Preparation of cultured cells for transmission electron microscope

Abstract

Researchers working on differentiated cells or cell-substrate interactions in vitro must prepare the cultures for transmission electron microscopy to image the cells at high resolution. In practice, workers have found that the procedures used for preparing plant and animal tissues are unreliable when applied to tissue cultures. These procedures often cause the plasma membrane and cytoplasmic membranes to appear as white spaces. We found that the extracted appearance of the membrane is typical of tissues that were never exposed to osmium tetroxide. This artifact is prevented by using hexylene glycol for dehydration, instead of ethanol. Previous workers have used hydroxypropylmethacrylate for dehydration (1). The grade of the epoxy constituent of the resin has been found to be important (2). Previous protocols lead to difficulty in separating the sample from the culture dish. This problem is remedied by making a thin, flexible layer of resin. Here, separate preparation procedures are given for situations in which the tissue culture sample is to be sectioned parallel or transverse to the attachment surface.