High resolution low voltage scanning electron microscopy of uncoated yeast cells fixed by the freeze-substitution method

Uncoated yeast cells prepared by the freeze-substltution fixation and cryo-fracturing method were studied witb an "In-lens " type field emission scanning electron microscope (FESEM) at low voltage (0.8-3 kV). The micrographs were compared with those obtained with a conventional FESEM and coated specimens, as well as witb transmission electron micrographs of ultrathin-sec-tioned yeast cells. The images of uncoated fractured cells taken at 1.2-1.5 kV seem to show the highest fidelity and thus they presented their three-dimensional structures more dynamically. The fine filaments produced on the outermost surface of the cell wall were also clearly observed witb a low-voltage scanning electron microscope. The filaments of a similar specimen taken at 20 kV after coating with Pt-C 2 nm in thickness, however, showed an enlarged and intertwined structure because of a masking and decoration effect due to coating. In addition to several intracellular organelles, free ribosomes in the cytoplasm and Intramembranous particles of the cell membrane were revealed by the "In-lens " FESEM and these image