Dual-laser homo-FRET on the cell surface

L. Bene, Tamás Ungvári, Roland Fedor, István Nagy, László Damjanovich

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1096-1112
Number of pages17
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1853
Issue number5
DOIs
Publication statusPublished - May 1 2015

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Anisotropy
Lasers
Fluorescence Polarization
Cell Surface Receptors
Spectrum Analysis
Fluorescence
Light

Keywords

  • Directed energy migration FRET (emFRET)
  • Flow cytometry
  • Fluorescence anisotropy
  • Fluorescence anisotropy lifetime imaging microscopy (rFLIM)
  • Inhomogeneous broadening
  • Red-edge effect

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Dual-laser homo-FRET on the cell surface. / Bene, L.; Ungvári, Tamás; Fedor, Roland; Nagy, István; Damjanovich, László.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1853, No. 5, 01.05.2015, p. 1096-1112.

Research output: Contribution to journalArticle

Bene, L. ; Ungvári, Tamás ; Fedor, Roland ; Nagy, István ; Damjanovich, László. / Dual-laser homo-FRET on the cell surface. In: Biochimica et Biophysica Acta - Molecular Cell Research. 2015 ; Vol. 1853, No. 5. pp. 1096-1112.
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