A preparation technique for observing cytoskeletons by high resolution scanning electron microscopy.

The three-dimensional architecture of the cytoskeleton in cultured fibroblasts was studied using a newly devised technique for revealing cell interiors and an ultrahigh resolution scanning electron microscope, the UHS-T1. Both the cytoskeleton and membranous structures such as the plasma membrane and endoplasmic reticulum were well preserved, and we could observe the relationship between both components. Actin filaments, intermediate filaments, and microtubules were identified by immunogold staining with 5-15 nm gold particles. Actin filaments, measuring 10 nm in diameter in material not metal coated, formed thick bundles (stress fibers), sheaths or meshworks. Just beneath the plasma membrane, actin filaments could be seen in a two-dimensional network, with fibers linked laterally to the membrane. Intermediate filaments, 12 nm in diameter in uncoated material, were observed mainly in the perikaryon. Microtubules (26 nm) and clathrin-coated vesicles were also clearly seen.