Identification of Cells by Backscattered Electron Imaging of Silver Stained Bulk Tissues in Scanning Electron Microscopy

Anuran tadpole tail muscle was stained en bloc by a modified light microscope silver stain for light microscopy and freeze-fractured in liquid nitrogen after partial dehydration with ethanol. The fractured specimens were observed in both secondary electron and backscattered electron modes in a scanning electron microscope. Since the cell nuclei specifically stained with silver provided high contrast against the unstained background due to atomic number contrast of backscattered electron image, various cells were easily identified by a comparison of secondary electron images and compositional images of backscattered electron signals.     

Scanning electron microscope histochemistry: The use of backscattered electrons to identify epidermal Langerhans cells in the scanning electron microscope

Summary Epidermal sheets reacted for ATPase were examined in the scanning electron microscope and images based on atomic number contrast formed by collecting the backscattered electrons. Langerhans cells lying within the tissue were revealed and could be related to surface structures by reference to conventional secondary electron images.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

Mapping gold-labeled IgE receptors on mast cells by scanning electron microscopy: receptor distributions revealed by silver enhancement, backscattered electron imaging, and digital image analysis

Immunogold labeling and silver enhancement techniques are widely used to determine density and distribution of cell membrane receptors by light and transmission electron microscopy. However, these techniques have not been widely used for receptor detection by scanning electron microscopy. We used antigen- or protein A-conjugated colloidal gold particles, together with silver enhancement, sequential secondary and back-scattered electron imaging (SEI and BEI), and digital image processing, to explore cell surface distribution of IgE-receptor complexes on RBL-2H3 cells, a rat leukemia line that provides a model for the study of mucosal mast cells. Cells were first incubated with a monoclonal antidinitrophenol IgE (anti-DNP-IgE) that binds with high affinity to cell surface IgE receptors. The resulting IgE-receptor complexes were cross-linked either with the multivalent antigen, DNP-BSA-gold, or with a polyclonal anti-IgE antibody. Antibody-treated cells were labeled after fixation with protein A-gold. Fixed, gold-labeled cell monolayers were silver enhanced (or not), dehydrated, critical point-dried, and coated with gold-palladium (for SEI analysis) or carbon (for combined SEI/BEI analysis). They were observed in an Hitachi S800 SEM equipped with a field emission tip and a Robinson backscattered electron detector. An image processor (MegaVision 1024XM) digitized images directly from the S800 microscope at 500-1000 line resolution. Silver enhancement significantly improves detection of gold particles in both SEI and BEI modes of SEM. On gold-palladium-coated samples, 20-nm particles are resolved by SEI after enhancement. BEI resolves 15-nm particles without enhancement and 5- or 10-nm particles are resolved by BEI on silver-enhanced, carbon-coated samples. Neither BEI nor SEI alone can yield high resolution topographical maps of receptor distribution (BEI forms images on the basis of atomic number contrast which reveals gold but not surface features). Image analysis techniques were therefore introduced to digitize, enhance, and process BEI and SEI images of the same field of view. The resulting high-contrast, high-resolution images were superimposed, yielding well-resolved maps of the distribution of antigen-IgE-receptor complexes on the surface of RBL-2H3 mast cells. The maps are stored in digital form, as required for computer-based quantitative morphometric analyses. These techniques of silver enhancement, combined BEI/SEI imaging, and digital image analysis can be applied to analyze density and distribution of any gold-labeled ligand on its target cell.     

Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.     

Scanning electron microscopy of macrophages in the tail musculature of the metamorphosing anuran tadpole,Rana japonica

Summary Scanning electron microscopy was used to investigate the morphological changes of the tail musculature of the metamorphosing anuran tadpole, attention being focused on phagocytosis by macrophages. Muscle fibers were stained en bloc with silver and freeze-fractured during dehydration, or torn after drying. Samples were sputter-coated with gold-palladium and observed in both secondary electron- and back-scattered electron modes with a scanning electron microscope.Various cells were identified by the methods of secondary electron- and back-scattered electron images. Some macrophages lying between muscle fibers at prometamorphic stages possessed numerous finger-like projections and well-developed ruffles. During degeneration of muscle fibers macrophages collected in the degenerating region and invaded the space between the disordering myofibrils. In advanced stages the numbers of macrophages clearly increased on or around the degenerating muscle fibers. At the climactic stage fragmented muscles were entrapped and then engulfed by the macrophages. With the completion of phagocytosis, the macrophages became globular with reduction of the ridge-like ruffles. Macrophages may play a role not only in scavenging the fragmented muscle fibers, but also using their long processes in active formation of the fragments.     

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.     

Imaging vesicular dispersions with cold-stage electron microscopy

A fast-freeze, cold-stage transmission electron microscopy technique which can incorporate in situ freeze-drying of the sample is described. Its use in elucidating structure in unstained and stained, hydrated and freeze-dried, aqueous vesicular dispersions of biological and chemical interest is demonstrated with vesicles of l-α-phosphatidylcholine (bovine phosphatidylcholine) and of the synthetic surfactant sodium 4-(1′-heptylnonyl)benzenesulfonate (SHBS). The contrast features observed in transmission electron microscope images of frozen, hydrated samples are identified and explained with the dynamical theory of electron diffraction. Radiolysis by the electron beam is shown to increase contrast in vesicle images and to change their structure and size. Micrographs illustrate the freeze-drying of a dispersion in the microscope; the process causes vesicles to shrink and collapse.     

