Visualization of Individual Reovirus Particles by Low-Temperature, High-Resolution Scanning Electron Microscopy

We used low-temperature, high-resolution scanning electron microscopy (cryo-HRSEM) to visualize surface structures on individual reovirus particles. Both intact virions and two forms of subvirion particles—infectious subvirion particles and cores—were examined, and despite some distortion of particles during specimen preparation and viewing in the microscope, the images obtained by cryo-HRSEM exhibited a level of interpretable detail not routinely achieved by other methods without image averaging. Cryo-HRSEM images of discrete reovirus particles were used to characterize and confirm features of the outer protein capsid of this virus by comparison with image reconstructions previously derived from cryotransmission electron microscopy. Distinct surface features attributable to each of the four outer-capsid proteins were identified. In addition, cryo-HRSEM images confirmed that significant changes occur on the surfaces of individual reovirus particles during disassembly and entry of cells and that the reovirus outer capsid is organized as a left-handed T=13 icosahedron. Several unique capabilities and potential uses suggest that cryo-HRSEM has a place alongside other, more established methods for molecular characterizations of virus particles.     

Ribonucleoside Triphosphate-Dependent Pyrophosphate Exchange of Reovirus Cores

Reovirus cores catalyze a ribonucleoside triphosphate (rNTP)-dependent pyrophosphate exchange reaction in the presence of all four rNTP species. When rNTP species are tested individually, only guanosine-5'-triphosphate supports pyrophosphate exchange.     

A Cellular Reaction to Antibody in Tissue Culture Studied with Electron Microscopy

The reaction of embryonic chick heart cells grown in tissue culture to specific guinea pig antiserum has been studied with electron microscopy. Heart fragments from chick embryos were cultured with a plasma clot. After being tested with antiserum or normal serum, they were fixed with buffered osmium tetroxide and embedded in butyl methacrylate before removal from the glass culture chamber. Thin cells found by phase microscopy to have reacted were sectioned in a plane parallel to the glass surface on which they had grown. The results confirm and extend observations made previously while the reactions were occurring. The plasma membrane, like that of the red cell, becomes disrupted or less resistant to trauma following the action of antiserum. The membranes of mitochondria and endoplasmic reticulum vesiculate and swell. Before nuclear shrinkage becomes prominent, the outer nuclear membrane separates over a large portion of the nuclear envelope and forms one or more large swollen blebs. Thus, the outer nuclear membrane shows a reactivity similar to endoplasmic reticulum. It is suggested that the various physical and chemical changes observed to follow the action of antibody and complement on fibroblasts may be explained by osmotic pressure differences between various cell components. Some basic similarities to the action of hemolytic agents on red cells are noted.     

Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of sections cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Cryogenic Transmission Electron Microscopy Nanostructural Study of Shed Microparticles

Microparticles (MPs) are sub-micron membrane vesicles (100–1000 nm) shed from normal and pathologic cells due to stimulation or apoptosis. MPs can be found in the peripheral blood circulation of healthy individuals, whereas elevated concentrations are found in pregnancy and in a variety of diseases. Also, MPs participate in physiological processes, e.g., coagulation, inflammation, and angiogenesis. Since their clinical properties are important, we have developed a new methodology based on nano-imaging that provides significant new data on MPs nanostructure, their composition and function. We are among the first to characterize by direct-imaging cryogenic transmitting electron microscopy (cryo-TEM) the near-to-native nanostructure of MP systems isolated from different cell types and stimulation procedures. We found that there are no major differences between the MP systems we have studied, as most particles were spherical, with diameters from 200 to 400 nm. However, each MP population is very heterogeneous, showing diverse morphologies. We investigated by cryo-TEM the effects of standard techniques used to isolate and store MPs, and found that either high-g centrifugation of MPs for isolation purposes, or slow freezing to –80°C for storage introduce morphological artifacts, which can influence MP nanostructure, and thus affect the efficiency of these particles as future diagnostic tools.     

