Fluorescence Resonance Energy Transfer Gating by Elliptically Polarized Light on the Cell Surface

Abstract

Enhancements of hetero-fluorescence resonance energy transfer (FRET) and homo-FRET have been observed upon excitation with circularly polarized light as compared to excitation with the linearly polarized one in the context of fluorescent dye-labeled cell surface receptors. The enhancement effect has been gradually evolved with changing ellipticity of the exciting light from zero of the linearly polarized light to unity of the circularly polarized one. The measurements have been carried out in a single-laser dual-anisotropy scheme in a flow cytometer, where polarized intensity components in the donor and acceptor channels have been measured simultaneously. For hetero-FRET, the FRET-enhancement effect revealed by a combined detection of donor quenching and sensitized emission of the acceptor has been corroborated by a parallel increase in donor anisotropy and a decrease in acceptor anisotropy. As to homo-FRET, the FRET enhancement, inferred from increased depolarizations of anisotropies measured in two different emission bands, centered at a shorter and a longer wavelength, has also been corroborated by a parallel reduction in “the longer to shorter wavelength” anisotropy ratio. The observed enhancement effects are explained by a relief of orientation mismatch between the FRET donors and acceptors, a phenomenon observable when excitation is depolarized as compared to the linearly polarized one, and a concomitant “homo-FRET-amplified hetero-FRET” phenomenon, both based on an increase in the orientation factor (κ2) for FRET. Circularly polarized light represents a depolarized mode of excitation leading to a photoselected orientational distribution of the donors matching better with that of the potential acceptors. The magnitude of this effect is dictated by the anisotropy of the orientational distributions of the donor and the acceptor. By comparing FRET with excitations of different helicities, a new steady-state method for the detection of molecular dynamics is presented. The method can equally be used in flow cytometers and fluorescence microscopes equipped with excitation with helical light even when the detection is not polarized. The conventional formalism of anisotropy detection in the T-format at vertically polarized excitation has been extended to linearly polarized excitation at different polarization angles and depolarized excitations.