Field emission scanning electron microscopy of plant cells

Abstract

Summary.Two basic specimen preparation protocols that allow field emission scanning electron microscope imaging of intracellular structures in a wide range of plants are described. Both protocols depend on freeze fracturing to reveal areas of interest and selective removal of cytosol. Removal of cytosol was achieved either by macerating fixed tissues in a dilute solution of osmium tetroxide after freeze fracturing or by permeabilizing the membranes in saponin before fixation and subsequent freeze fracturing. Images of a variety of intracellular structures including all the main organelles as well as cytoskeletal components are presented. The permeabilization protocol can be combined with immunogold labelling to identify specific components such as microtubules. High-resolution threedimensional imaging was combined with immunogold labelling of microtubules and actin cables in cell-free systems. This approach should be especially valuable for the study of dynamic cellular processes (such as cytoplasmic streaming) in live cells when used in conjunction with modern fluorescence microscopical techniques.

High-Resolution and Low-Voltage SEM of Plant Cells

“…This technique has shown some promise on young spinach leaves, with chloroplasts and microtubules present though the thylakoid membranes of the chloroplasts are somewhat swollen. However, in most cells very much poorer results were obtained, with few fractures through organelles and extensive vesiculation of both the chloroplast and mitochondrial envelopes (Vesk et al 2000). Poor diffusion of digitonin through plant tissue seems the likely culprit, and there does remain the possibility that further technical development could give improved results.…”

“…If a "standard" TEM fixation of 2-3 % glutaraldehyde is used, the time needed for sufficient extraction of cytosol is greatly extended, whereas a dilute aldehyde prefixation followed by OsO 4 can make extraction easier (Lea et al 1992). Vesk et al [2000] found that a relatively weak primary fixative of 0.5% glutaraldehyde + 0.5% paraformaldehyde in 0.05M sodium phosphate buffer reduced the time of maceration needed for plant tissue to 48-72 hours at room temperature. The final stage is provision of a conductive stain by mordanting in 2% tannic acid and final staining in 1% OsO 4 .…”

“…The later protocol, as shown in Table 9.1, gives fewer problems with charging in the SEM, and also makes it possible to carry out immunogold labelling; it has since become the starting point for schedules for many diverse tissues. Only minor changes (mainly to the saponin permeabilization) were needed to prepare such varied specimens as onion-root tips, Chara internodal cells and the unicellular alga Chlamydomonas (Vesk et al 2000). Fowke et al [1999] have used the same basic protocol, with slight modifications, to image, and immunogold label, microtubules in highly vacuolated suspensor cells in white spruce somatic embryos.…”

“…Unicellular algae require more drastic fracturing since the cells are both separate and resistant. The pellet is spread on a piece of aluminium foil, which is folded, plunge-frozen in liquid nitrogen and fractured by repeated hammer blows (Vesk et al 2000).…”

“…The topic of preparing plant cells for field-emission SEM was reviewed extensively by Vesk et al [2000], and this chapter draws heavily on that review.…”