STED Super-Resolution Microscopy for Studying Actin Cytoskeleton Organization

Actin is one component of the intracellular cytoskeleton, forming filaments that are thin, flexible fibers approximately 7 nm in diameter and extending up to several micrometers in length. These filaments organize into higher-order assemblies, creating bundles of three-dimensional networks that behave like semisolid gels. Individual actin filaments (~7 nm) cannot be resolved with conventional light microscopy; however, super-resolution techniques such as STED, PALM, and STORM might increase resolution up to ~20-30 nm, enabling visualization of single filaments and fine structural details within cellular networks. For even higher resolution and ultrastructural detail, electron microscopy remains indispensable. Here, we describe STED procedures for imaging immunostained actin, including critical steps for laser alignment, a method for determining the actual spatial resolution, and criteria for selecting fluorophores and preparing samples.