Super-resolution microscopy represents a new evolution of optical microscopy that allows images to be obtained at resolutions higher than those imposed by the light diffraction limit. The 3rd edition of the Scuola di Microscopia is focused on the understanding of optical problems related to the use of such techniques, including Structured illumination microscopy (SIM), STochastic Optical Reconstruction Microscopy (STORM), STimulated Emission Depletion (STED) microscopy, Total Internal Reflection Fluorescence (TIRF) microscopy as well as deconvolution algorithms. These different approaches fall into the concept of “nanoscopy”, and represent new frontiers in biomedical research, allowing researchers to study subcellular structures more in-depth with respect to standard microscopes.