A comparative fine structure study of rat cerebral cortex following ultra-rapid freezing and conventional chemical fixation procedures

Abstract

Adult rat (Sprague-Dawley) cerebral cortex was processed by ultra-rapid freezing at liquid helium temperature followed by freeze-substitution, osmium fixation and by other chemical, post-osmication procedures at 0-4 degrees and ambient temperatures as a comparative study for purposes of identifying differences and/or similarities in fine structure following these techniques. Five methods of processing were used: 1) rapid, slam-freezing at liquid helium temperature followed by osmium tetroxide/acetone freeze substitution; 2) perfusion with buffered, 2% glutaraldehyde at ambient temperature followed by post-osmication (2%); 3) en-bloc, buffered 2% glutaraldehyde fixation at 0-5 degrees centigrade and post-osmication (2%); 4) buffered, 2% osmium tetroxide perfusion at ambient temperature; and 5) en-bloc, buffered 2% osmium tetroxide fixation at 0-5 degrees centigrade. In ultra-rapid-frozen cortex good preservation was seen to a depth of 10-15 microns from the surface of the initial, copper-block contact. The tissue processed by ultra-rapid-freeze, freeze substitution demonstrates a general 'smoothness' of plasmalemmal and organelle membranes not observed in tissue prepared by chemical fixation alone. Cellular and organelle morphological differences were minor beyond the general 'smoothness' of membranes and a more intense background, electron density found in tissue prepared by rapid-freeze. Of particular interest was the practically identical images found in the four, chemical techniques not preceded by ultra-rapid freezing. High magnification images also revealed rather minor differences following ultra-rapid-freezing compared to tissue fixed by chemical fixation alone. Although these morphological differences are minimal, there can be no question of the fact that ultra-rapid-freeze followed by freeze-substitution is morphologically superior to chemical fixation alone. Ultra-rapid-freeze, eventually utilizing other substitution agents than osmium tetroxide, will offer several advantages and should be particularly useful for investigators involved in cytochemical and immunochemical methods.