Chemical extraction of the cytosol using osmium tetroxide for high resolution scanning electron microscopy

Abstract

Detailed examination of subcellular structures in three dimensions (3D) by high resolution scanning electron microscopy (HRSEM) is now possible due to improvements in the design of the scanning electron microscope and the introduction of methods of specimen preparation using chemical removal of the cytosol and cytoskeleton by dilute osmium tetroxide. Cells which have been fixed, frozen, cleaved, thawed, and subjected to cytosol extraction display intact intracellular structures in 3D including nuclear chromatin, endoplasmic reticulum, mitochondria, and the Golgi complex at a resolution close to that of conventional biological transmission electron microscopy (TEM). Small changes in the 3D structure of subcellular components can be conveniently examined in this way in development, in a variety of physiological processes and in disease. Broad areas of the specimen can be quickly surveyed by HRSEM since sectioning is not required and specimens of comparatively large size (up to 5 mm3) can be placed in the microscope. Extraction of the cytosol with dilute osmium tetroxide (OsO4) exposes subcellular structures in relief, permitting their examination in 3D from several aspects. However, the OsO4 extraction technique is limited, since significant intracellular structures, such as the cytoskeleton, vesicles, and antibody binding sites can be removed or inactivated during the cytosol removal steps.

Field‐emission scanning electron microscopy of the internal cellular organization of fungi

“…Fixation and postfixation of fungi allowed us to cut the colony into segments for freeze-fracturing. In animal cells, osmium tetroxide is preferred (e.g., Tanaka 1980); glutaraldehyde, which affects the maceration process, is omitted (reviewed by Lea et al 1992). However, when we omitted glutaraldehyde, septal pore caps were not exposed in the fractured samples.…”

“…In our studies, the maceration time required for R. solani was about 13 days while that for S. commune was only 5 days. Lea et al (1992) suggested that the extraction times are influenced by concentration of the fixative, fixing time, and temperature. The longer the fixation, the longer a maceration time is required.…”

“…To open up tissues and cells, investigators have employed (1) a tissue sectioner , (2) manually fracturing (El Shennawy et al 1982), (3) oxygen plasma etching (Lee andNicholls 1983, Richards et al 1993), (4) cryofracturing of tissue embedded in paraffin (Dalen et al 1978), and (5) freeze fracturing (Lea et al 1992). In the latter procedure, the chemically fixed material is first cryoprotected in dimethyl sulfoxide (DMSO), then frozen on a metal plate chilled with liquid nitrogen and fractured with a razor blade and a hammer (Barnes andBlackmore 1984a, 1984b;Haggis 1982;Müller et al 1998a, b;Tanaka 1981).…”

New technique for examining invertebrate membranes using high resolution scanning electron microscopy

“…Malpighian tubules (Mts) were taken from adult female Acheta domesticus and prepared using a modified version of the protocol described by Lea et al, (1992). Specimens were fixed in (4°C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer for approximately 18 hours.…”

“…By employing High Resolution Scanning Electron Microscopy (HRSEM) to observe cells that have been fixed, frozen, cleaved, thawed, and subjected to cytosol extraction, we were able to study broad areas of the cells in three-dimensional depiction. Although a similar technique has been applied to mammalian tissue (Tanaka and Naguro, 1981;Lea et al, 1992), this is the first demonstration of its usefulness in studying the membrane dynamics of invertebrates. …”