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1.

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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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
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3.

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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4.

001-es BibID:BIBFORM004858
Első szerző:Bene László (biofizikus)
Cím:Detection of receptor trimers on the cell surface by flow cytometric fluorescence energy homotransfer measurements / László Bene, János Szöllősi, Gergely Szentesi, László Damjanovich, Rezső Gaspar, Thomas A. Waldmann, Sándor Damjanovich
Dátum:2005
ISSN:0006-3002
Megjegyzések:Fluorescence energy homotransfer offers a powerful tool for the investigation of the state of oligomerization of cell surface receptors on a cell-by-cell basis by measuring the polarized components of fluorescence intensity of cells labeled with fluorescently stained antibodies. Here we describe homotransfer-based methods for the flow cytometric detection and analysis of hetero- and homo-associations of cell surface receptors. Homotransfer efficiencies for two- and three-body energy transfer interactions are defined and their frequency distribution curves are computed from the fluorescence anisotropy distributions of multiple-labeled cells. The fractions of receptors involved in homo-clustering is calculated based on the dependence of the fluorescence anisotropy on the surface concentration of the fluorescently stained antibodies. A homotransfer analysis of the homo- and hetero-clustering of the MHCI and MHCII glycoproteins, the cytokine receptor IL-2Ralpha, transferrin receptor and the receptor-type tyrosine phosphatase CD45 on JY B and Kit-225-K6 T cells is presented. We investigated how various factors such as the type of dye, rotational mobility of the dye and dye-targeting antibody, as well as the wavelength of the exciting light affect the homotransfer. We show that the homotransfer technique combined with the high statistical resolution of flow cytometry is an effective tool for detecting different oligomeric states of receptors by using fluorophores having restricted rotational mobility on the time scale of fluorescence
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Fluorescence anisotropy
Receptor clustering
MHCI and MHCII glycoprotein
IL-2Rα
Transferrin receptor
CD45
Megjelenés:Biochimica et Biophysica Acta (BBA). Molecular Cell Research. - 1744 : 2 (2005), p. 176-198. -
További szerzők:Szöllősi János (1953-) (biofizikus) Szentesi Gergely (1976-) (kémia-fizika tanár) Damjanovich László (1960-) (általános sebész) Gáspár Rezső (1944-) (biofizikus) Waldmann, Thomas A. Damjanovich Sándor (1936-2017) (biofizikus)
Internet cím:Intézményi repozitóriumban (DEA) tárolt változat
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5.

001-es BibID:BIBFORM069692
Első szerző:Ungvári Tamás
Cím:Perrin and Förster unified : dual-laser triple-polarization FRET (3polFRET) for interactions at the Förster-distance and beyond / Tamás Ungvári, Péter Gogolák, Miklós Bagdány, László Damjanovich, László Bene
Dátum:2016
ISSN:0167-4889
Megjegyzések:Dual laser flow cytometric energy transfer (FCET)--elaborated by Trón et al. in 1984--is an efficient and rapid way of measuring FRET on large cell populations. FRET efficiency and the donor and acceptor concentrations are determined from one donor and two acceptor signals. In this communication this method is extended towards the domain of receptor dynamics by the detection of polarized components of the three intensities. By enabling a complete description of the proximity and dynamics of FRET-systems, the new measuring scheme allows a more refined description of both the structure and dynamics of cell surface receptor clusters at the nano-scale and beyond. Associated donor fraction, limiting anisotropy and rotational correlation time of the donor, acceptor anisotropy and cell-by-cell estimation of the orientation factor for FRET (K2) are available in the steady state on a single FRET sample in a very rapid and statistically efficient way offered by flow cytometry. For a more sensitive detection of conformational changes the "polarized FRET indices"--quantities composed from FRET efficiency and anisotropies--are proposed. The method is illustrated by measurements on a FRET system with changing FRET-fraction and on a two donor-one acceptor-system, when the existence of receptor trimers are proven by the detection of "hetero-FRET induced homo-FRET relief", i.e. the diminishing of homo-FRET between the two donors in the presence of a donor quencher. The method also offers higher sensitivity for assessing conformational changes at the nano-scale, due to its capability for the simultaneous detection of changes of proximity and relative orientations of the FRET donor and acceptor. Although the method has been introduced in the context of FRET, it is more general: It can be used for monitoring triple-anisotropy correlations also in those cases when FRET actually does not occur, e.g. for interactions occuring beyond the Förster-distance R0. Interpretation of K2 has been extended.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Triple-anisotropy correlations
Donor anisotropy
Acceptor anisotropy
Orientation factor for FRET
Homo-FRET relief
FRET-fraction
Megjelenés:Biochimica et Biophysica Acta (BBA). Molecular Cell Research. - 1863 : 4 (2016), p. 703-716. -
További szerzők:Gogolák Péter (1968-) (biológus, immunológus) Bagdány Miklós Damjanovich László (1960-) (általános sebész) Bene László (1963-) (biofizikus)
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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