Efficient Photoconversion Distorts the Fluorescence Lifetime of GFP in Confocal Microscopy: A Model Kinetic Study on Mutant Thr203Val

Abstract

Phototransformations of autofluorescent proteins are applied in high-resolution microscopy and in studying cellular transport, but they are detrimental when accidentally occurring in blinking or photobleaching (BL). Here, we investigate the kinetics of phototransformations of a photoactivatable green fluorescent protein (GFP) in confocal microscopy. Photoconversion (PC) is achieved by excitation of the barely present anionic chromophore state R(eq) (-) in the GFP mutant Thr203Val. Besides the shift of the equilibrium between the neutral chromophore state RH and R(eq) (-), the photoconverted anionic chromophore R(PC) (-) exhibits a reduced fluorescence lifetime tau(fl)=2.2 ns. In fluorescence lifetime imaging microscopy, tau(fl) is found to depend, however, on the excitation conditions and history. The underlying photochemistry is described by the kinetic scheme of consecutive reactions, R(eq) (-)-->R(PC) (-)-->P(dark), in which the anionic chromophore species and the dark protein P(dark) are coupled by PC and BL. Time-correlated single-photon-counting detection in a confocal geometry of freely diffusing species is used to compute the quantum yields for PC and BL, Phi(PC) and Phi(BL). The assessed values are Phi(PC)=5.5 x 10(-4) and Phi(BL)>1 x 10(-5). Based on these values, PC provokes misinterpretation in fluorescence resonance energy transfer experiments and is responsible for spectroscopic peculiarities in single-molecule detection.

Ultrafast Photoconversion of the Green Fluorescent Protein Studied by Accumulative Femtosecond Spectroscopy

“…It rather seems likely that under those conditions the long-living R I -* state, which is created after excited state proton transfer, is responsible for photoconversion with a fairly low efficiency. This, however, can yield significant amounts of Rpc through prolonged (quasi-)CW irradiation (19,25).…”

“…This mutant is particularly appropriate for investigating photoconversion due to the preponderance of RH ( Fig. 2) and the detailed knowledge about its fluorescence dynamics (11,(17)(18)(19).…”

Visualization of Cu2+ uptake and release in plant cells by fluorescence lifetime imaging microscopy

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