Beating Nyquist Limits for the Measurement of Fluorophore Blinking Rates using Image Correlation and Camera Detection

Abstract

We present an ensemble autocorrelation technique for rapidly measuring on- and off-blinking rate constants from an image time series of fluorescent probes that is significantly faster than individual single-molecule trajectory analysis approaches. We demonstrate that the use of this technique allows for the extraction of characteristic blinking times which are faster than camera detector exposure times, which cannot be accessed by threshold based single molecule approaches due to aliasing. We test our technique on a wide set of computer simulations, as well as on TIRF image series of surface-immobilized labeled DNA. We analyzed DNA-Cy5 complex in the presence of varying concentrations of Ni(II) ions, which act as triplet-state quenchers. In general, we have found good agreement between our technique and a single-molecule based method. Furthermore, we recover blinking times which are shorter than the detector exposure time from one of the datasets. Finally, we analyze STORM data, and show it is possible to recover the characteristic on-times, using the autocorrelation analysis.