Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

Brittle fracture of epidermal cells by liquid shear.

A suspension of epidermal cells obtained from pig tail skin by trypsinization was subjected to high liquid-shear forces in a French press. The material issuing from the press was examined by phase-contrast microscopy, transmission electron microscopy and scanning electron microscopy. The cytoskeleton of tonofibrils retained the shape of cell fragments, and subcellular organelles remained enmeshed in the network of tonofibrils. Examination of some cell fragments by scanning electron microscopy revealed the internal organization of the tonofibrils. The relevance of these findings to the problem of isolating subcellular fractions from epidermis is discussed.     

Ultrastructure of Cells Cultured on Polycarbonate Membranes

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 pm or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Ultrastructure of cells cultured on polycarbonate membranes.

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 micrometer or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

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.     

Preparation techniques for the electron microscopy of granular sludge from an anaerobic digester

Several fixation and dehydration techniques for scanning and transmission electron microscopy of glycocalyx and microbial populations within granules from an upflow anaerobic sludge blanket digester purifying a brewery wastewater were compared. Sputter-cryo and freeze-drying techniques prior to scanning electron microscopy (SEM) allowed viewing of the glycocalyx. In contrast standard fixation and dehydration techniques were suitable for examination of underlying microbial populations by both SEM and transmission electron microscopy.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Comparison between direct methods for determination of microbial cell volume: electron microscopy and electronic particle sizing.

Size frequency distributions of different phototrophic and heterotrophic microorganisms were determined by means of scanning and transmission electron microscopy and electronic particle sizing. Statistically significant differences existed among the three techniques used in this study. Cells processed for electron microscopy showed lower mean cellular volumes than those processed for electronic particle sizing, reflecting a shrinkage by factors ranging from 1.1 to 6.2 (mean, 2.3). Processing of cells for scanning electron microscopy caused higher shrinkage than processing for transmission electron microscopy. Shrinkage was dependent neither on the size nor on the cell wall type of the microorganism. When processed for scanning electron microscopy, phototrophic bacteria were strongly shrunken, whereas heterotrophic microorganisms were less affected. A direct relationship existed among phototrophic bacteria between percentage of shrinkage and specific pigment content. This was probably a consequence of the pigment extraction by organic solvents during the dehydration process, previous to the critical point drying, necessary to examine the specimens under the scanning electron microscope.     

Ultrastructural changes in murine lymphocytes induced by aflatoxin B1

This investigation sought to determine whether splenic lymphocytes obtained from Balb/C mice exposed to aflatoxin B1 (AFB1) showed any ultrastructural changes which could account for the immunodysfunction attributable to aflatoxins. Lymphocytes obtained from Balb/C mice administered aflatoxin B1 in olive oil daily for three weeks were studied using both transmission and scanning electron microscopy. The lymphocytes demonstrated ultrastructural changes primarily in the mitochondria where marked internal dissociation of the cristae was revealed by transmission electron microscopy. All other cellular organelles were unaffected. No significant alterations in external structure were observed under scanning electron microscopy. The findings of this study indicate that AFB1 administration does not affect the surface topography of lymphocytes, but AFB1, by causing extensive mitochondrial damage, may affect the way in which these cells function. This could be a possible explanation for the immunodysfunction associated with AFB1.Abbreviations AFB1 Aflatoxin B1 - SEM scanning electron microscopy - TEM transmission electron microscopy     

Development of alveolar septa and formation of alveolar pores during the early postnatal period in the rat lung

In order to investigate the formation of alveolar pores, lungs of rats, after intratracheal perfusion of glutaraldehyde, are processed at postnatal days 1, 7, 14, 16 and 21 for light and transmission electron microscopy and at days 7 and 16 for scanning electron microscopy. The initial low secondary crests of day 1 rapidly elongate to pleats subdividing the primary saccules. The ledges of some pleats partly grow toward each other as ring like diaphragms, leaving openings whose boundary is composed of alveolar epithelium separated by a basal lamina from a connective tissue sheath with capillaries. At day 7, in scanning electron microscopy the lumina of some rudimentary alveoli communicate by apertures of different sizes, as a result of the outgrowth of curved alveolar pleats which narrow to a ring-like aperture. The interalveolar openings observed in scanning electron microscopy resemble those investigated by light and transmission electron microscopy. The number of interalveolar pores increases from day 7 on; they become more and more frequent at days 14, 16 and 21, respectively. It appears that alveolar multiplication in newborn rats proceeds not only by segmentation of terminal respiratory units but also by compoundment of septa. The difference between genuine pores and transsections of folds in transmission electron microscopy will be given closer attention in this study. Also, the incidence and location of type II pneumocytes during rapid enlargement of the alveolar surface area is discussed.     

Use of electron microscopy to characterize the surfaces of flocculent and nonflocculent yeast cells

The surfaces of flocculent and nonflocculent yeast cells have been examined by electron microscopy. Nonextractive preparative procedures for scanning electron microscopy allow comparison in which sharp or softened images of surface details (scars, etc.) are the criteria for relative abundance of flocculum material. Asexually flocculent budding-yeast cells cannot be distinguished from nonflocculent budding-yeast cells in scanning electron micrographs because the scar details of both are well resolved, being hard and sharp. On the other hand, flocculent fission-yeast cells are readily distinguished from nonflocculent cells because fission scars are mostly soft or obscured on flocculent cells, but sharp on nonflocculent cells. Sexually and asexually flocculent fission-yeast cells cannot be distinguished from one another as both are heavily clad in "mucilaginous" or "hairy" coverings. Examination of lightly extracted and heavily extracted flocculent fission-yeast cells by transmission electron microscopy provides micrographs consistent with the scanning electron micrographs.     

