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 res olution scanning electron microscopy. A visualization test at x 300,00 0 was made using a gold-evaporated magnetic tape, and the resolution w as found to be about 1 nm at 2.5 kV. The ultrastructure of the cell wa ll, especially the reverting glucan network, from the protoplast of Sc hizosaccharomyces pombe was studied using this improved ultrahigh-reso lution low-voltage SEM (UHR-LVSEM). The results with uncoated revertin g protoplasts observed with this microscope revealed that the network was originally formed as secreted particles scattered on the protoplas t 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 ri bbon-shaped network of glucans about 16-nm-thick which covered the ent ire protoplast surface. The UHR-LVSEM images of reverting protoplasts treated with glucanase confirmed that the particles scattered on the p rotoplast surface in the initial stage of regeneration were glucan in nature.