title: Dual-laser homo-FRET on the cell surface.
abstract: Inhomogeneous broadening and red-edge effects have been detected on a highly mobile system of fluorescently conjugated mAbs targeted to cell surface receptors. By exploiting site-selective spectroscopy and the characteristic loss of homo-FRET on increasing excitation and decreasing emission wavelengths, contributions of physical rotation and homo-FRET to the depolarization of fluorescence anisotropy have been separated. Absolute homo-FRET efficiency has been determined by ratioing two anisotropies: a homo-FRET-sensitive one, which is excited at the absorption main band and detected at the long wavelength region of emission, and a homo-FRET-insensitive one, which is excited at the long wavelength region of absorption and detected at the short wavelength region of emission. Because the anisotropies are simultaneously detected in a unified detection scheme of a dual T-format arrangement, the method is applicable for the real-time tracking of dynamical changes of physical rotations and proximities. The utility of the method is demonstrated in the context of the MHCII molecule and the heavy and light chains of the MHCI molecule, a system of three receptors with well-characterized close mutual proximities. Although the method is presented for a flow cytometer, it can also be realized in a fluorescence microscope capable for dual-laser excitation and dual-anisotropy detection.
authors: Department of Surgery, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary. Electronic address: bene@med.unideb.hu.
authors: László
authors: L
authors: Bene
authors: Department of Biophysics and Cell Biology, University of Debrecen, Medical and Health Science Centre, Faculty of Medicine, Hungary.
authors: Tamás
authors: T
authors: Ungvári
authors: Department of Surgery, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
authors: Roland
authors: R
authors: Fedor
authors: Division of Electronics, Research Center for Nuclear Physics of the Hungarian Academy of Sciences, Debrecen, Hungary.
authors: István
authors: I
authors: Nagy
authors: Department of Surgery, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
authors: László
authors: L
authors: Damjanovich
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007834:Lasers; D055503:Protein Multimerization; D011956:Receptors, Cell Surface / Q000378:metabolism; D012815:Signal Processing, Computer-Assisted; D013050:Spectrometry, Fluorescence; D017186:Titrimetry
chemical_list: D000911:Antibodies, Monoclonal; D011956:Receptors, Cell Surface
keywords: Directed energy migration FRET (emFRET); Flow cytometry; Fluorescence anisotropy; Fluorescence anisotropy lifetime imaging microscopy (rFLIM); Inhomogeneous broadening; Red-edge effect