Three-dimensional super-resolution imaging of the cytoskeleton in hippocampal neurons using selective plane illumination

Recent advances in single-molecule-based super-resolution imaging have been in the forefront of biological research for the visualization of the detail structures in cellular and molecular biology. A number of super-resolution optical microscopy techniques have been reported; however, several challenges such as the use of high-activation sources resulting in photobleaching and photodamage effects, restrictions in three-dimensional imaging, long data acquisition, and limited field of view remain unresolved. To address these concerns, a rapid, large-scale, and three-dimensional super-resolution fluorescence microscopy has been developed through the introduction of selective plane illumination microscopy based on scanning Bessel beam and a spontaneously blinking dye HMSiR as a reporter. This localization-based super-resolution microscope offers several advantages. namely minuscule levels of photodamage and phototoxicity effects due to low activation source, good optical sectioning suitable for three-dimensional imaging, large field of view, and fast data acquisition. In this chapter, protocols on the three-dimensional super-resolution imaging of neurons through the application of selective plane illumination technique are discussed.