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July 18th, 2025
Version: 2
University of Amsterdam
cell biology
biorxiv

mScarlet3-H with low brightness and fluorescence lifetime has potential for cellular lifetime-unmixing and lifetime-based pH-sensing applications

Gadella, T. W. J.Open in Google Scholar•van Weeren, L.Open in Google Scholar

An independent evaluation of the spectroscopic properties, cellular performance, merits and pitfalls of the red fluorescent protein mScarlet3-H as compared to mScarlet3 is reported. mScarlet3-H was generated from mScarlet3 by a single M163H mutation. Purified mScarlet3-H is characterized by a molar coefficient of 79,040 M-1cm-1, a fluorescence quantum yield of 17.8%, molecular brightness of 14.1 and a heterogeneous multiexponential decay with an average fluorescence lifetime of 1 ns. Evaluation in living mammalian cells revealed a comparable maturation speed and efficiency of mScarlet3 and mScarlet3-H, but the overall cellular brightness of mScarlet3-H was 5-fold lower than that of mScarlet3. Photobleaching analysis in live cells revealed identical photobleaching kinetics of mScarlet3-H and mScarlet-H. The fluorescence intensity, fluorescence spectra and fluorescence lifetime of mScarlet3-H were found to be strongly pH-dependent between pH 4-8. The fluorescence lifetime increased from 1 ns to 3 ns in lowering the pH from 8 to 4 with a pK of ~6. The much lower lifetime of mScarlet3-H (~1 ns) as compared to mScarlet3 (~4 ns) allows dual fluorescence lifetime unmixing applications in single channel FLIM recordings in compartments with neutral to slightly alkaline pH. Furthermore, the strongly pH-dependent fluorescence lifetime of mScarlet3-H enables fluorescence lifetime-based pH sensing in a pH region between pH 5 to 7. Using this property, a pH of 6.5 was measured in the lumen of the Golgi system in living cells and motile endomembrane vesicles revealed pH values between 5.5 and 6. Also autophagy of the cytoplasm can be visualized by the pH-dependent fluorescence lifetime with mScarlet3-H accumulation in lysosomes. Potential useful applications and pitfalls regarding the special properties of mScarlet3-H are discussed.

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