A High-Throughput Image Correlation Method for Rapid Analysis of Fluorophore Photoblinking and Photobleaching Rates

ACS Nano. 2019 Oct 22;13(10):11955-11966. doi: 10.1021/acsnano.9b06033. Epub 2019 Sep 16.


Super-resolution fluorescence imaging based on localization microscopy requires tuning the photoblinking properties of fluorescent dyes employed. Missing is a rapid way to analyze the blinking rates of the fluorophore probes. Herein we present an ensemble autocorrelation technique for rapidly and simultaneously measuring photoblinking and bleaching rate constants from a microscopy image time series of fluorescent probes that is significantly faster than individual single-molecule trajectory analysis approaches. Our method is accurate for probe densities typically encountered in single-molecule studies as well as for higher density systems which cannot be analyzed by standard single-molecule techniques. We also show that we can resolve characteristic blinking times that are faster than camera detector exposure times, which cannot be accessed by threshold-based single-molecule approaches due to aliasing. We confirm this through computer simulation and single-molecule imaging data of DNA-Cy5 complexes. Finally, we demonstrate that with sufficient sampling our technique can accurately recover rates from stochastic optical reconstruction microscopy super-resolution data.

Keywords: fluorescence microscopy; image correlation; photoblinking; photophysical and photochemical rates; single-molecule; super-resolution.

Publication types

  • Research Support, Non-U.S. Gov't