MINSTED fluorescence localization and nanoscopy

We introduce MINSTED, a stimulated-emission-depletion (STED) based fluorescence localization and super-resolution microscopy concept providing spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED donut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background, and the required number of fluorescence detections are low compared to most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200-1000 detections per fluorophore provide a localization precision of 1-3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement of far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.