Inspired by recent publications doubtful of the FCS technique, we scrutinize how irreversible (static) and reversible (dynamic) quenching can influence the interpretation of such data. We consider intermediate cases where the assessment of photophysics (static quenching, blinking-like triplet state relaxation) influence on autocorrelation curves can be delicate if dye-labeled objects diffuse on comparably-rapid time scales and use tryptophan as the quencher. As our example of small-molecule dye that diffuses rapidly, we mix quencher with Alexa 488 dye, and quenching is reflected in the fact that the data become exceptionally noisy. This reflects the bidisperse population of quenched and unquenched dye when the time scales overlap between the processes of translational diffusion, quenching, and blinking. As our example of large-molecule dye-labeled object, we mixed quencher with dye-labeled bovine serum albumin. Diffusion, static quenching and blinking time scales are now separated, and inferred translational diffusion now depends weakly on quencher. We conclude that when the diffusing molecule is substantially slower to diffuse than the time scale of photophysical processes of the fluorescent dye to which it is attached, influence of quenching is self-evident and the FCS autocorrelation curves give appropriate diffusion coefficient if correct fitting functions are chosen in the analysis.
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