Requirements for Samples in Super-Resolution Fluorescence Microscopy

The preparation of samples and the choice of appropriate labeling techniques have become instrumental for the development of light microscopy techniques with increasingly high resolution. Both localization microscopy and STED approaches require fluorophores with specific features, including high photostability, specific excitation–emission spectra, and selective switching of single molecules to “on” and “off” state. Additionally, at higher resolutions the limits of conventional immunostaining often become apparent, as clearly exemplified by rather fragmented stainings of continuous cellular components such as microtubules and membranous organelles. Hence, the correct exploitation of fluorescent probes is of crucial importance for successful super-resolution imaging.