Efficient photoconversion distorts the fluorescence lifetime of GFP in confocal microscopy: a model kinetic study on mutant Thr203Val.

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.