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The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.

Comprehensive correlation analysis for super-resolution dynamic fingerprinting of cellular compartments using the Zeiss Airyscan detector