Abstract
We present a method for exploring proteins in living cells. In a single measurement, the method enables to reveal a significant number of quantitative properties of the proteins dynamics including the diffusion coefficient, bound and free fraction, protein concentration and binding-unbinding kinetics. The method combines in a single measurement quantitative photobleaching, fluorescence correlation spectroscopy and fluorescence lifetime imaging. After labeling the protein of interest with a fluorescent protein, we use time correlation single photon counting to measure the fluorescence intensity at fixed points in the sample. The information provides both the fluorescent intensity as a function of time for ∼60 seconds, the rapid intensity fluctuations (KHz) and the decay time of each arriving photon. This information is processed according to a procedure that takes the biophysical dynamic model of a protein into account and the full kinetic behavior of the protein is revealed.
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