Excitation polarization angle-resolved single-laser dual-polarization energy transfer on the cell surface

Abstract

A quick and robust approach for the simultaneous detection of donor and acceptor anisotropies and FRET efficiency is presented based on changing the angle of polarization of the exciting light in a single-laser dual channel ratiometric detection scheme of FRET in a flow cytometer. The convenience of the method lies in that for the extra assessment of anisotropies it does not necessitates alteration of the detection side of the optics conventionally used in measuring FRET in the steady state. The detection of anisotropies is pushed to the side of excitation where anisotropies are enabled by just a few sequential measurements at different polarization angles of the exciting light. The power of the method is illustrated by comparing results on the homo-associations and inter-subunit FRET on the MHCI receptor on the surface of Kit-225K6 T cells obtained with the angle-resolved approach to those obtained by the dual-T format approach as a validation. In flow conditions, but not in imaging microscopy, a minor drawback of this method is that due to the sequential nature of anisotropy detection, the mutual cell-by-cell correlations of anisotropies with each other and with FRET are lost. The merit of the approach is that besides the degree of molecular associations probed by the FRET efficiency, information can also be gained on the rotational dynamics and extent of homo-FRET prevailing in the the donor and acceptor dye systems during the hetero-FRET process, and as a consequence, orientation factor for FRET can also be estimated. FRET efficiencies at the different polarization angles are shifted depending on the donor and acceptor anisotropy, enabling quantitation of anisotropy changes by FRET efficiency. Attention is called for polarization biases when using even depolarized excitation and detecting not concerning with fluorescence polarization. When established in polarization contrast microscopy, the approach might be applied for also controlling FRET.A new formula for the determination of the sensitivity calibration factor for FRET α (or Get) has also been deduced based on the donor and acceptor anisotropies.