Super-Resolution Imaging Through Stochastic Switching and Localization of Single Molecules: An Overview

Abstract

The resolution of fluorescence microscopy had traditionally been limited to ~200–300 nm due to the diffraction of light. Recently, this resolution limit has been broken using mainly two classes of methods, one of which utilizes photoswitching of fluorophores to temporally separate the spatially overlapping images of individual molecules such that the positions of these molecules can be precisely determined. A sub-diffraction-limit image can then be reconstructed from these molecular coordinates. With relatively simple optical instrumentation and sample preparation, this class of methods has improved the spatial resolution of far-field optical microscopy by more than an order of magnitude, achieving resolutions down to sub-10 nm range for biological specimens. Three-dimensional, multicolor, and live-cell super-resolution imaging has been demonstrated. In this chapter, we provide an overview of this class of optical nanoscopy, primarily in terms of the imaging principle, the spatial/temporal resolution, the different imaging schemes, the photoswitchable probes, the applications, and future directions.