High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

Abstract

The feasibility of plasma coating of a thin osmium layer for high-resolution immuno-scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro-muscular preparations were fixed with formaldehyde and labelled with a neurone-specific antibody and 10 or 5 nm colloidal gold-conjugated secondary antibodies. The specimens were post-fixed with osmium tetroxide and freeze-dried. Then they were coated with a 1-2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three-dimensionally complex surface structures of cellular processes with a resolution of several nanometres.