Superresolution Imaging of Cytoskeletal Networks in Fixed Brain Tissue

Emerging evidence suggests that neurodegeneration is directly linked to dysfunction of cytoskeleton; however, visualizing the organization of cytoskeletal structures in brain tissues remains challenging due to the limitation of resolution of light microscopy. Superresolution imaging overcomes this limitation and resolves subcellular structures below the diffraction barrier of light (20-200 nm), while retaining the advantages of fluorescent microscopy such as simultaneous visualization of multiple proteins and increased signal sensitivity and contrast. However, superresolution imaging approaches have been largely limited to very thin samples such as cultured cells growing as a single monolayer. Analysis of thicker tissue sections represents a technical challenge due to high background fluorescence and quality of the tissue preservation methods. Among superresolution microscopy approaches, structured illumination microscopy is one of the most compatible methods for analyzing thicker native tissue samples. We have developed a methodology that allows maximal preservation and quantitative analyses of cytoskeletal networks in tissue sections from a rodent brain. This methodology includes a specialized fixation protocol, tissue preparation, and image acquisition procedures optimized for the characterization of subcellular cytoskeletal structures using superresolution with structured illumination microscopy.