Practical Aspects of Super-Resolution Imaging and Segmentation of Macromolecular Complexes by dSTORM

Super-resolution fluorescence microscopy allows imaging macromolecular complexes down to the nanoscopic scale and thus is a great tool to combine and integrate cellular imaging in the native cellular environment with structural analysis by X-ray crystallography or high-resolution cryo electron microscopy or tomography. Here we describe practical aspects of SMLM imaging by dSTORM, from the initial sample preparation using mounting media, antibodies and fluorescent markers, the experimental setup for data acquisition including multi-color colocalization and 3D data acquisition, and finally tips and clues on advanced data processing that includes image reconstruction and data segmentation using 2D or 3D clustering methods. This approach opens the path toward multi-resolution integration in cellular structural biology.