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.     

3D imaging of diatoms with ion-abrasion scanning electron microscopy

Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at 30 nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems. Our study provides new insights into the architecture and assembly principles of both the “hard” (siliceous) and “soft” (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we show that both micro- and nanoscale siliceous structures can be visualized at specific stages in their formation. We show that not only are structures visualized in a whole-cell context, but demonstrate that fragile, early-stage structures are visible, and that this can be combined with elemental mapping in the exposed slice. We demonstrate that the 3D architectures of silica structures, and the cellular components that mediate their creation and positioning can be visualized simultaneously, providing new opportunities to study and manipulate mineral nanostructures in a genetically tractable system.     

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.     

Method of preparing suspended cells for scanning electron microscopy]

To preserve the lifetime morphology of the surface of suspended cells, these must be fixed in suspensions. The subsequent stages of cell preparation for scanning electron microscopy (dehydratation, critical point drying, coating) are considerably facilitated if fixed cells are preliminary attached to some substrate surface. An effective substrate should provide a firm rather than selective attachment of the overwhelming majority of fixed cells; the substrate should be also available for a wide application. The trial of different types of substrates showed a sufficient effectivity of plates made of commercial aluminium foil. In tests with murine embryonal and transformed fibroblasts as well as with human blood leukocytes, in average 90 per cent of cells fixed with glutaraldehyde in suspensions got attached to foil substrate surfaces; the fixed cells both settled from suspension and attached were seen distributed evenly on the substrate surface. The use of aluminium foil substrates made it possible to study the surface topography of some types of suspended cells.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

A method for preservation of soft tissues of marine teleosts for scanning electron microscopy

Soft, internal tissues of marine teleosts have been preserved for scanning electron microscopy in 2% glutaraldehyde in half-strength flounder saline; the osmolality of this fixative approximated the plasma osmolality of marine teleosts. Fixation was followed by washing, dehydration and Freon critical point drying. This procedure has revealed surface features of renal tubules of Anoplogaster and swimbladder of Melanonus     

High magnification scanning electron microscopy of bacterial cells and virions

When freeze-dried or critical point-dried cells of Escherichia coli and Bacillus sphaericus were coated with a 15 nm thick gold layer by means of sputtering, the surface of the bacteria appeared in the scanning electron microscope to consist of globules having a diameter of about 10–25nm. When the cells were coated with 3–5 nm of tungsten by sputtering, their surface appeared smooth or slightly grainy.Freeze-dried adenovirus type 2 virions sputtered with 15 nm gold exhibited an irregular surface, whereas virions sputtered with 3 nm tungsten looked smooth and had a more or less hexagonal shape. No capsomeres could be discerned.     

A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM)

This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3-5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.     

A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

Fracture of plant tissues and walls as visualized by environmental scanning electron microscopy

The environmental scanning electron microscope (ESEM) provides a highly relevant and controllable environment in which to study hydrated systems without the artefacts of other highly prepared specimens. The instrument facilitates control of turgor through hydration using different chamber vapour pressures. Deformation of a simple plant tissue-upper epidermal layers in Allium cepa (onion)-was observed at the scale of the two principal failure mechanisms: cell breakage; and cell separation induced by treatment with a chelating agent. Cell rupture and release of contents occurred at cellular junctions ahead of an imposed growing notch, indicating that disruption of cells occurred remotely from the creation of a new surface. Cells that separated usually maintained their turgor and the separation process took place through progressive failure of middle lamellar material seen as strands between separating cells. These mechanisms were compared with the rupture of excised Chara corallina walls that occurred by formation and breakage of strands between separating wall layers. This study provides in situ visual characterization of wall rupture and cell separation at the microscopic level in hydrated plant material.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Light and scanning electron microscopy of fallow deer (Dama dama) spermatozoa

No basic differences in size (mean +/- s.d. for at least 300 spermatozoa), shape and ultrastructure of the spermatozoa of fallow deer were detected (1) in comparison to other artiodactyls, (2) between different fallow bucks, and (3) between different months of the fertile season. The total length of the normal spermatozoon was 67.2 +/- 1.2 microns. The flat, paddle-shaped head was 8.2 +/- 0.3 microns long, 4.4 +/- 0.2 microns for the greatest width, 1.9 +/- 0.2 microns for basal width and, approximately 0.7 microns in thickness. The tail measurements were 13.7 +/- 0.3 microns for the midpiece, 0.5 +/- 0.1 microns for the diameter of the midpiece, 42.6 +/- 0.9 microns for the principal piece, and 2.7 +/- 0.6 microns for the endpiece. Spermatozoa with abnormalities such as cytoplasmic remnants and droplets, bent and coiled tails, as well as microcephalic forms were observed.     

Examination of immobilized fungal cells by phase-contrast and scanning electron microscopy

Conidia of Penicillium urticae were immobilized in kappa-carrageenan beads and then shaken, in a growth-supporting medium to yield an in situ grown population of mycelia. The physical stability of these beads and the degree of mycelial growth inside the beads were significantly affected by the concentrations of kappa-carrageenan and locust bean gum (LBG) in the bead matrix and by the porous or nonporous nature of the interior. Thus 16-h-old porous and nonporous beads, prepared from 1.25% kappa-carrageenan, 0.5% LBG, and conidia, possessed a very dense mycelial mass at the surface. Only the porous beads possessed a moderately dense mycelial mass at the centre. The conidia at the centre of nonporous beads either failed to germinate or formed very small germ tubes. When washed, 36-h-old porous beads were repeatedly (i.e., 48 h) transferred into nitrogen-free medium, the density of mycelia at the centre increased to equal that at the surface after three transfers or 8 days. Mycelia at the surface exhibited signs of physical damage, while those in the centre did not. The addition of 100 micrograms/mL of cycloheximide to these replacement cultures was reflected by the distortion of interior mycelia.     

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.     

Improved preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM)

Various fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR-LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OsO₄ and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing. © 1993 Wiley-Liss, Inc.