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Optical super-resolution microscopy is a key technology for structural biology that optimally complements electron microscopy because of its high contrast and live-cell compatibility. MINFLUX is an emerging super-resolution technique that measures the precise position of single fluorophores by targeted scanning of a patterned excitation beam featuring a minimum. It achieves unprecedented localization precision for a given number of detected photons. In combination with switchable fluorophores, it can be used for super-resolution imaging with nanometer resolution. For tracking of single fluorophores, it greatly outperforms camera-based techniques and achieves nanometer spatial and sub-millisecond temporal resolution over long tracks. Here, we introduce the basic principle of MINFLUX and explain how it can reach such high photon efficiencies. We then discuss the potential and limitations of MINFLUX for super-resolution imaging and single-fluorophore tracking, describe recent extensions and variations of MINFLUX and give an outlook to future developments.

MINFLUX is an emerging super-resolution technology that measures the position of single fluorophores with nanometer precision using fewer photons than any other fluorescence imaging or tracking technique. Here, we derive simple and instructive analytical equations for MINFLUX localization precision with a special focus on background photons. A fluorescence background, either arising from an imperfect zero of the MINFLUX excitation point spread function (PSF) or from auto- or out-of-focus fluorescence, ultimately limits the resolution achievable with MINFLUX. Building on previous work, we try to improve our understanding of the influence of background, especially when it is unknown, through a new set of expressions for the localization precision, based on an explicit background term instead of signal-to-background ratio. We use these equations to generate an intuitive understanding of how fluorescence background affects MINFLUX measurements and illustrate that: - The precision of an emitter position estimate depends on the gradient of its excitation profile. - Knowledge of the fluorescence background, obtained during post-processing of MINFLUX traces or through separate measurements, provides a better localization precision than in the case of unknown background. - In diffraction-limited systems, localization with a PSF that features a near-zero minimum outperforms localization with a maximum. We also present an analytical expression for the localization precision in orbital tracking, which we use for comparison to MINFLUX.