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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
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2.

001-es BibID:BIBFORM056493
Első szerző:Bene László (biofizikus)
Cím:Single-laser polarization FRET (polFRET) on the cell surface / László Bene, Tamás Ungvári, Roland Fedor, László Damjanovich
Dátum:2014
ISSN:0167-4889
Megjegyzések:A new method for the simultaneous detection of rotational mobility and proximity of cell surface receptors is presented based on cell-by-cell basis measurement of polarized fluorescence intensity components of the donor and acceptor of a FRET system. In addition to the FRET efficiency and the donor and acceptor concentrations, the method makes also possible the determination of the rotational characteristics and the associated fraction of the donors (FRET-fraction). The method is illustrated with flow cytometric and rFLIM measurements on donor-acceptor systems comprising fluorescently labeled whole antibodies and their Fab fragments against epitopes of the MHCI and MHCII cell surface receptors on human lymphoblast cells. Fluorescence anisotropy of donor and acceptor and FRET efficiency were measured for samples of different acceptor-to-donor concentration ratios. Acceptor anisotropy proved to be more sensitive than the donor anisotropy for sensing FRET. After determining the rotational constants of the donor-conjugated antibodies by measurements of FRET in the steady state, and by rFLIM as a reference, the associated fractions of the MHCI and MHCII molecules in their clusters were determined. Besides the flow cytometer and the wide-field rFLIM used in this study, the method can be applied also in other devices capable of dual-anisotropy detection.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Fluorescence anisotropy
Rotational mobility
Proximity
Receptor cluster
FRET-fraction
rFLIM
Megjelenés:Biochimica et Biophysica Acta (BBA). Molecular Cell Research. - 1843 : 12 (2014), p. 3047-3064. -
További szerzők:Ungvári Tamás Fedor Roland (1975-) (sebész) Damjanovich László (1960-) (általános sebész)
Internet cím:Intézményi repozitóriumban (DEA) tárolt változat
DOI
Szerző által megadott URL
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3.

001-es BibID:BIBFORM049426
Első szerző:Bene László (biofizikus)
Cím:Intensity correlation-based calibration of FRET / László Bene, Tamás Ungvári, Roland Fedor, László Sasi Szabó, László Damjanovich
Dátum:2013
ISSN:0006-3495
Megjegyzések:ABSTRACT Dual-laser flow cytometric resonance energy transfer (FCET) is a statistically efficient and accurate way of determiningproximity relationships for molecules of cells even under living conditions. In the framework of this algorithm, absolutefluorescence resonance energy transfer (FRET) efficiency is determined by the simultaneous measurement of donor-quenchingand sensitized emission. A crucial point is the determination of the scaling factor a responsible for balancing the differentsensitivities of the donor and acceptor signal channels. The determination of a is not simple, requiring preparation of specialsamples that are generally different from a double-labeled FRET sample, or by the use of sophisticated statistical estimation(least-squares) procedures. We present an alternative, free-from-spectral-constants approach for the determination of a andthe absolute FRET efficiency, by an extension of the presented framework of the FCET algorithm with an analysis of the secondmoments (variances and covariances) of the detected intensity distributions. A quadratic equation for a is formulated withthe intensity fluctuations, which is proved sufficiently robust to give accurate a-values on a cell-by-cell basis in a wide systemof conditions using the same double-labeled sample from which the FRET efficiency itself is determined. This seemingly newapproach is illustrated by FRET measurements between epitopes of the MHCI receptor on the cell surface of two cell lines,FT and LS174T. The figures show that whereas the common way of a determination fails at large dye-per-protein labeling ratiosof mAbs, this presented-as-new approach has sufficient ability to give accurate results. Although introduced in a flow cytometer,the new approach can also be straightforwardly used with fluorescence microscopes.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
FRET
statistically efficient
Megjelenés:Biophysical Journal. - 105 : 9 (2013), p. 2024-2035. -
További szerzők:Ungvári Tamás Fedor Roland (1975-) (sebész) Sasi Szabó László András (1974-) (sebész) Damjanovich László (1960-) (általános sebész)
Pályázati támogatás:TÁMOP-4.2.2.A-11/1/KONV-2012-0045
TÁMOP
No. ASTF No 201-06
Egyéb
Short-term EMBO fellowship
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