Focussed ion beam milling and scanning electron microscopy of brain tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.     

Three-dimensional image of tubulin in zinc-induced sheets, reconstructed from electron micrographs

A three-dimensional image of zinc-induced brain tubulin sheets has been reconstructed by computer from digitized electron microscope images of negatively stained specimens. Different views of the sheets were obtained by tilting the specimens in the microscope and also by sectioning normal to the plane of the sheets. The overall resolution of the data is about 2 nm. The features of the resulting three-dimensional model suggest an explanation for the somewhat variable appearance of tubulin subunits in electron microscope images of negatively stained intact and opened-out microtubules.     

Scanning electron microscopy: Identification of mast cells by indirect cathodoluminescence

Summary The epithelial tissues of the rabbit gall bladder reacted for acid mucosaccharides were studied with the electron microscope. A series of acid mucosaccharide-containing ultrastructures of the gall bladder epithelium were observed in specimens treated with dialyzed iron, colloidal thorium and ruthenium red. In the epithelium stained with dialyzed iron, reactive ultrastructures are not only extra- but intracellular; the surface coat of the plasma membrane, pinocytotic vesicles, granules of secretion and certain elements of the Golgi apparatus. In the epithelial tissues stained by colloidal thorium or ruthenium red, the surface coat of the plasma membrane is the only ultrastructure which is reacted positively for the acid mucosaccharide stains. The present images of ultrastructural elements containing acid mucosaccharides are taken to indicate a multiple function of the substances in rabbit gall bladder epithelium and are well correlated with the results of previous light and electron microscopic studies on the gall bladder epithelium of various vertebrate species.     

The prophase stages in human foetal oocytes studied by light and electron microscopy

Summary The progression of the prophase stages of meiosis in human foetal ovaries is reported from a series of aborted foetuses spanning the first two trimesters of gestation. A surface-spreading technique allowed cells to be examined at both light and electron microscope levels. The pachytene stage was specifically examined for evidence of synaptic or other abnormalities. Two observations of interest are the relatively high incidence with which errors of pairing occur and second the state of thickening observed when such unpaired axes are stained with silver.     

Enzyme-histochemical identification of lymphatic vessels by light and backscattered image scanning electron microscopy

The walls of lymphatics are characterized by strong 5'-nucleotidase activity, whereas those of blood capillaries reveal significantly lower or no activity. Alkaline phosphatase activity, on the other hand, is markedly higher in blood capillaries than in lymphatic vessels. On the basis of such characteristics, lymphatics and blood capillaries were distinguished histochemically in rat stomach using 5'-nucleotidase-alkaline phosphatase double staining. The distribution and intensity of lead-demonstrated 5'-nucleotidase activity in lymphatic vessels could be determined by comparing the images of the same histochemically stained cryostat section as seen by light and backscattered image scanning electron microscopy. The specificity of the 5'-nucleotidase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the 5'-nucleotidase incubation medium. The products of the 5'-nucleotidase activity were deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells.     

Observations on charging effects of non-conductive and uncoated materials by ion beam pre-bombardment in scanning electron microscopy

The specimen charging defects of non-conductive materials in scanning electron microscopy are discussed with reference to the surface electric field generated by the illuminating electron beam dose. If the charge density depends on the relaxation time constant as defined by a product of the permittivity and resistivity when known or available, the electric field can be evaluated by the incident dose stored when illuminated by an electron scanning beam.It was found by observation that uncoated or non-conductive materials pre-bombarded by a positive ion beam, which contributes to the generated negative field, together with the charging effects, could be eliminated at the optimum time of neutralization.In the normal process of double fixation and staining of biological specimens, the local electric field produces increased contrast due to polarization effects. The dark and bright images of secondary and backscattered electrons, respectively, can be analysed by taking into account local polarization, in addition to voltage contrast.     

Ultrastructural cytochemical localizations by back-scattered electron imaging of white blood cells

White blood cells have been studied in the back-scattered electron imaging (BEI) mode of scanning electron microscopy (SEM) with cytochemical methods for endogenous peroxidase, acid phosphatase, and a silver-staining method for nuclei. Peroxidase-positive granules were seen with good contrast and resolution in myeloid precursor cells and acid phosphatase activity was easily detected in macrophages and monoblasts. Silver staining permitted recognition of the shapes and location of the nuclei. In spite of the cytochemical procedures, cell surface structures were reasonably well-preserved in all methods, making direct correlation of BEI and secondary electron imaging (SEI) images an attractive feature in cell research with the scanning electron microscope.     

Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.     

Silver staining of synaptonemal complexes in surface spreads for light and electron microscopy

Synaptonemal complexes (SCs), axes of the X and Y chromosomes, and nucleoli in surface-spread preparations of spermatocytes are selectively stained for both light and electron microscopy by ammoniacal silver. Combined with a simple technique for transferring material from glass slides to grids, sequential light and electron microscopic analysis of full SC complements is possible with no further preparation. This new method has potential for both basic and clinical cytogenetic research.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.