Field-emission scanning electron microscopy of the internal cellular organization of fungi

Internal viewing of the cellular organization of hyphae by scanning electro n microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutarald ehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Followi ng this procedure, the colonies were frozen and fractured on a liquid nitro gen-precooled metal block. Next, the fractured samples were macerated in di luted osmium tetroxide to remove the cytoplasmic matrix and subsequently de hydrated by freeze substitution in methanol. After critical point drying, m ounting, and sputter coating, fractured cells of several basidiomycetes wer e imaged with field-emission SEM. This procedure produced clear images of e longated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of sept al pore caps. This method is a powerful approach for studying the intracell ular ultrastructure of fungi by SEM.