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Simultaneous time resolution of the emission spectra of fluorescent proteins and zooxanthellar chlorophyll in reef-building corals[para][dagger]
Photochemistry and Photobiology, May 2003 by Gilmore, Adam M, Larkum, Anthony W D, Salih, Anya, Itoh, Shigeru, Et al
RESULTS
Simultaneous time resolution of FP and zooxanthellae PSII fluorescence emission spectra
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Figure 2 illustrates a comprehensive view of the time and wavelength dependence of both the FP and zooxanthellae fluorescence from a green color morph specimen of P. versipora harvested from Sydney Harbor, Australia. Figure 2a illustrates the decay kinetics of the clearly separate FP and chl emission components. The FP fluorescence bands (peak ~515 nm) emitted with stronger amplitudes (left side of main panel) and slower decay times compared with the algae's chl fluorescence bands (peak 683 nm, upper right of main panel). Figure 2b profiles the data and model fits on a log scale to illustrate the slower main FP emission component in direct comparison with the more rapid PSII component. Figure 2c illustrates the same spectral data as in Fig. 2a,b plotted as a function of time, where each time channel is normalized to the peak FP emission wavelength intensity. It is clear that all components of the chl exhibit kinetics that are independent of the FP emission. Figure 2d shows the main fluorescence lifetime decay distribution components with positive amplitudes used to simulate the chl (red) and FP emission (green) in Fig. 2b. The major chl distribution mode was significantly broader and centered at a much faster lifetime value compared with the narrow FP emission component. The model simulations all clearly indicated that there were no kinetic components resolved in the analysis to show that the excited-state population of the FP exhibited any significant correlation with that of the algae's chl, i.e. except for the original laser excitation pulse event. Interestingly, however, the raw data normalization procedure in Fig. 2c did clearly reveal that the initial kinetic phase of the FP rise, during and immediately after the laser excitation (0-300 ps), is associated with a small re-producible greenshift in the fluorescence. We interpret this shift to be consistent with rapid resonance energy transfer (FRET) from blue to greener FP proteins. The evidence for FRET is examined in more detail later.
Overall, Fig. 2 clearly indicates that there is little or no evidence in the form of reciprocal amplitudes or sustained excitation for either direct resonance or indirect radiative excitation of the chl bands by the FP emission, respectively. We assume that resonance energy transfer would be evident as a reciprocal decay of the FP emission and a rise and decay in emission from the zooxanthellae, whereas an indirect radiative transfer would exhibit kinetic behavior similar to a sustained excitation pulse with the same shape profile (as a function of time) as the long-lived FP decay component. As mentioned previously, we were able to simulate the zooxanthellae emission kinetics assuming that the only convolved excitation event, beginning from time t = 0, was from the laser. The caption of Fig. 2 provides evidence for the goodness of fit by the global Durbin-Watson d-statistic (an indicator of autocorrelation trends in the time series) and Runs test (an indicator of the randomness of sign and symmetric distribution of the errors around O) (28,29).
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