Zooming in on biological processes with fluorescence nanoscopy

Fluorescence nanoscopy enables the study of biological phenomena at nanometer scale spatial resolution. Recent biological studies using fluorescence nanoscopy have showcased the ability of these techniques to directly observe protein organization, subcellular molecular interactions, structural dynamics, electrical signaling, and diffusion of cytosolic proteins at unprecedented spatial resolution. Super-resolution imaging techniques critically rely on bright fluorescent probes such as organic dyes or fluorescent proteins. Recently, these methods have been extended to live cells and multicolor, three-dimensional imaging, thereby providing exquisite spatiotemporal resolutions of the order of 10–20 nm and 1–2 s for subcellular imaging. Further improvements in image processing algorithms, labeling techniques, correlative microscopy, and development of advanced fluorescent probes will be required to achieve true molecular-scale resolution using these techniques.

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► Fluorescence nanoscopy can be used to decipher subcellular structures in biological systems.
► We review the underlying principles and biological application of STED, PALM, and STORM.
► For super-resolution imaging methods, there is a tradeoff between temporal and spatial resolution.
► Improving probe brightness and probe photostability are key factors for achieving higher resolution.