title: Depolarized FRET (depolFRET) on the cell surface: FRET control by photoselection.
abstract: Sensitivity of FRET in hetero- and homo-FRET systems on the photoselected orientation distribution of donors has been proven by using polarized and depolarized light for excitation. FRET as well as donor and acceptor anisotropies have been simultaneously measured in a dual emission-polarization scheme realized in a conventional flow cytometer by using single laser excitation and applying fluorophore-conjugated mAbs against the MHCI and MHCII cell surface receptors. Depolarization of the originally polarized light have been achieved by using crystal depolarizers based on Cornu's principle, a quarter-wave plate for circular polarization, and a parallel beam splitter acting as a diagonal-polarizer for dual-polarization excitation. Simultaneous analysis of intensity-based FRET efficiency and acceptor depolarization equivocally report that depolarization of light may increase FRET in an amount depending on the acceptor-to-donor concentration ratio. Acceptor depolarization turned to be more sensitive to FRET than donor hyper-polarization and even than intensity-based FRET efficiency. It can be used as a sensitive tool for monitoring changes in the dynamics of the donor-acceptor pairs. The basic observations of FRET enhancement and increased acceptor depolarization obtained for hetero-FRET are paralleled by analog observations of homo-FRET enhancements under depolarized excitation. In terms of the orientation factor for FRET, the FRET enhancements on depolarization in the condition of the macroscopically isotropic orientation distributions such as those of the cell surface bound fluorophores report on the presence of local orientation mismatches of the donor and acceptor preventing the optimal FRET in the polarized case, which may be eliminated by the excitation depolarization. A theory of fluorescence anisotropy for depolarized excitation is also presented.
authors.affiliation: Department of Surgery, Faculty of Medicine, University of Debrecen, Debrecen, Hungary. Electronic address: bene@med.unideb.hu.
authors.forename: László
authors.initials: L
authors.lastname: Bene
authors.affiliation: Department of Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Gogolák
authors.affiliation: Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Ungvári
authors.affiliation: Department of Physiology, McGill University, Montreal, Canada.
authors.forename: Miklós
authors.initials: M
authors.lastname: Bagdány
authors.affiliation: Division of Electronics, Research Center for Nuclear Physics of the Hungarian Academy of Sciences, Debrecen, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Nagy
authors.affiliation: Department of Surgery, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Damjanovich
mesh_terms: D000465:Algorithms; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008859:Microscopy, Polarization / Q000379:methods; D008962:Models, Theoretical
chemical_list: D005456:Fluorescent Dyes
keywords: CORNU-depolarizer; Circularly polarized light; Diagonal-polarization; Homo-FRET; Orientation factor for FRET; Polarizationally structured light