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Inspired by recent publications doubtful of the FCS technique, we scrutinizehow irreversible (static) and reversible (dynamic) quenching can influence theinterpretation of such data. We consider intermediate cases where theassessment of photophysics (static quenching, blinking-like triplet staterelaxation) influence on autocorrelation curves can be delicate if dye-labeledobjects diffuse on comparably-rapid time scales and use tryptophan as thequencher. As our example of small-molecule dye that diffuses rapidly, we mixquencher with Alexa 488 dye, and quenching is reflected in the fact that thedata become exceptionally noisy. This reflects the bidisperse population ofquenched and unquenched dye when the time scales overlap between the processesof translational diffusion, quenching, and blinking. As our example oflarge-molecule dye-labeled object, we mixed quencher with dye-labeled bovineserum albumin. Diffusion, static quenching and blinking time scales are nowseparated, and inferred translational diffusion now depends weakly on quencher.We conclude that when the diffusing molecule is substantially slower to diffusethan the time scale of photophysical processes of the fluorescent dye to whichit is attached, influence of quenching is self-evident and the FCSautocorrelation curves give appropriate diffusion coefficient if correctfitting functions are chosen in the analysis.

Robustness of FCS (Fluorescence Correlation Spectroscopy) with Quenchers Present