A High-Throughput Image Correlation Method for Rapid Analysis of Fluorophore Photoblinking and Photobleaching Rates
| Autor: | Simon Sehayek, Hugo B. Brandão, Gonzalo Cosa, Paul François, Viktorija Glembockyte, Yasser Gidi, Paul W. Wiseman |
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| Rok vydání: | 2019 |
| Předmět: |
0303 health sciences
Fluorescence-lifetime imaging microscopy Digital image correlation Materials science Fluorophore Biophysics General Engineering General Physics and Astronomy 02 engineering and technology 010402 general chemistry 021001 nanoscience & nanotechnology 01 natural sciences Photobleaching Fluorescence 0104 chemical sciences 03 medical and health sciences chemistry.chemical_compound chemistry Microscopy Fluorescence microscope General Materials Science 0210 nano-technology Throughput (business) 030304 developmental biology |
| Zdroj: | ACS Nano. 13:11955-11966 |
| ISSN: | 1936-086X 1936-0851 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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