The fixation, dehydration, drying and coating of cultured cells for SEM

Abstract

Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues. Studies on cultivated cells indicate that the glutaraldehyde component of a glutaraldehyde-based fixative does not contribute to the effective osmotic pressure of the fixative and thus the osmolarity of the buffer, and other components, must be equalized to that of the medium in which the cells grow. Even small deviations from this ideal effective osmotic pressure will result in osmotically induced artefacts. Disturbances of pH and temperature of the cultures prior to and during fixation will result in changes in the appearance of many cellular structures such as microspikes and ruffles. We find that osmium fixation is advisable in most instances for best possible membrance preservation and that even long periods of glutaraldehyde fixation do not compensate for osmium fixation. Dehydration always results in shrinkage. Freeze drying (FD) and critical point drying (CPD) also give rise to shrinkage, the former to a lesser degree than the latter. A gold-palladium alloy gives a less granular coating that does gold alone. When cultured cells are studied, a metal thickness of between 5 and 15 nm is usually sufficient to give rise to an adequate secondary electron production and to avoid charging even at accelerating voltages of 30-40 kV. Without treatment with OsO4 a thicker metal coating is required.