Application of Electron Diffraction to Biological Electron Microscopy

Three methods by which electron diffraction may be applied to problems in electron microscopy are discussed from a fundamental point of view, and experimental applications with biological specimens are demonstrated for each case. It is shown that wide-angle electron diffraction provides valuable information for evaluating specimen damage that can occur either during specimen preparation or while in the electron beam. Dark-field electron microscopy can be used both to enhance the image contrast and to provide highly restricted and therefore highly specific information about the object. Low-angle electron diffraction provides quantitative information about the object structure in the range from 20 A to ~ 1000 A. Lowangle electron diffraction also demonstrates the important role of Fourier contrast with biological specimens, which are usually characterized by structural features with dimensions of 20 A or larger.     

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.     

Immune Electron Microscopy of Rhinoviruses

Aggregates of three rhinovirus serotypes have been readily observed by utilizing the technique of immune electron microscopy. The aggregates generally fell into three categories: those with complete virions, those with "empty" capsids, and those in which the two types of particles were mixed.     

Electron Microscopy of Mummified Material

It has proved possible to cut ultrathin sections of mummified material obtained from an American Indian burial (approximate age unknown). Small pieces of tissue were placed for 48 hr in a softening fluid consisting of 96% ethyl alcohol, 30 vol.; 1% aqueous formalin, 50 vol.; 5% aqueous Na₂CO₃, 20 vol. During this period the fluid was changed twice. The tissue was then cut with a razor blade into cubes of 1 mm per side or less, dehydrated in graded ethanols, infiltrated and embedded in methacrylate and the plastic polymerised by placing in the oven at 58°C overnight. The blocks were trimmed to a truncated cone leaving a surface area of 0.5 mm² or less, and cut on a Porter Blum ultramicrotome using a glass or a diamond knife.     

Epoxy Resins in Electron Microscopy

A method of embedding biological specimens in araldite 502 (Ciba) has been developed for materials available in the United States. Araldite-embedded tissues are suitable for electron microscopy, but the cutting qualities of the resin necessitates more than routine attention during microtomy. The rather high viscosity of araldite 502 also seems to be an unnecessary handicap. The less viscous epoxy epon 812 (Shell) produces specimens with improved cutting qualities, and has several features—low shrinkage and absence of specimen damage during cure, minimal compression of sections, relative absence of electron beam-induced section damage, etc.—which recommends it as a routine embedding material. The hardness of the cured resin can be easily adjusted by several methods to suit the materials embedded in it. Several problems and advantages of working with sections of epoxy resins are also discussed.     

Poststaining Sections for Electron Microscopy

“Dirt” on electron microscopic sections can generally be avoided by the simple strategem of preventing dry sections from coming in contract with any solution with a dirty surface layer. Wet sections can be pushed through the surface layer of such solutions without ill effect. The first and last solutions to touch a section must be dean, preferably distilled water from a plastic wash bottle.

Mention of a trademark name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA, nor does it imply its approval to the exclurion of other products that may also be suitable.     

Epon-Maraglas Embedment for Electron Microscopy

There can be little doubt that Epon (Luft 1961) is currently the most widely used embedding medium for electron microscopy. While for the most part this embedding material is reliable, it has the disquieting tendency to fail occasionally to polymerize properly for no apparent reason. to counter this problem, several investigators have proposed that epoxide-anhydride ratios be taken into account to arrive at the best balance of ingredients for the final embedding medium (Coulter 1967, Burke and Geiselman 1971, Chang 1973). in an alternative solution, Mollenhauer (1964) suggested that the Epon be mixed with Araldite, and indeed, this combination has achieved some popularity. Epon-Araldite mixtures, however, have a relatively high viscosity in the unpolymerized state; this may slow permeation of tissue specimens.     

Ultra-Thin Sectioning for Electron Microscopy

A technic is described for obtaining thin sections of animal tissue suitable for electron microscopy. Fixation is accomplished by perfusion of the whole animal with neutral formalin or alcohol formalin followed by immersion of pieces to be examined in neutralized osmium tetroxide. The embedding medium is a mixture of equal parts of n-butyl and ethyl methacrylate polymerized by ultra-violet light. Sectioning is done by means of a glass knife on an International ultra-thin sectioning microtome set at 0.1 μ. The sections are floated on warm water to spread, then placed on Formvar-coated grids, dried, and put into toluene to dissolve the plastic. The technic produces routinely usable, thin sections that show a minimum of damage owing to fixation, embedding, and sectioning.