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TitleA High-Throughput Image Correlation Method for Rapid Analysis of Fluorophore Photoblinking and Photobleaching Rates.
Publication TypeJournal Article
Year of Publication2019
AuthorsSehayek, Simon, Yasser Gidi, Viktorija Glembockyte, Hugo B. Brandão, Paul François, Gonzalo Cosa, and Paul W. Wiseman
JournalACS Nano
Date Published2019 Sep 16
ISSN1936-086X
Abstract

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.

DOI10.1021/acsnano.9b06033
Alternate JournalACS Nano
PubMed ID31513377