STED Microscopy

The concept of stimulated emission depletion (STED) microscopy demonstrated that the diffraction barrier could, in fact, be surpassed. In STED microscopy, the diffraction limit is overcome by exploiting the inherent photophysical properties of the fluorescent molecules to reduce the size of the effective focal volume of a laser scanning microscope (LSM). The two major classes of fluorophores available for STED microscopy are fluorescent proteins (FPs) and small-molecule organic dyes. In recent years, STED microscopy has been extended to three-dimensional, multicolor, and live-cell imaging. These innovations are already proving to be invaluable tools for cell biology. Reversible saturable optical fluorescence transitions (RESOLFT) microscopy using reversibly switching FPs or other fluorophores with on/off transitions that require low light intensities is a compelling alternative to STED microscopy. Given recent advances in imaging speed for this super-resolution modality, the ability to image subcellular dynamics with reduced phototoxicity should be of broad appeal.