Ultrahigh-resolution low-voltage SEM reveals ultrastructure of the glucan network formation from fission yeast protoplast.

The refined field emission SEM, S-900 LV which gives better resolution especially at low voltages below 5 kV was developed for ultrahigh resolution scanning electron microscopy. A visualization test at x 300,000 was made using a gold-evaporated magnetic tape, and the resolution was found to be about 1 nm at 2.5 kV. The ultrastructure of the cell wall, especially the reverting glucan network, from the protoplast of Schizosaccharomyces pombe was studied using this improved ultrahigh-resolution low-voltage SEM (UHR-LVSEM). The results with uncoated reverting protoplasts observed with this microscope revealed that the network was originally formed as secreted particles scattered on the protoplast surface and these were subsequently stretched to microfibrils about 2 nm thick. The microfibrils were twisted around each other and joined together so that they developed into 8-nm-thick fibrils, forming a ribbon-shaped network of glucans about 16-nm-thick which covered the entire protoplast surface. The UHR-LVSEM images of reverting protoplasts treated with glucanase confirmed that the particles scattered on the protoplast surface in the initial stage of regeneration were glucan in nature.