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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
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
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
No. ASTF No 201-06
Short-term EMBO fellowship
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