Adaptive-illumination STED nanoscopy

Significance We demonstrate DyMIN (Dynamic Intensity Minimum), a versatile illumination concept for coordinate-targeted superresolution fluorescence imaging. Dynamically adapting the illumination (and therefore resolution) for the probing of molecular signals to local structural features entails major reductions in the light dose applied to the sample over the duration of the image scan. The resulting strong photobleaching reduction benefits signal and image contrast, as well as resolution, in biological nanoscale imaging. We show DyMIN stimulated emission depletion nanoscopy of different challenging samples in two and three dimensions. The concepts called STED/RESOLFT superresolve features by a light-driven transfer of closely packed molecules between two different states, typically a nonfluorescent “off” state and a fluorescent “on” state at well-defined coordinates on subdiffraction scales. For this, the applied light intensity must be sufficient to guarantee the state difference for molecules spaced at the resolution sought. Relatively high intensities have therefore been applied throughout the imaging to obtain the highest resolutions. At regions where features are far enough apart that molecules could be separated with lower intensity, the excess intensity just adds to photobleaching. Here, we introduce DyMIN (standing for Dynamic Intensity Minimum) scanning, generalizing and expanding on earlier concepts of RESCue and MINFIELD to reduce sample exposure. The principle of DyMIN is that it only uses as much on/off-switching light as needed to image at the desired resolution. Fluorescence can be recorded at those positions where fluorophores are found within a subresolution neighborhood. By tuning the intensity (and thus resolution) during the acquisition of each pixel/voxel, we match the size of this neighborhood to the structures being imaged. DyMIN is shown to lower the dose of STED light on the scanned region up to ∼20-fold under common biological imaging conditions, and >100-fold for sparser 2D and 3D samples. The bleaching reduction can be converted into accordingly brighter images at <30-nm resolution.