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

Figure 5c illustrates the correspondence between the spectral kinetic contours of the model donor (blue) and acceptor (green) species and the actual image data (dotted line). The rapid decay of the donor that behaves as one component is evident when plotted on this linear (unnormalized) scale and complete to the ground state in less than a 1 ns time frame. The bands assigned to the acceptor(s) indicated some kinetic heterogeneity in the form of bluer components accepting from the donor and other greener species, apparently directly excited by the laser pulse. The differences in rise components for the acceptor's component bands were accounted for by allowing model variation for the relative amplitudes of the negative rise components and the main decay component. Figure 5d shows a normalized format for the image (dotted lines) and model (colored contour and lines) similar to that shown in Fig. 2c. It is evident from both the normalized spectral contour plot (Fig. 5d) and the natural spectral contour plot (Fig. 5c) that the obvious spectral shift is reasonably well simulated by the FRET model. However, because of the nature of the tendency for reciprocal amplitude components to cancel each other in the same spectral region, we cannot prove that the solutions obtained by the model are kinetically unique. Nonetheless, we conclude that the data and model simulation, especially when considered together, provide irrefutable evidence for FRET among the FP in vivo.

DISCUSSION

Inefficient transfer of light energy from the FP to the zooxanthellae

This study documents that in the shallow water corals analyzed, the relative quantum yield of energy transfer of the blue to green FP fluorescence to the light-harvesting apparatus of the zooxanthellae is negligible. This is in comparison with the direct excitation by absorption of light energy of wavelengths greater than 350 nm. The result is clear despite the fact that both the blue and green FP emission spectra clearly overlap with the excitation spectra of the zooxanthellae. The three main pieces of kinetic evidence obtained from the simultaneous acquisition analysis supporting the main conclusion include that (1) the symbionts' spectral components from PSII indicate distinctly more rapid decay kinetic modes and also different fluorescence lifetime distribution width and center properties; (2) the model simulation is consistent with only one convolved excitation source for both the FP and zooxanthellae components, namely, the laser pulse; the kinetic model thus indicates a low quantum yield for reabsorption of the FP emission by the zooxanthellae; and (3) there is no evidence of reciprocal kinetic amplitudes between the FP and zooxanthellae to indicate dipole-dipole interactions required for FRET between the two emitters.

The lack of FRET between algal PSII and the FP is most likely explained by prohibitive distance factors between the chromophores in the respective organelles (A. Salih, unpublished). Consistent with the kinetic data and modeling, the steady-state spectral data clearly show that the FP emission and excitation contour maps and integral profiles do not correlate with those of the algae's chl components. The data thus indicate that under natural illumination conditions the blue to green FP fluorescence would not make any substantial contributions to light harvesting in shallow water-dwelling corals. One important technical note, as an indication for the relative quantum yield of direct excitation, compared with indirect or FRET mechanisms, is that the excitation pulse profiles were acquired under conditions where the laser emission was attenuated with neutral density filters by a factor of 2 x 10^sup -4^. In these experiments, the peak laser signal intensity was still acquired in roughly 10-25% of the time required to resolve a strong FP emission signal. Moreover, it was also obvious that the yield of the zooxanthellae PSII emission was considerably lower than the FP emission.