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

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.

Estimating the Dynamic Range of Quantitative Single-Molecule Localization Microscopy

“…4C (Supporting Material). Although a more careful analysis is needed to precisely extract the photokinetic rates [38], our inference of these rates from the localization table is sufficient for the present analysis. Finally, the relative number of missed localizations was measured for the experimentally acquired data sets and agreed well with our theoretical estimates.…”

Kinetics of Palladium(0)‐Allyl Interactions in the Tsuji‐Trost Reaction, derived from Single‐Molecule Fluorescence Microscopy

“…Labeled ligands, used for enantioselective chemistry, may be employed as well . With this development and some technical improvements, e. g. for accessing the ms‐time regime, we head for resolving this widely applied metal‐organic reaction on the SM‐level.…”

Quantitative, super-resolution localization of small RNAs with sRNA-PAINT

“…Furthermore, quantification with a dSTORM probe using a single fluorophore molecule would be greatly hindered by photobleaching. Bleached dye molecules would result in an underestimation of the number of sRNA targets ( 20 , 35 ). In contrast, qPAINT-based methods are mostly unaffected by fluorophore photobleaching because fluorophore molecules are constantly being replenished by the binding of new imager strands ( 23 ).…”