Larger sections and larger grids for diagnostic histopathological transmission electron microscopy

Sampling errors and orientation difficulties are common problems in diagnostic histopathological transmission electron microscopy (TEM). The use of larger sections could greatly reduce these. The practicability of using larger sections for diagnostic TEM was investigated. It required the construction of a larger grid and a minor modification to a standard grid holder. The results of the study show that it is feasible to use larger sections for TEM. Areas in which larger sections could be of particular diagnostic value are discussed.     

Preparation of ultra-thin frozen sections of plant tissues for electron microscopy

Summary A method is described, for the first time, by which ultra-thin frozen sections of plant tissues may be prepared for electron microscopy. Sections of both plant and animal tissues were prepared from either unfixed or fixed tissues, without prior dehydration or infiltration of the tissue with a support medium, and with the aid of a Reichert OmU 2 ultra-microtome with freezing attachment.     

A rapid method for resectioning of semithin large epoxy sections for electron microscopy

A simple and rapid method is described for resectioning semithin large epoxy tissue sections. Six-micron thick sections are cut, floated on a polystyrene coverslip, and stained with toluidine blue. The coverslip is trimmed to the size of a BEEM capsule cap and placed section face-up inside the cap. A BEEM capsule with the conical end cut off is fitted onto the cap, filled with plastic and polymerized. The capsule and coverslip are trimmed off and thin sectioning is performed. This method allows the study of multiple tissue lesions in their entirety, i.e. serially from beginning to end, without sacrificing any part during trimming as in conventional thin sectioning.     

Visualization of nucleosomes in thin sections by stereo electron microscopy

Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding.     

Fluorescent labeling of resin-embedded sections for correlative electron microscopy using tomography-based contrast enhancement

Locating areas of interest by electron microscopy can be laborious. This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in the projection images. We evaluated the applicability of fluorescent probes to thin plastic sections, in combination with fluorescence microscopy, as an aid in selecting areas for subsequent electron microscopic analysis. We show that pre-embedding labeling of DNA and RNA with acridine orange yielded a predominant nuclear stain. The stain greatly reduced the time needed to scan sections for mitotic cells, or cells with characteristic nuclei such as neutrophils. Post-embedding labeling with SYTOX green yielded a nuclear stain comparable to acridine orange, and wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 labeled mucous granules and the Golgi area in intestinal goblet cells. The fluorescent labels were visualized directly on sections on electron microscope grids. It was therefore possible to establish a coordinate system based on the position of the grid bars, allowing for easy retrieval of selected areas. Because the fluorescent probes were incompatible with osmium tetroxide treatment, contrast in the sections was faint. We propose a simplified electron tomography procedure for the generation of 2D views with enhanced contrast and resolution.     

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.

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