Photobleaching Correction in Fluorescence Correlation Spectroscopy

Abstract

Fluorescence correlation spectroscopy (FCS) is a fluorescence technique conventionally used to study the kinetics of fluorescent molecules in a dilute solution. Being a non-invasive technique, FCS is now finding wider applications in the study of more complex systems like the dynamics of DNA or proteins in living cells and cell membranes. Unlike an ordinary dye solution, the dynamics of macromolecules like proteins or entangled DNA, in crowded environments is often slow and subdiffusive in nature. This in turn leads to longer residence times of the attached fluorophores in the excitation volume of the microscope. As a result, photobleaching becomes a problem: the number of photons available to calculate the intensity autocorrelation function is limited, and the decay of the fluorescence intensity itself contributes a spurious signal to the autocorrelation function that can easily obscure the signature of the molecular dynamics of interest.We present a method of calculating the intensity autocorrelation function from the arrival times of the photons on the detector that maximizes the information content while correcting for the effect of photobleaching to yield an autocorrelation function that reflects only the underlying dynamics of the sample. For this purpose, we determine the overall photobleaching rate by fitting a multi-exponential decay to the fluorescence intensity. Then we assign a weight to each photon using the reciprocal of this fit function. The autocorrelation function is then calculated from all photon pairs using the product of the individual photon weights. This gives late-arriving fluorescence photons that come from a partially photobleached sample a higher weight than those photons that arrived earlier, compensating for the loss of some of the fluorophores. We demonstrate the utility of this technique by acquiring artifact-free FCS data from entangled DNA solutions and from chromosomal DNA in E. coli.