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001-es BibID:BIBFORM065560
Első szerző:Bene László (biofizikus)
Cím:Depolarized FRET (depolFRET) on the cell surface : FRET control by photoselection / László Bene, Péter Gogolák, Tamás Ungvári, Miklós Bagdány, István Nagy, László Damjanovich
Dátum:2016
ISSN:0167-4889
Megjegyzések: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.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Polarizationally structured light
Circularly polarized light
Diagonal-polarization
CORNU-depolarizer
Orientation factor for FRET
Homo-FRET
Megjelenés:Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. - 1863 : 2 (2016), p. 322-334. -
További szerzők:Gogolák Péter (1968-) (biológus, immunológus) Ungvári Tamás Bagdány Miklós Nagy István (1957-) (villamosmérnök) Damjanovich László (1960-) (általános sebész)
Pályázati támogatás:TÁMOP-4.2.2.A-11/1/KONV-2012-0045
TÁMOP
Sebészet Kutatócsoport
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DOI
Intézményi repozitóriumban (DEA) tárolt változat
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2.

001-es BibID:BIBFORM057905
Első szerző:Bene László (biofizikus)
Cím:Dual-laser homo-FRET on the cell surface / László Bene, Tamás Ungvári, Roland Fedor, István Nagy, László Damjanovich
Dátum:2015
ISSN:0167-4889
Megjegyzések: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.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Inhomogeneous broadening
Red-edge effect
Directed energy migration FRET (emFRET)
Fluorescence anisotropy
Fluorescence anisotropy lifetime imaging microscopy (rFLIM)
Megjelenés:Biochimica et Biophysica Acta (BBA). Molecular Cell Research. - 1853 : 5 (2015), p. 1096-1112. -
További szerzők:Ungvári Tamás Fedor Roland (1975-) (sebész) Nagy István (1957-) (villamosmérnök) Damjanovich László (1960-) (általános sebész)
Pályázati támogatás:TÁMOP-4.2.2.A-11/1/KONV-2012-0045
TÁMOP
Kardiológia Kutatócsoport
OTKA Bridging Fund support OSTRAT/ 810/213 by the University of Debrecen
OTKA
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
Borító:
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