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bioRxiv · 10.1101/2025.07.15.664898

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

Gadella, T. W. J.·van Weeren, L.
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Abstract

An independent evaluation of the spectroscopic properties, cellular performance, merits and pitfalls of the redfluorescent 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 coeWicient of 79,040 M-1cm-1, afluorescence quantum yield of 17.8%, molecular brightness of 14.1 and a heterogeneous multiexponential decay with an averagefluorescence lifetime of 1 ns. Evaluation in living mammalian cells revealed a comparable maturation speed and eWiciency 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. Thefluorescence intensity,fluorescence spectra andfluorescence lifetime of mScarlet3-H were found to be strongly pH-dependent between pH 4-8. Thefluorescence 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 dualfluorescence lifetime unmixing applications in single channel FLIM recordings in compartments with neutral to slightly alkaline pH. Furthermore, the strongly pH-dependentfluorescence lifetime of mScarlet3-H enablesfluorescence lifetime-based pH sensing in a pH region between pH 5 to 7. With this property autophagy of the cytoplasm can be visualized by the pH-dependentfluorescence 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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Gadella, T. W. J., van Weeren, L.. 2025-07-16. mScarlet3-H with low brightness and fluorescence lifetime has potential for cellular lifetime-unmixing and lifetime-based pH-sensing applications. https://doi.org/10.1101/2025.07.15.664898

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