Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

In situ flat embedding of monolayers and cell relocation in the acrylic resin LR White for comparative light and electron microscopy studies

Summary A simple and reliable method has been developed for the in situ LR White embedding of cell monolayers grown on glass cover-slips. Combined with cytochemical or immunological procedures, this technique allows light and/or electron microscopy investigations of a large number of cells in the same horizontal plane within a relatively short period of time. It can be applied to cells grown on microgrid finder cover-slips which allows a distinct site of even an individual cell of a monolayer to be studied at first at the light microscope level and subsequently at the electron microscope level. Hence, it is also suitable for controlling manipulation of single cells, followed by their serial sectioning after relocation in the electron microscope.     

Ultrastructure of the bacterial spores and crystals of various Bacillus thuringiensis serotypes

Spores and crystals of various Bacillus thuringiensis serotypes were studied by electron microscopy. The serotypes were shown to differ in the fine structure of the exosporium and crystals. The packing parameters of morphological subunits were determined by analysing electron photomicrographs by the technique of optical diffraction and filtration. Some of the strains are characterised by hypersynthesis of surface spore structures. The individual morphological properties of crystals can be used for their differentiation.     

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron‐microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high‐pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non‐correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron‐microscopic protocols, making them more accessible to a wider range of researchers. Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time‐demanding procedure of specimen preparation for any kind of TEM.     

Epoxy-Slide Embedment of Cytochemically Stained Tissues and Cultured Cells for Light and Electron Microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cost-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

Epoxy-slide embedment of cytochemically stained tissues and cultured cells for light and electron microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cast-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

Electron microscopy of free-floating cells: thin-layering technique, and selection of individual cells for ultramicrotomy.

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily be relocated by the aid of the pre-embedding reference micrographs.     

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.     

Electron Microscopy of Free-Floating Cells: Thin-Layering Technique, and Selection of Individual Cells for Ultramicrotomy

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily he relocated by the aid of the pre-embedding reference micrographs.     

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads.     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

A rapid technique for preparing microorganisms for transmission electron microscopy.

A rapid and efficient method of preparing microorganisms for transmission electron microscopy is reported. In developing the method Salmonella, streptococcal, and protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

A Single Method for Cryofixation and Correlative Light, Electron Microscopy and Tomography of Zebrafish Embryos

The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples.     

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated ^1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated ^4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot ^8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.     

A Simple Screening Procedure for Evaluating Central Nervous System Tissue Sections Showing Structural and Cytochemical Alterations of the Blood-Brain Barrier

A simple method for rapidly screening and evaluating many areas of central nervous system tissue before and after flat embedding in Beem capsules is described. This method uses light microscopy to select regions surrounding needle track injuries of brain tissue for subsequent fine structural and enzyme cytochemical analysis of the blood-brain barrier. The mouse cerebral cortex was sectioned with a tissue chopper at 40-50 μm and reacted with diaminobenzidine to demonstrate the presence of exogenous horseradish peroxidase near an injured central nervous system site. Following the enzyme reaction, both osmicated and unosmicated tissue slices were processed for routine electron microscopy, infiltrated with unpolymerized resin, and evaluated on glass slides by light microscopy prior to flat embedding and polymerization. Numerous tissue specimens can be screened in this way for maximum information per tissue slice, and extra tissue samples can be polymerized on the glass slides and conveniently stored for future sectioning.     

Demonstration of ricin within the mammalian para-aortic lymph node I. Comparison of the localization, after intramuscular injection, with three immunocytochemical methods

Summary Following a supralethal injection of ricin into thigh muscle of the adult rat, the toxin was demonstrated post-mortem in the para-aortic lymph node, ipsilateral to the side of injection. The relative merits of two immunoenzyme methods, peroxidase anti-peroxidase (PAP) and avidin—biotin—peroxidase complex (ABC) and a silver-enhanced immunogold method (IGSS) were assessed in the detection of ricin in the lymph node tissue. The toxin was clearly seen to be located in association with histiocytes found both within and lining the sinuses of the nodes and also, in some cases, in the subcapsular sinus of the node; the toxin was not demonstrable within lymphoid follicles by light microscopy. However, using electron microscopy and the IGSS technique, cells carrying discrete particles of gold could be visualized within follicular areas. The IGSS and ABC-peroxidase methods were both found to give excellent results without background staining at the light microscopy level. However, when these techniques were used prior to embedding and viewing by electron microscopy, the IGSS technique proved to be far superior.