A new technique for visualization of internal structure by SEM

Abstract

K E Y WORDS. SEM, fracture technique, paraffin embedding, soft animal tissues, internal structure. S U M M A R YA simple, rapid, economic and reliable method was used for visualization of internal structure of soft tissues by fracturing paraffin embedded samples at room temperature. The same sample which was used for light microscopy was then fractured for SEM. Most of the cellular and extracellular details are exposed without any need of special equipment. The technique preserves the architecture of tissues and can be used in routine diagnostic pathology. I N R O D U C T I O NThe SEM has greatly expanded the knowledge of the anatomy of various organs and tissues but a limiting factor in its use is the difficulty of visualization of internal structure at both histological and cytological levels. Also tissue preparation for SEM is still difficult and expensive (Buss & Hollweg, 1980). In this work a simple and economic technique was used by fracturing (at room temperature) paraffin embedded tissues originally prepared for light microscopy examination. MATERIALS A N D M E T H O D SNormal male Sprague Dawley rats (250-350 g) were used. All animals were killed by decapitation. Tissue samples were taken within 5 min of death, and the blocks were subsequently trimmed to size 5 x 5 x 2 mm. All tissues were fixed by immersion in either phosphate buffered formalin (pH 7-2) or 31,'0 phosphate buffered glutaraldehyde (pH 7.2) for at least 24 h. Tissues were then embedded in paraffin using the routine procedures. Preparation for SEMAfter taking sections for light microscopy, the wax blocks were treated as follows: 1. Wax blocks were removed from the plastic casettes. The wax was cut with a hot razor blade on either side of the block without cutting into the tissue. The direction of the cuts was either from side to side or from corner to corner. To avoid cracking of the wax prematurely during cutting, minimal pressure with the heated blade was applied. The blade was reheated when excessive resistance was felt during cutting.2. The block was held between fingers and manually fractured along the plane of cuts. 3. Thin pieces of tissue (0.5 mm thickness) bearing the fractured surfaces were cut from each fractured piece of tissue using a warm razor blade. The remaining tissue in the blocks was labelled for future use if needed.

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

“…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).…”