Time and frequency-domain measurement of ground-state recovery times in red fluorescent proteins

The Journal of Physical Chemistry. B
Premashis Manna, Ralph Jimenez

Abstract

The field of bioimaging and biosensors has been revolutionized by the discovery of fluorescent proteins (FPs) and their use in live cells. FPs are characterized with rich photodynamics due to the presence of nonfluorescent or dark states which are responsible for fluorescence intermittency or "blinking", which has been exploited in several localization-based super-resolution techniques that surpass the diffraction-limited resolution of conventional microscopy. Molecules that convert to these dark states recover to the ground states either spontaneously or upon absorption of another photon, depending on the particular FP and the structural transition that is involved. In this work, we demonstrate time- and frequency-domain methods for the measurement of the ground-state recovery (GSR) times of FPs both in live cells and in solutions. In the time-domain method, we excited the sample with millisecond pulses at varying dark times to obtain percent-recovery. In the frequency-domain method, dark-state hysteresis was employed to obtain the positive phase shift or "phase advance". We extracted the GSR time constants from our measurements using calculations and simulations based on a three-state model system. The GSR time constants of t...Continue Reading

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Citations

Dec 18, 2015·Journal of the American Chemical Society·Romain BerardozziDominique Bourgeois
Apr 22, 2015·Analytical Chemistry·Kevin M DeanRalph Jimenez
Jan 27, 2017·The Journal of Physical Chemistry Letters·Yen-Cheng Chen, Robert M Dickson
Aug 11, 2018·Integrative Biology : Quantitative Biosciences From Nano to Macro·Premashis MannaRalph Jimenez
Jun 5, 2021·Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photobiology·Elena A ProtasovaAlexey M Bogdanov

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