Fluorescence fluctuation based super resolution microscopy, basic concepts for an easy start

Abstract

Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of about half of the wavelength of the detected light, i.e., within the range of 200 to 300 nm. The Fluorescence Fluctuation based Super Resolution Microscopy (FF-SRM) encompases a collection of image analysis techniques which rely on the statistical processing of temporal variations of fluorescence to reduce the uncertainty about the fluorophore positions within a sample, hence, bringing spatial resolution down to several tens of nm. The FF-SRM is known to be suitable for live-cell imaging due to its compatibility with most fluorescent probes and lower 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 revisited. Their operational parameters are explained and guidance for its selection is provided.