Low Temperature Ultramicroincineration of Thin-Sectioned Tissue

Abstract

Low temperature ultramicroincineration was employed to determine the morphological localization of "structure-bound" mineral and/or metallic elements within biological cells at the electron microscope level . This technique chemically removes organic material from thin sections of tissues with reactive, excited oxygen instead of heat as used in a furnace . The remaining ash representing the mineral/metallic ultrastructure of cells is advantageous for ultrastructural studies because incineration without applying heat is less destructive than the burning associated with high temperatures . This low temperature incineration method was applied to thin-sectioned avian shell gland mucosa, a calcium transporting system, as a sample tissue . The results include : recognition of many subcellular organelles in the ash patterns, identification of dense, ash-containing granules (possibly organic-metallic complexes) in epithelial cells which may be involved in calcium transport, description of ashed erythrocytes and collagen, comparison of ashed glutaraldehyde fixed tissue with and without osmium postfixation, description of lead-stained cells after ashing, demonstration that ash preservation is dependent upon section thickness, illustration of the fine resolution obtainable because the ash residues remain relatively near their in situ origins, discussion of technical problems in this relatively new field, and demonstration of the presence of Ca and P in the ash with electron microprobe X-ray analysis .

Ultrastructural comparison of ion beam and radiofrequency plasma etching effects on biological tissue sections

“…In contrast, Hohman & Schraer (1972) found that lack of Os04 fixation made no difference in the SEM appearance of most cellular structures in totally ashed sections of avian shell gland tissue. Similarly, Yoshino et al (1982) found no difference in the post plasma SEM appearances of nuclear membranes and zymogen granules for Os04-stained versus unstained pancreas cell sections.…”

X‐ray microanalysis of epon sections after oxygen plasma microincineration

“…Applied to thin sections of plasticembedded, fixed material, this treatment has been shown to produce ash patterns with considerable ultrastructural detail (Thomas, 1964;Thomas & Greenawalt, 1968). However, X-ray microanalytical studies of such preparations have so far used conventional microprobes (Frazier, 1971 ;Hohman & Schraer, 1972;Thomas & Corlett, 1973) and questions relating to the sensitivity increase and the spatial resolution achievable by the total system were not considered. We have examined the 0022-2720/78/0800-0269 932.00 0 1978 The Royal Microscopical Society 269 ash patterns produced by plasma microincineration of thin, fixed, plastic sections using a high-resolution electron microscope interfaced to an X-ray spectrometer as an initial step towards applying plasma microincineration to thin, frozen-dried cryosections.…”

Microincineration and elemental X-ray microanalysis of single Bacillus cereus T spores

“…megaterium spores in bulk; however, no X-ray microanalyses of single spores before and after low-temperature ashing are available. Low-temperature ashing has also been used as a preconcentration step for elemental detection and localization (Thomas 1974;Hohman 1974;Barnard and Thomas 1978;Brenna and Morrison 1984).…”

“…Ashing generally increases elemental X-ray intensities of biological specimens (Albright and Lewis 1965;Hohman and Schraer 1972;Scherrer and Gerhardt 1972;Meyer and Stewart 1977; Barnard and Thomas 1978;Adamec et al 1979).…”

“…Recently, oxygen plasma ashing performed at lower temperature in a radiofrequency field of activated oxygen has also been used in biology (Thomas 1974;Hohman 1974;Brenna and Morrison 1984). Microincineration has been adapted successfully to transmission electron microscopic specimens (Thomas 1964(Thomas , 1969Boothroyd 1968; Thomas and Greenawalt 1968;Hohman and Schraer 1972). Partial ashing, i.e., differential '~u t h o r to whom all correspondence should be addressed.…”