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

Evidence for FRET among fluorescent proteins

Figure 4 outlines evidence for FRET among the FP obtained by analyzing the kinetic sequences of the normalized spectral profiles from the FP emission regions. The data in the left panels correspond to 10 spectral profiles resolved at time intervals of 31.25 ps for specimens of A. digitiferti (Fig. 4a,b), A. nasuta (Fig. 4c,d) and both a blue (Fig. 4e,f) and green (Fig. 4g,h) morph of P. versipora. The spectral profiles referred to as 0 ps (black lines and shaded areas) correspond to the initial time frame of the analysis that coincided closely with the rise of the emission when a spectral shape was first clearly resolved. The difference spectra in the right panels correspond to the difference between the final spectrum at 281.25 ps and all the previous spectra and hence represent the time-resolved difference between the initial donor species and the final acceptor species of the FP. It is clear that each specimen exhibited a similar pattern with a time-dependent shift from blue to greener fluorescence that appeared to be complete, when viewed in this normalized format, in less than 200 ps. The shift was strongest in the two acroporids (Fig. 4a-d) and weakest in the green P. versipora (Fig. 4g,h). The peak of the difference spectra coincided with the peak of the FP emission, being greenest in the green P. versipora morph. In general, the spectral change coincided with a 3-10 nm shift in the peak emission and a filling in of the greener wing of the spectral profile. Once complete, there was little, if any, evidence for further spectral changes in the FP region, suggesting excitation had transferred to a terminal acceptor pool.

Figure 5 outlines a model FRET simulation of the FP streak-camera image obtained with A. digitifera and corresponds to the data presented in Fig. 4a,b. The image was simulated assuming two types of FP species, namely a donor species (blue) associated with the spectral contours in the early rise and rapid decay components and acceptor species (green) associated with the main decay spectral contour. The kinetic profiles plotted on a linear scale in Fig. 5a correspond to the complete wavelength-dependent integral of the measured image for each of the FP species, the total data, the model and the laser excitation pulse. The subplot displays the randomly distributed weighted residual errors to show that there were no remarkable systematic deviations to indicate a significant kinetic discrepancy between the data and model simulation. As explained in the caption of Fig. 5, the goodness of fit was further reflected by the acceptable global Durbin-Watson (-d-statistic and Runs test (28,29).

It is clear from Fig. 5a that there is a kinetic correspondence between the decay of the donor and initial rise of the acceptor. Figure 5b plots the corresponding distributions of m([tau]) for the spectral bands assigned to the donor and acceptor species; the lifetime distribution data in Fig. 5b represent the total integral for all bands from both species. The donor and acceptor were modeled with four and five Gaussian spectral bands, respectively; the sums but not the individual bands for both species are illustrated for clarity. The rapid decay of the donor was accounted for by a minor rapid (