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001-es BibID:	BIBFORM075129
Első szerző:	Bene László (biofizikus)
Cím:	Confocal microscopic dual-laser dual-polarization FRET (2polFRET) at the acceptor side for correlating rotations at different distances on the cell surface / László Bene, Matthias Gralle, László Damjanovich
Dátum:	2018
ISSN:	0304-4165
Megjegyzések:	Relationship of donor and acceptor fluorescence anisotropies as well as efficiency of fluorescence resonanceenergy transfer (FRET) has been investigated in a confocal microscope in the context of FRET systems comprisedof donor and acceptor-labeled MHCI and MHCII receptors on the surface of Kit-225 K6 human T-cells. Themeasurements have been carried out in a 2-laser, 5-signal platform where the total donor fluorescence intensityand 2 acceptor fluorescence intensities with their anisotropies ? one at the donor's excitation wavelength, theother at the acceptor's excitation wavelength ? have been detected. This configuration enabled the determinationof FRET efficiency and correlating it with the two acceptor fluorescence anisotropies as a kind of calibration.Estimations for the FRET-enhanced donor fluorescence anisotropy, the directly excited acceptor fluorescenceanisotropy, and the fluorescence anisotropy of sensitized emission have been obtained. Procedures for determiningFRET by measuring only the total donor intensity and the acceptor intensity and its anisotropy, or twoacceptor intensities and their anisotropies have been elaborated, the errors of which have been estimated basedon the fluorescence anisotropy values obtained in the calibration with the method of flow cytometric energytransfer (FCET).The combined detection of the donor and acceptor fluorescence anisotropies enabled also the determinationof the lower and upper limits of the orientation factor for FRET (?2). An increase in range for ?2 with increasingFRET efficiency has been observed, with average ?2 values different from the dynamic random average of 2/3.These observations call for the need of ?2 determination in proximity measurements, where the donor andacceptor orientations are not predictable.An increasing range of ?2 with increasing intermolecular proximity of the MHCI and MHCII receptors hasbeen observed. This indicates that molecular flexibility in the clusters of the MHCI and MHCII receptors reduceswith increasing cluster density, i.e. a "fluidity gradient" exists in the clusters. More specifically, the local densitydependent flexibility can also be taken as a direct proof for that the association of these receptors is non-random,but mediated by some type of physical interaction, a finding as a benefit of FRET detection by polarizationspectroscopy.Two new quantities ? the quenched donor fluorescence anisotropy and a fluorescence anisotropy analogue,the "dissymmetry index" of the polarized FRET efficiency components ? have also been introduced for thecharacterization of the orientational dynamics of the excited state during FRET.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban Dual-anisotropy correlations Donor anisotropy Acceptor anisotropy Orientation factor (k2)-imaging Anisotropy multiplexing rFLIM
Megjelenés:	Biochimica et Biophysica Acta (BBA). General Subjects. - 1862 : 4 (2018), p. 1050-1068. -
További szerzők:	Gralle, Matthias Damjanovich László (1960-) (általános sebész)
Pályázati támogatás:	TÁMOP-4.2.2.A-11/1/KONV-2012-0045 TÁMOP Sebészet Kutatócsoport
Internet cím:	DOI Intézményi repozitóriumban (DEA) tárolt változat
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