Abstract
Some years ago we introduced a thaw-fix technique for SEM viewing of internal cell structure1. In thisptechnique cells or tissues are frozen unfixed, fractured at -170 to -190°C and thawed into fixative at room temperature. In early work we infiltrated cells with glycerol or dimethylsulphoxide (to 25-30%) prior to freezing, to avoid ice crystal artifact during freezing and thawing. This infiltration took 20 mins, to 1 hr. with the possibility of ultrastuctural change during this time, since the cells were unfixed. Results for interphase chromatin were interesting but cytoplasmic structure was not well preserved. We have now improved our techniques for rapid freezing and rapid thawing and report results here for fresh material, rapidly frozen without cryoprotectant, showing excellent cytoplasmic preservation. Interphase chromatin shows structural detail comparable to that obtained earlier with glycerol cryoprotection.3T3 cells were grown on 20μm fibrin films, rapidly frozen by propane-jet freezing, fractured in a Balzers BAF 400 freeze-etch unit at -170°C and thawed into a fixative of Bershadsky buffer3 + 1.5% glutaraldehyde.
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