool to optimize the sol-gel chemistry of hybrid polymer membranes for fuel cell

Proton-Exchange Membrane Fuel Cells is a promising emission free energy technology. New generation of hybrid membrane for hydrogen fuel cells were produced from commercial sPEEK membranes, chemically and mechanically stabilized with a Sol-Gel (SG) phase. To study the process-structure-properties relationship of our alternative membranes we coupled Atomic Force Microscopy and Raman microspectroscopy on membranes prepared by cryo-ultramicrotomy without epoxy embedding. This paper demonstrates the powerfulness of co-localized AFM-Raman analysis, revealing the inner structure of hybrid membranes. We obtained quantitative data on the diffusion/condensation of SG precursors inside the sPEEK membrane. Co-localized analyses revealed the formation of skin layers with lower SG concentration on both sides of the membrane. The diffusion of species from the SG phase at the cross-section surface was observed at the first step of fabrication (insufficient SG condensation) and disappeared after hydrothermal treatments (improved SG condensation). The nano-mechanical data revealed a densification of the SG phase through the fabrication process.

Improving FIB-SEM Reconstructions by Using Epoxy Resin Embedding

Segmentation of FIB-SEM data remains a major challenge to achieving accurate reconstructions. Epoxy embedding is used in this article to eliminate background data from SEM images in order to aid in segmentation and generate 3D reconstructions of conventional high surface area CLs from FIB-SEM images. Two CL samples with the same composition but different hours of operation, namely, beginning of life (BOL), i.e. post conditioning, and end of life (EOL), i.e., post failure, were used. The samples were imaged with and without epoxy embedding to characterize the difference in the structure of the sample due to sample preparation. Further, the use of two CL samples allowed the quantification of the differences in the structure of the CL due to degradation and ensure validity of the sample preparation across multiple CL samples. Image analysis showed that a simple thresholding algorithm, such as Otsu, was sufficient to accurately segment the images for the sample with epoxy embedding due to the lack of background features. Statistical descriptors, such as porosity, two-point correlation function, chord length function, specific interface area and pore size distribution, and effective diffusion coefficient are used to quantify the differences in the reconstructed structures of the CLs with different sample preparation. Results showed that the CL reconstructions of the epoxy embedded samples had a higher porosity, larger pore sizes and lower specific interface area due to the more accurate segmentation and removal of background features.

Structural Features of the Nucleolus in the Mouse Germinal Vesicle Oocyte Revealed by AFM, SEM, and ToF-SIMS

The structural organization of the biological material in the nucleoli (Nucleolus-Like Bodies (NLBs)) in mouse GV oocytes has been studied with atomic force microscopy (AFM), scanning electron microscopy (SEM), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Experimental samples are prepared by quick plunge freezing, followed by freeze-drying, embedding in Epon epoxy resin, and sectioning with a microtome. The biological material in the NLBs is found to be organized as a domain structure of densely packed granules with sizes from tenths of a micron to a micron.

Improving FIB-SEM Reconstructions By Using Epoxy Resin Embedding

Focus ion beam-scanning electron microscopy (FIB-SEM) is a viable technique to visualize and reconstruct the morphology of polymer electrolyte membrane fuel cell (PEMFC) porous media, such as catalyst layers (CLs) (1,2) and micro-porous layers (MPLs) (3,4), with a high resolution of few nanometers. FIB-SEM provides morphological information about the solid and pore phase from which several statistical descriptors and effective transport properties (2) can be extracted to characterize the random heterogeneous structure. A major challenge in the analysis of the FIB-SEM images is the binarization of the grey scale images into the solid and pore phases (5). This problem arises because the SEM detects the solid particles inside the pores which are not in the plane of the image. This adds to the complexity of the segmentation by introducing an additional dependency to not only distinguish between the pore and solid phase but also, distinguish between solid phase in the plane of the image and solid phase in the background. In order to tackle this problem, advanced segmentation algorithms have been developed (5,6). However, these algorithms lack consistency across datasets and often require manual intervention which limits their robustness. Binarization can be improved by enhancing the sample preparation for the image acquisition. This has been done by penetrating the pores with filling material by embedding the sample in an epoxy based resin (7,8) or silicon based resin (9), platinum vapor deposition (10) and atomic layer deposition (ALD) of zinc oxide (11). ALD and platinum vapor deposition result in partial filling of the pores but provide a higher contrast from the carbon rich backbone. However, due to the partial filling manual corrections and additional image filtering operations are required post segmentation of the images. Epoxy and silicon based resin embedding results in complete filling of the pores but provides a very low contrast for the images. Ghosh et al. (8) recently showed that images obtained using the backscattered electrons (BSE) provide a better contrast than the secondary electron (SE) images (8,9) for a PEMFC CL embedded with an epoxy based resin. However, Ghosh et al. (8) only examined and compared 2D images of the CL. Comparison of a full 3D reconstruction of the PEMFC CL using FIB-SEM with and without epoxy embedding has yet to be performed. In the present article, 3D reconstructions of a conventional high surface area CL are performed using FIB-SEM images of the same sample treated with and without epoxy embedding to analyze the differences in image analysis and structure for both the modes of sample preparation. Figure 1 below shows the raw images obtained using SE mode for the sample without epoxy embedding and BSE mode for the sample with epoxy embedding. Multiple stacks of images are processed from both the datasets to ensure global validity of the results. Image processing operations are performed to further enhance the raw images before segmentation. Image analysis reveals that a simple thresholding algorithm, such as Otsu, is sufficient to accurately segment the images for the sample with epoxy embedding due to the lack of background features. Comparison of the reconstructions for the two modes suggests an increase in porosity and chord length function (12) for the sample imaged using epoxy embedding due to a more accurate segmentation. Transport simulations are being carried out to compute the effective transport properties.

3D reconstruction of biological structures: automated procedures for alignment and reconstruction of multiple tilt series in electron tomography.

Transmission electron microscopy allows the collection of multiple views of specimens and their computerized three-dimensional reconstruction and analysis with electron tomography. Here we describe development of methods for automated multi-tilt data acquisition, tilt-series processing, and alignment which allow assembly of electron tomographic data from a greater number of tilt series, yielding enhanced data quality and increasing contrast associated with weakly stained structures. This scheme facilitates visualization of nanometer scale details of fine structure in volumes taken from plastic-embedded samples of biological specimens in all dimensions. As heavy metal-contrasted plastic-embedded samples are less sensitive to the overall dose rather than the electron dose rate, an optimal resampling of the reconstruction space can be achieved by accumulating lower dose electron micrographs of the same area over a wider range of specimen orientations. The computerized multiple tilt series collection scheme is implemented together with automated advanced procedures making collection, image alignment, and processing of multi-tilt tomography data a seamless process. We demonstrate high-quality reconstructions from samples of well-described biological structures. These include the giant Mimivirus and clathrin-coated vesicles, imaged in situ in their normal intracellular contexts. Examples are provided from samples of cultured cells prepared by high-pressure freezing and freeze-substitution as well as by chemical fixation before epoxy resin embedding.

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

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.