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The MINFLUX concept: molecular resolution in fluorescence nanoscopy. (a) Implementation of MINFLUX in 2D fluorescence imaging and tracking. (Top) Diagrams of the positions of the doughnut in the focal plane and resulting fluorescence photon counts. (Bottom) Basic application modalities of MINFLUX. (Left) Nanoscopy: A nanoscale object features molecules whose fluorescence can be switched on and off, such that only one of the molecules is on within the detection range. They are distinguished by abrupt changes in the ratios between the different n _0,1,2,3 or by intermissions in emission. (Middle) Nanometer-scale (short-range) tracking: The same procedure can be applied to a single emitter that moves within the localization region of size L. As the emitter moves, different fluorescence ratios are observed that allow the localization. (Right) Micron-scale (long-range) tracking: If the emitter leaves the initial L-sized field of view, the triangular set of positions of the doughnut zeros is (iteratively) displaced to the last estimated position of the molecule. By keeping it around r ₀ by means of a feedback loop, photon emission is expected to be minimal for n ₀ and balanced between n ₁, n ₂, and n ₃, as shown. (b) With MINFLUX nanoscopy one can, for the first time, separate molecules optically which are only a few nanometers apart from each other. On the left, a schematic of the molecules is presented. Whereas the ultra-high resolution PALM/STORM microscopy at the same molecular brightness (Right) delivers a diffuse image of the molecules (here in a simulation under ideal technical conditions), the position of the individual molecules can be easily discerned with the practically realized MINFLUX (middle). (c) Many much faster movements can be followed than is possible with STED or PALM/STORM microscopy. Left: Movement pattern of 30S ribosomes (colored) in an E. coli bacterium (gray scale). Right: Movement pattern of a single 30S ribosome (green) shown enlarged. (d) MINFLUX tracking of rapid movements of a custom-designed DNA origami. (Top left) Diagram of the DNA origami construct with a single ATTO 647N fluorophore attached at the center of the bridge (10 nm from the origami base). By design, the emitter can move on a half-circle above the origami and is thus ideally restricted to a 1D movement. (Bottom left) Histogram of 6118 localizations of the sample with δt = 400 μs time resolution and a 1.5 × 1.5-nm binning. The predominant motion is along a single direction. (Right, Upper) A 300-ms excerpt of the photon count trace (time resolution δt = 400 μs per localization). The color coding corresponds to the zero positions shown to the left. (Right, Lower) Mean-subtracted trajectory. Figure reproduced from [72] (a) (reprinted with permission from AAAS) and [73] (d)