Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins

Jord C. Prangsma, Robert Molenaar, Laura Van Weeren, Daphne S. Bindels, Lindsay Haarbosch, Jente Stouthamer, Theodorus W.J. Gadella, Vinod Subramaniam, Willem L. Vos, Christian Blum*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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The fluorescence quantum yield of four representative red fluorescent proteins mCherry, mKate2, mRuby2, and the recently introduced mScarlet was investigated. The excited state lifetimes were measured as a function of the distance to a gold mirror in order to control the local density of optical states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate and the nonradiative rate of the bright states. We thus obtain for the first time the bright state quantum yield of the proteins without interference from dark, nonemitting states. The bright state quantum yields are considerably higher than previously reported quantum yields that average over both bright and dark states. We determine that mCherry, mKate2, and mRuby2 have a considerable fraction of dark chromophores up to 45%, which explains both the low measured quantum yields of red emitting proteins reported in the literature and the difficulties in developing high quantum yield variants of such proteins. For the recently developed bright mScarlet, we find a much smaller dark fraction of 14%, accompanied by a very high quantum yield of the bright state of 81%. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from quantitative fluorescence microscopy to FRET studies to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should pay more attention to minimizing the fraction of dark proteins.

Original languageEnglish
Pages (from-to)1383-1391
Number of pages9
JournalThe Journal of physical chemistry B
Issue number8
Early online date3 Feb 2020
Publication statusPublished - 27 Feb 2020


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