Time-resolved fluorescence anisotropy imaging

Siegel, J., Suhling, K., Leveque-Fort, S., Webb, S. E. D., Davis, D. M., Phillips, D., Sabharwal, Y. and French, P. M. W. (2003). Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) Imaging the rotational mobility of a fluorophore. Rev. Sci. Instrum. 74, 182-92. 

Fig. 4.9. Schematic of time-resolved fluorescence anisotropy sample is excited with linearly polarized light and time-resolved fluorescence images are acquired with polarization analyzed parallel and perpendicular to excitation polarization. Assuming a spherical fluorophore, the temporal decay of the fluorescence anisotropy, r(t), can be fitted to an exponential decay model from which the rotational correlation time, 6, can be calculated.

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

Fig. 2c. It can be seen that at 530 nm, the fluorescence decays mono-exponentially with the fluorescence lifetime of 3.24 ns. The rise of the emission seen below 50 ps in the corresponding FlUp data is obviously not resolved here. In contrast, the TCSP data at 450 nm is described by a triple-exponential decay whose dominant component has a correlation time well below the time resolution. This component is obviously equivalent to the fluorescence decay observed in the FlUp experiment. A minor contribution has a correlation time of about 3.2 ns and reflects again the fluorescence lifetime that was also detected at 530 nm. The most characteristic component at 450 nm however has a time constant of about 300 ps. It is important to emphasize that this 300 ps decay does not have a rising counterpart when emission near the maximum of the stationary fluorescence spectrum is recorded. In other words, the above mentioned mirror image correspondence of the fluorescence dynamics between 450 nm and 530 nm holds only on time scales shorter than 20 ps. Finally, in contrast to picosecond time scales, the anisotropy deduced from the TCSPC data displays a pronounced decay. This decay is reminiscent of the rotational diffusion of the entire protein indicating that the optical chromophore is rigidly embedded in the core of the 6-barrel protein.

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