Recognition of Single Fluorescence Events by Temporal Pixel Intensity Fluctuation

Single-molecule localization microscopy circumvents the diffraction limit of traditional fluorescence microscopy by detecting the photoemission signals of individual fluorescent molecules. The accurate recognitions of fluorescence molecules/events are critical to single-molecule/super-resolution imaging experiments, which determine the precision of molecular localizations and the quality of the image reconstruction. Herein, we presented a single-molecule detection method which relied on the temporal pixel intensity fluctuation. The method was capable of quickly determining the approximate localizations of fluorescence events with high sensitivity. We evaluated the performance of the method under a series of signal-to-noise ratios (SNR) and discussed the criterion of setting the temporal fluctuation threshold to achieve the optimized spots recognition results.