Abstract
Single-molecule FRET has greatly contributed to our detailed mechanistic understanding of many bio-molecular systems. While reactions occurring in the range of several minutes can be readily studied, data acquisition for longer time scales is hindered by accumulated focal drift of a high numerical aperture objective, which should be corrected in real time. Here, we develop an autofocusing system based on the analysis of optical astigmatism of single-fluorophore images. Compared to the other autofocusing methods, our approach has a merit of simplicity that neither fiduciary makers nor additional light sources and detectors are required. As a demonstration of the new autofocusing system, we observed slow B-Z transition dynamics occurring in several hours using single molecule FRET.
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