4:45 PM - 5:00 PM
▲ [16p-2C-10] Dark state dynamics of eGFP investigated by temporally-modulated excitation
Keywords:Fluorescent protein
Photobleaching of fluorescent proteins prevents proper measurements and efficient image acquisition. For instance, the ratio measurements in FRET imaging are severely affected by photobleaching because donor and accepter molecules bleach at different rates. The mechanism of photobleaching has been explored by a number of researchers. In two photon excitation (TPE), the broad spectrum of an excitation laser such as Ti:S laser causes excited state absorption (ESA), which may trigger photobleaching. Dark states of fluorescence molecules have much longer life time than the singlet state which emits fluorescence. Thus, ESA from a dark state is a plausible candidate as a photobleaching process. In this work, we investigated the dark state dynamics of eGFP.
We assumed that there are three energy states (S0, S1, and T1) in the molecules of the eGFP in one photon excitation. While the fluorescent molecules are being excited, the fluorescence intensity decays with the transition to T1. When the light exposure is off, the fluorescence intensity recovers due to the relaxation from the T1. These processes happen in a timescale of µs to ms and thus the observation of these processes through fluorescence requires temporal modulation of the excitation light in this time scale. Experiments were conducted with a home-built confocal setup. Temporal modulation was done by an acousto-optic modulator.
If the energy state dynamics could be described by a three state model, the fluorescence decay curve had to be well fitted to a single exponential function. However, the decay curve was fitted to a biexponential function. We also investigated fluorescence recovery during the excitation intervals and the recovery curve was also fitted to a biexponential function.
Biexponential behaviors of the fluorescence imply the existence of another transient dark state other than T1. Assuming there is another dark state, we performed numerical simulations of the energy state dynamics and determined kinetic parameters involving these states.
In summary, the dark state dynamics of eGFP was explored by temporally-modulated excitation. The fluorescence variations due to the transitions between the singlet states and the dark states were observed, and the results imply the existence of the dark state other than T1. The kinetic parameters involving these states were determined by numerical simulations. Branching ratios of photobleaching through the ESA from these dark states will be reported at the conference.
We assumed that there are three energy states (S0, S1, and T1) in the molecules of the eGFP in one photon excitation. While the fluorescent molecules are being excited, the fluorescence intensity decays with the transition to T1. When the light exposure is off, the fluorescence intensity recovers due to the relaxation from the T1. These processes happen in a timescale of µs to ms and thus the observation of these processes through fluorescence requires temporal modulation of the excitation light in this time scale. Experiments were conducted with a home-built confocal setup. Temporal modulation was done by an acousto-optic modulator.
If the energy state dynamics could be described by a three state model, the fluorescence decay curve had to be well fitted to a single exponential function. However, the decay curve was fitted to a biexponential function. We also investigated fluorescence recovery during the excitation intervals and the recovery curve was also fitted to a biexponential function.
Biexponential behaviors of the fluorescence imply the existence of another transient dark state other than T1. Assuming there is another dark state, we performed numerical simulations of the energy state dynamics and determined kinetic parameters involving these states.
In summary, the dark state dynamics of eGFP was explored by temporally-modulated excitation. The fluorescence variations due to the transitions between the singlet states and the dark states were observed, and the results imply the existence of the dark state other than T1. The kinetic parameters involving these states were determined by numerical simulations. Branching ratios of photobleaching through the ESA from these dark states will be reported at the conference.