Nanoscale imaging by superresolution fluorescence microscopy and its emerging applications in biomedical research.

Superresolution microscopy, an ensemble of light microscopy methods developed with the aim of surpassing the resolution limit imposed by diffraction, has been at the forefront of imaging technology innovations in recent years. By harnessing advances in fluorophore photophysics, fluorescent protein engineering, optics, and image processing, rapid strides have been made in enhancing imaging resolution via 3 major approaches: structured illumination microscopy, stimulated emission depletion microscopy, and single-molecule localization microscopy. From a diffraction-limited resolution of ~250 nm, an improvement of more than an order of magnitude down to ~10 nm can now be attained, converging upon the size scale of the macromolecular building blocks of cells. This opens up the possibility of direct visualization of molecular-scale architecture and interactions of specific proteins in biological structures that are important to health and disease. Here, theoretical foundations and practical considerations of superresolution microscopy in 2- and 3-dimensional imaging are discussed, along with their recent applications in addressing biological questions.