The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm 
playing critical roles in cell motility, shape generation, and mechanical 
properties of a cell. Fibrillar polymers-actin filaments, microtubules, and 
intermediate filaments-are major constituents of the cytoskeleton, which 
constantly change their organization during cellular activities. The actin 
cytoskeleton is especially polymorphic, as actin filaments can form multiple 
higher order assemblies performing different functions. Structural information 
about cytoskeleton organization is critical for understanding its functions and 
mechanisms underlying various forms of cellular activity. Because of the 
nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a 
key tool to determine the structure of the cytoskeleton. This article describes 
application of rotary shadowing (or metal replica) EM for visualization of the 
cytoskeleton. The procedure is applicable to thin cultured cells growing on 
glass coverslips and consists of detergent extraction of cells to expose their 
cytoskeleton, chemical fixation to provide stability, ethanol dehydration and 
critical point drying to preserve three-dimensionality, rotary shadowing with 
platinum to create contrast, and carbon coating to stabilize replicas. This 
technique provides easily interpretable three-dimensional images, in which 
individual cytoskeletal fibers are clearly resolved, and individual proteins can 
be identified by immunogold labeling. More importantly, replica EM is easily 
compatible with live cell imaging, so that one can correlate the dynamics of a 
cell or its components, e.g., expressed fluorescent proteins, with high 
resolution structural organization of the cytoskeleton in the same cell.