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.     

Structure and association of wall fibrils produced by regenerating tobacco protoplasts

A study has been made of the wall fibrils produced by tobacco protoplasts, using scanning electron microscopy in conjunction with negative staining. It has been shown that the fibres seen in scanning electron microscopy correspond to aggregates of microfibrils. These aggregates are only visible where they are lifted clear of the protoplast surface. Negative staining of fixed protoplasts shows that the aggregation of microfibrils into the fibres visible in scanning electron microscopy is probably produced by air-drying. Gentle disruption of microfibrils produces both random broken fragments and bundles of short pieces of fibrillar material about 60 nm in length. This material is present in undisrupted young walls, but not in undisrupted older walls. The microfibrils in young walls seem much more fragile and liable to breakage than those in older walls. These results are discussed in terms of the interpretation of scanning electron microscope images and the mechanism of cellulose microfibril formation by higher plants.Abbreviations SEM Scanning electron microscopy     

Scanning and transmission electron microscopy of human ejaculate spermatozoa with special reference to their abnormal forms

Summary Spermatozoa from fertile and infertile human ejaculates were observed under the scanning electron microscope. A parallel study of sections was performed by transmission electron microscope.The normal head shows under the scanning electron microscope vesicular elevations in the region of the acrosome and a smooth and rigid appearance corresponding to the postnuclear cap whose occurrence is confirmed under the transmission electron microscope. Immediately anterior to this cap a shallow furrow transverses the head. Duplicated, unusually large or small and deformed heads are found under the scanning electron microscope. Most of these abnormal heads show no surface structure suggesting an acrosome.The neck and middle piece are occasionally, though frequently in abnormal spermatozoa, covered by a cytoplasmic droplet. Otherwise, the mitochondrial sheath is recognized under the scanning electron microscope as a beaded thickening in the middle piece. The lack of mitochondria is manifested by a smooth middle piece thinner than the principal portion. Transmission electron microscopy of sections reveals various types of anomalies in the number of cores, core filaments and mitochondria embedded in the cytoplasmic droplets.Abnormalities in the principal portion of the tail such as duplication, unusual thickness and length are shown under the scanning electron microscope.The investigation indicates that scanning electron microscopy is suited for the clinical as well as cytological examination of human ejaculate spermatozoa.     

The three-dimensional morphology of aggregates of native cotton cellulose microfibrils

The three-dimensional morphology of native bacterial cellulose is confirmed by scanning electron microscopy. In addition, it is shown by scanning electron microscopy, and transmission electron microscopy with positive staining by phosphotungstic acid ions that aggregates of microfibrils of native cotton cellulose have a similar structure. The results are consistent with previous reports on microfibrils of algal cellulose. These observations exclude a simple spinneret process as a mechanism of formation of the microfibrils of these sources of cellulose.     

The optical near-field and cell biology

A new form of scanning light microscopy is described in which the lens is replaced by a point of light that is smaller than the wavelength. Resolution is obtained that is defined not by the wavelength but by the size of the spot of light. This is the case so long as the point of light is within the dimension of a wavelength from the surface that is to imaged or within the optical near-field. This new form of light microscopy is called near-field scanning optical microscopy (NSOM). Resolutions are being obtained with NSOM that are similar to scanning electron microscopy but without the destructive effects of a vacuum or of an electron beam. In addition such a microscope is readily interfaced with fluorescent and non-fluorescent contrast enhancing stains that are commonly used in cell biology. The possibility of a near-field/far-field microscope is discussed with overlapping resolutions from a few hundred of a conventional microscope to the tens of thousand that can be obtained with NSOM.     

A Method for the Examination of the Same Cell Using Light, Scanning and Transmission Electron Microscopy

A method is described for preparing the same cell from a cytospin preparation for comparative investigation by light microscopy, scanning electron microscopy and transmission electron microscopy. A permanent numbered grid pattern was etched on a glass microscope slide to facilitate cell location in each microscopic mode. Data from one cell or group of cells was thus obtained from three sources. This method provides a useful adjunct to routine cytological diagnosis.     

The use of backscattered electrons to examine selectively stained nerve fibers in the scanning electron microscope

Selective staining of neuronal tissues using standard light microscopic techniques has been combined with backscattered electron scanning electron microscopy. This technique allows neurons to be readily distinguished from their surrounding tissues and examined at high resolution. The technique overcomes some of the problems involved in scanning electron microscopy of nervous tissue in situ.     

The Use of Backscattered Electrons to Examine Selectively Stained Nerve Fibers in the Scanning Electron Microscope

Selective staining of neuronal tissues using standard light microscopic techniques has been combined with backscattered electron scanning electron microscopy. This technique allows neurons to be readily distinguished from their surrounding tissues and examined at high resolution. The technique overcomes some of the problems involved in scanning electron microscopy of nervous tissue in situ.     

Development and morphology of rat synovial membrane

The development of the knee joint and synovial membrane in Wistar rats was studied from the 13th fetal day until adulthood by means of light microscopy, transmission electron microscopy and scanning electron microscopy. Using acid phosphatase and peroxidase methods, the different cell types of synovial membrane could be identified.