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Efficient Photoconversion Distorts the Fluorescence Lifetime of GFP in Confocal Microscopy: A Model Kinetic Study on Mutant Thr203Val
Author(s) -
Jung Gregor,
Werner Michael,
Schneider Marc
Publication year - 2008
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.200800276
Subject(s) - photobleaching , chromophore , fluorescence , chemistry , förster resonance energy transfer , green fluorescent protein , confocal microscopy , photochemistry , microscopy , fluorescence microscope , fluorescence recovery after photobleaching , confocal , fluorescence lifetime imaging microscopy , two photon excitation microscopy , kinetics , biophysics , optics , physics , biochemistry , quantum mechanics , biology , gene
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 τ fl =2.2 ns. In fluorescence lifetime imaging microscopy, τ 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, Φ PC and Φ BL . The assessed values are Φ PC =5.5×10 −4 and Φ BL >1×10 −5 . Based on these values, PC provokes misinterpretation in fluorescence resonance energy transfer experiments and is responsible for spectroscopic peculiarities in single‐molecule detection.