Efficient photoconversion distorts the fluorescence lifetime of GFP in confocal microscopy: a model kinetic study on
Gregor Jung1, Michael Werner, Marc Schneider
1Biophysical Chemistry, Saarland University, Campus, Building B2 2, 66123 Saarbrücken, Germany. g.jung@mx.uni-saarland.de
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.
Insights
Accidental phototransformations in green fluorescent proteins (GFPs) can interfere with microscopy. This study quantifies photoconversion (PC) and photobleaching (BL) in a specific GFP mutant, revealing their kinetics and impact on experiments.
Area of Science:
- Biophysics
- Photochemistry
- Microscopy
Background:
- Phototransformations of autofluorescent proteins are crucial for high-resolution microscopy and cellular transport studies.
- Unintended phototransformations like blinking and photobleaching (BL) can negatively impact experimental outcomes.
- Photoactivatable green fluorescent proteins (GFPs) are widely used but their photochemistry requires detailed understanding.
Purpose of the Study:
- To investigate the kinetics of phototransformations in a photoactivatable green fluorescent protein (GFP) mutant using confocal microscopy.
- To quantify the quantum yields of photoconversion (PC) and photobleaching (BL) for this GFP mutant.
- To understand how excitation conditions affect fluorescence lifetime and the implications for experimental data.
Main Methods:
- Utilized confocal microscopy to study phototransformations in a GFP mutant (Thr203Val).
- Employed time-correlated single-photon-counting detection in a confocal geometry for freely diffusing species.
- Analyzed the kinetic scheme of consecutive reactions involving chromophore states and dark protein species.
Main Results:
- Determined the quantum yields for photoconversion (Phi(PC) = 5.5 x 10(-4)) and photobleaching (Phi(BL) > 1 x 10(-5)).
- Observed that the fluorescence lifetime (tau(fl) = 2.2 ns) of the photoconverted state depends on excitation conditions and history.
- Established a kinetic model describing the interplay between PC and BL.
Conclusions:
- Photoconversion (PC) in GFPs can lead to misinterpretations in fluorescence resonance energy transfer (FRET) experiments.
- Spectroscopic peculiarities observed in single-molecule detection can be attributed to these phototransformation processes.
- Understanding the kinetics of PC and BL is essential for accurate interpretation of GFP-based experimental data.
