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FESEM and Cryo-Techniques in the Study of Marine Diatom A. Longipes

Field emission scanning electron microscopy (FESEM) provides a direct view of a biological sample with high spacial resolution. Combining FESEM with cryo-techniques, macromolecular structures have been obtained from single SEM image successfully (Hermann & Müller, 1992; Chen et al., 1995, 1997). This protocol is now applied to study the extracellular matrix of the diatom A. longipes. Stalk, which is originated from the terminal region of diatom cells, consists of 3 regions: a surfaceadhered pad, a shaft that separates the cell from the substratum, and a ring-like collar (Fig. 1). Investigating the structure and function of the stalk will help to understand the adhesion and interaction of diatom with the external environment. The extracellular matrix of diatom is highly hydrated, it is difficult to preserve them for EM. It will be easily extracted and collapse during specimen preparation (e.g. fixation, dehydration). In order to preserver the stalk structures close to their native condition and observer them with high fidelity, cryo-preparation techniques are implemented. Cryo-technique is widely used in the plant biology. However, to our knowledge, it has not been used in the diatom research.