Fixation of Platelets for Scanning and Transmission Electron Microscopy

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

Fixation of platelets for scanning and transmission electron microscopy.

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

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.     

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     

Transmission electron microscopy of small numbers of sorted cells

A new method that allows the transmission electron microscopic examination of as few as 1 x 10(4) cells obtained by flow cytometric sorting is described. The approach involves "sandwiching" fixed cells in an agarose case by a microcentrifugation system consisting of small-diameter cell-centrifugation tubes and subsequent processing of the cells by conventional techniques. The advantages offered by this method are discussed.     

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.     

Negative staining of freshwater bacterioneuston sampled directly with electron microscope specimen support grids

A technique for observation of surface microlayer bacteria (bacterioneuston) is described, utilizing direct sampling of the air-water interface with carbon-stabilized electron microscope specimen support grids, followed by negative staining and transmission electron microscopy. The method resulted in excellent preservation of forms of microcolonial association, regular surface arrays, surface appendages, and prosthecae in the bacterioneuston of a freshwater pond.     

A Simple Method for Fixing, Dehydrating and Embedding Pollen Tubes Cultivated in Vitro for Optical and Transmission Electron Microscopy

A simple method to cultivate pollen tubes in a gelatin medium is presented. After the growth of the pollen tubes in the culture medium, they are fixed, dehydrated, and embedded in resin for ultramicrotomy. The method is easy and does not require the purchase of special materials beyond those needed for the usual techniques for studying biological specimens under transmission electron microscopy.     

Modification of a scanning transmission electron microscope specimen holder for large section viewing.

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter "giant" grids is explained and the procedure for sample preparation is outlined. The modification aids the microscopist in his evaluation of tissue structural relationships by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm2 electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research

In transmission electron microscopy (TEM), electrons are transmitted through a plastic-embedded specimen, and an image is formed. TEM enables the resolution and visualization of detail not apparent via light microscopy, even when combined with immunohistochemical analysis. Ultrastructural examination of tissues, cells and microorganisms plays a vital role in diagnostic pathology and biologic research. TEM is used to study the morphology of cells and their organelles, and in the identification and characterization of viruses, bacteria, protozoa and fungi. In this protocol, we present a TEM method for preparing specimens obtained in clinical or research settings, discussing the particular requirements for tissue and cell preparation and analysis, the need for rapid fixation and the possibility of analysis of tissue already fixed in formalin or processed into paraffin blocks. Details of fixation, embedding and how to prepare thin and semi-thin sections, which can be used for analysis complementary to that performed ultimately using TEM, are also described.     

Observations on the Preparation of Epoxy Embedded Tissue Samples for Energy-Dispersive X-Ray Analysis

Elemental X-ray microanalysis of biological tissues by energy-dispersive detectors attached to conventional transmission or scanning electron microscopes is a technique with many potential applications. Proper specimen preparation and consideration of problems inherent with the method are necessary to achieve satisfactory results. This report concerns some of the problems encountered in analyzing tissue samples embedded for electron microscopy in epoxy resins.     

Procoagulant platelet balloons: evidence from cryopreparation and electron microscopy

Visualisation of the procoagulant transformation of human platelets has recently become possible through use of an in vitro approach combined with fluorescence and phase contrast microscopy. Here, we extended these studies to the ultrastructural level by employing both rapid freezing/freeze-substitution and conventional ambient-temperature chemical fixation for transmission and scanning electron microscopy. Procoagulant transformation was only inducible by adhering platelets to collagen fibrils or to the collagen-related peptide and exposing them to physiological extracellular Ca2+ levels. Under these conditions prominent, 2- to 4-micron-wide balloon-like structures were regularly observed, regardless of the specimen fixation protocol. In strong contrast to normal platelets in their vicinity, the balloons' subcellular architecture proved remarkably poor: dilute cytoplasm, no cytoskeleton, only a few, randomly distributed organelles and/or their remnants. Cryofixed balloons displayed intact and smooth surfaces whereas conventional specimen processing caused plasma membrane perforations and shrinkage of the balloons. Our results clearly show that neither the balloons themselves, nor their simple ultrastructure reflect fixation artefacts caused by inadequate membrane stabilisation. The balloons are interpreted as to be transformed and/or fragmented procoagulant platelets. Thus, the generation of balloons represents a genuine, final stage of platelet ontogenesis, presumably occurring alternatively to aggregate formation.     

Modification of a Scanning Transmission Electron Microscope Specimen Holder for Large Section Viewing

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter “giant” grids is explained and the procedure for sample preparation is outlined The modification aids the microscopist in his evaluation of tissue structural relationship by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm² electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

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.     

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.     

Atomic force microscopy imaging of dried or living bacteria.

Atomic force microscopy (AFM) was used to obtain micrographs of dried bacteria in air, and of living ones in their culture medium. Images of dried bacteria were very similar to images obtained elsewhere by the much more complicated cryoetching preparation technique for transmission electron microscopy. Living bacteria were immobilized on a poly-L-lysine film, and directly observed in their culture medium at a resolution unattainable by any other technique applicable to living material. The images were similar to those obtained in scanning electron microscopy where the specimen must be fixed, dried and coated with conductive material, and as a result, no longer viable.     

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.     

A Rapid Technique for Preparing Microorganisms for Transmission Electron Microscopy

A rapid and efficient method of preparing microorganisms for transmission electors microscopy is reported. In developing the method Salmonella, streptococcal, ad 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.     

Oriented Embedding of Single-Cell Organisms

The dexribed technique facilitates oriented embedding of individual cells in various media for both light and electron microscopy. A fixed Specimen is embedded in a small cube of 2% agar at 40 C and subsequently sealed in the desired orientation to a strip of black paper which then serves as a tab for transferring the specimen during dehydrating and embedding procedures. The beveled ends of the strip indicate the exact location of the specimen in the cube. This technique can be employed for the embedding media used in both light and electron microscopy. It ah permits photomicrographs of the whole specimen to be made which can be compared with photomicrographs of individual sections cut from the specimen in a selected plane.     

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.