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Based on recently published parameters (Rane et al. 2023, JPCB 127, 5046-5054) for (rs)EGFP triplet state formation and decay rates and yields, we consider the power density dependence of triplet state population dynamics and its consequences for the application of green fluorescent proteins in biological single molecule fluorescence microscopy. We find that under certain conditions, the photon budget of GFP type fluorescent proteins can be linearly dependent on power density and we propose a possible explanation for such a non-Hirschfeld photobleaching behavior. Moreover, illumination with ms pulses at sub-kHz rates is shown to improve photostability. We stipulate that a judicious choice of excitation wavelength should take into account the triplet state absorption spectrum along with the singlet state absorption spectrum. Formulas are given for the estimation of the effects of such choice as function of the experimental parameters.

Abstract Figure

Hirschfeld predicted that photobleaching rates should scale with fluorescence brightness, which is obviously not the case for fluorescent proteins found in the fpbase.org . We investigate a modified theory considering the triplet state as precursor for photobleaching.