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

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).

Table 1 compares the spectral band and fluorescence lifetime distribution parameters for the main decay functions of both the FP emission peaks and algal PSII emission peaks that were resolved simultaneously. The experimental conditions were as described in Fig. 2 and include data from images for the blue and green color morphs of P. versipora plus a specimen of A. digitifera. In several cases the symbionts' emission was too weak to easily resolve from the strong background FP emission. Positioning of the sample with the three-axis stage allowed us to focus the laser spot (3-5 mm^sup 2^ area), in the cases presented in Table 1, on areas enriched in chl emission, which were visually brown (30) as opposed to blue or green in color. The data indicate that the FP emission bands (after completion of FRET) are broader with respect to wavelength and significantly longer in lifetime center values (> 1990 ps) than PSII, which was under the laser illumination conditions less than 800 ps. Interestingly, Table 1 indicates that the main FP emission bands were all resolved with a very narrow lifetime distribution width (100 ps).

Excitation-emission contour maps for FP and zooxanthellae PSII

Figure 3 illustrates the relationship between the wavelength of exciting light and the fluorescence emission for both FP (right) and algal PSII (left) measured at steady state with a photon-counting fluorimeter. The data compare blue and green color morphs of P. versipora. The left panels correspond to contour maps of the chl emission as a function of excitation wavelength, whereas the right panels correspond to the FP emission for the same excitation spectral region. The excitation wavelength region (350-450 nm) was chosen for two reasons: (1) because it spans both the area of peak excitation for the blue FP (Fig. 3a,b) and an area where little or no excitation is observed for the green FP (Fig. 3c,d); and (2) because it spans an area of considerable spontaneous excitation corresponding to major chl and carotenoid absorption bands. Hence, we reasoned that if energy transfer were to occur between the FP and algal PSII, then there should be a correspondence between the excitation contours and integral profiles for the FP and zooxanthellae maps. The contour maps in the left panels (Fig. 3a,c) indicate that the excitation contours for PSII (683 nm region) show little, if any, difference aside from an overlap (~20% at 650 nm) of the broad blue FP emission tail with the entire chl emission spectrum. The overlap of FP and PSII emission was considerably less in the green morph with its narrower FP emission band. The normalized integral profiles (Fig. 3e,f) for both the excitation (solid lines) and emission (clashed lines) of the zooxanthellae and FP emission reinforce the observations in the contour plots. Most importantly, the chl emission and excitation profiles are virtually indistinguishable when comparing morphs, indicating that little, if any, chl excitation can be attributed to the FP.