Optimization and characterization of toroidal foci for super-resolution fluorescence microscopy: tutorial

Abstract

Single-molecule localization microscopy (SMLM) has become an essential tool to investigate phenomena at the nanoscale. Among the different SMLM approaches, methods that interrogate the molecular position with an intensity minimum, such as minimal emission fluxes (MINFLUX) or the more recent raster scanning a minimum of light (RASTMIN), stand out for reaching true molecular resolution. To implement these methods, the phase of the excitation beam needs to be modulated to obtain a focus with a central minimum, i.e., a so-called toroidal or doughnut-shaped focus. In this tutorial,we explain the basis and experimental tricks to generate and optimize such beams, particularly in raster-scanning microscopes.