A comparison of heating modes in rapid fixation techniques for electron microscopy.

Abstract

Recent studies have established that microwave irradiation during aldehyde fixation of electron microscopy specimens can reduce fixation times substantially. Similar reductions in duration of histochemical and light microscopy procedures have been reported. Both thermal and non-thermal effects of microwaves have been proposed to explain these dramatic decreases in processing time. Possible thermal effects include increases in fixative diffusion and reaction rates and increased formation of glutaraldehyde monomers. Proposed non-thermal effects include preferential orientation of fixative molecules by the microwave field and other more speculative direct microwave effects. Several reported attempts to produce rapid fixation without temperature increase by cooling specimens during irradiation have produced conflicting results. If rapid fixation is a thermal effect, other heating modes in addition to microwave exposure should produce similar effects. We show that for mouse liver samples (< or = 1 mm3) comparable fixation can be obtained with microwave irradiation, conductive and convective heating in a waterbath, and resistive heating with a low-frequency (1 kHz) current passed through the fixative solution. We also show that using an efficient convective cooling method to prevent temperature increase during microwave exposure produces unsatisfactory fixation. These results are consistent with thermal mechanisms for rapid fixation.