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

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.