Superresolution Microscopy

Abstract

Abstract More than a century after Ernst Abbe defined the optical diffraction limit and thus seemed to rule out the accurate visualisation of subcellular details below its threshold forever, several approaches now achieve resolutions significantly below it. Structured illumination microscopy, stimulated emission depletion microscopy and single‐molecule localisation‐based methods use different strategies to separate and visualise the cellular and molecular details that until now were lost in the blur of diffraction. This opens up new possibilities in the observation of biological processes at the smallest scales. The new field is only developing and still poses exciting challenges that require new instrumentation and new labeling strategies. Key Concepts Resolution in light microscopy is not limited anymore by diffraction even though the optical diffraction limit still exists. Superresolution microscopy requires local contrast between structures that can be achieved through different mechanisms. All superresolution concepts are based on the control of ‘on’ and ‘off’ states of fluorescence. Label properties contribute to the achievable resolution gain, and labelling is an essential part of the superresolution result. Most superresolution images require postprocessing, and care needs to be taken to avoid image artefacts.