Fluorescence fluctuation‐based super‐resolution microscopy: Basic concepts for an easy start

Due to the wave nature of light, optical microscopy has a lower‐bound lateral resolution limit of approximately half of the wavelength of visible light, that is, within the range of 200 to 350 nm. Fluorescence fluctuation‐based super‐resolution microscopy (FF‐SRM) is a term used to encompass a collection of image analysis techniques that rely on the statistical processing of temporal variations of the fluorescence signal. FF‐SRM aims to reduce the uncertainty of the location of fluorophores within an image, often improving spatial resolution by several tens of nanometers. FF‐SRM is suitable for live‐cell imaging due to its compatibility with most fluorescent probes and relatively simple instrumental and experimental requirements, which are mostly camera‐based epifluorescence instruments. Each FF‐SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a range of FF‐SRM methods published to date are described. Their operational parameters are explained and guidance for their selection is provided.