Two-Photon Fluorescence Resonance Energy Transfer in Protein Fluorophore Complexes

Abstract

Summary Fluorescence resonance enerm transfer (HUi7) is B technique now widely applied to probe biological and other complex systems for the determination of fluorophore separation and structore Recently Galanin's seminal results for the residual anisotropy following resonance energy transfer have been enended beyond the Foister limit to include the radiative long-range (wave-zone) transfer' These results are now funher extended to accommodate two-photon excitation, revealing not only novel polarisation charaneristics but also a distance dependence whose analysir doer not require opriorr knowledge ofthe donor-acceptor spectral overlap. Multi-photon microscopy is now a commercially available technique. used infer alia to probe deep into biological systems Very recently there have been repons of three-photon excitation of lerotonin in leukemia infected rat cells Here the use of multi-photon nearinfrared excitation circumvents the ofhenvise destructive use of high energy ultraviolet photons and dla~ the technique to be used for in vivo imaging This non-destructive technique taken together with the well-known use of FRET as B spectroscopic mlule, reflects the practical utility ofthe theory to be developed. In thispaper we consider the modified anisotropy oftwo-photon induced fluorescence, resulting following resonant energy transfer from the site of excitation, to an acceptor tluorophore from which the luminescence signal emerges A mmber of novel features emerge For example ir vanspires that the fluorescence cm be injtiated with either plane or circularly polarised light and that the collected signal displays a residual polarisation anisotropy that increaser by afmor of precisely seven as the transfer distance increases from the near-me to wave-mne range. Due to the non-destructive naNn of the nonlinear scattering pre-jing the photon migration this may be a technique ideally suited to biological tissues. The anisotropy is earily calwlated. using the results to be presented by simple ratios of the intensity of the fluorescence &er pa&g through light palarisers Inherent in the redti is a separation funetion that links near and far-rone asymptotes. For main fluorophore pairs it may be possible to tez the hypothesis that these asymptotes are simply the limits of a unified transfer process.