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

Sehayek, Simon; Gidi, Yasser; Glembockyte, Viktorija; Brandao, Hugo B.; Francois, Paul; Cosa, Gonzalo und Wiseman, Paul W. (2019): A High-Throughput Image Correlation Method for Rapid Analysis of Fluorophore Photoblinking and Photobleaching Rates. In: ACS Nano, Bd. 13, Nr. 10: S. 11955-11966

Abstract

Super-resolution fluorescence imaging based on localization microscopy requires tuning the photo-blinking 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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Chemie und Pharmazie > Department Chemie
• Fakultätsübergreifende Einrichtungen: Center for NanoScience (CENS)
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie ; 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
• ISSN: 1936-0851
• Sprache: Englisch