Depolarized FRET (depolFRET) on the cell surface: FRET control by photoselection

L. Bene, P. Gogolák, Tamás Ungvári, Miklós Bagdány, István Nagy, László Damjanovich

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)322-334
Number of pages13
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1863
Issue number2
DOIs
Publication statusPublished - Feb 1 2016

Fingerprint

Light
Fluorescence Polarization
Anisotropy
Cell Surface Receptors
Lasers

Keywords

  • Circularly polarized light
  • CORNU-depolarizer
  • Diagonal-polarization
  • Homo-FRET
  • Orientation factor for FRET
  • Polarizationally structured light

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

Depolarized FRET (depolFRET) on the cell surface : FRET control by photoselection. / Bene, L.; Gogolák, P.; Ungvári, Tamás; Bagdány, Miklós; Nagy, István; Damjanovich, László.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1863, No. 2, 01.02.2016, p. 322-334.

Research output: Contribution to journalArticle

Bene, L. ; Gogolák, P. ; Ungvári, Tamás ; Bagdány, Miklós ; Nagy, István ; Damjanovich, László. / Depolarized FRET (depolFRET) on the cell surface : FRET control by photoselection. In: Biochimica et Biophysica Acta - Molecular Cell Research. 2016 ; Vol. 1863, No. 2. pp. 322-334.
@article{10f88f7a994e4929afef53e180597ec6,
title = "Depolarized FRET (depolFRET) on the cell surface: FRET control by photoselection",
abstract = "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.",
keywords = "Circularly polarized light, CORNU-depolarizer, Diagonal-polarization, Homo-FRET, Orientation factor for FRET, Polarizationally structured light",
author = "L. Bene and P. Gogol{\'a}k and Tam{\'a}s Ungv{\'a}ri and Mikl{\'o}s Bagd{\'a}ny and Istv{\'a}n Nagy and L{\'a}szl{\'o} Damjanovich",
year = "2016",
month = "2",
day = "1",
doi = "10.1016/j.bbamcr.2015.12.003",
language = "English",
volume = "1863",
pages = "322--334",
journal = "Biochimica et Biophysica Acta - Molecular Cell Research",
issn = "0167-4889",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Depolarized FRET (depolFRET) on the cell surface

T2 - FRET control by photoselection

AU - Bene, L.

AU - Gogolák, P.

AU - Ungvári, Tamás

AU - Bagdány, Miklós

AU - Nagy, István

AU - Damjanovich, László

PY - 2016/2/1

Y1 - 2016/2/1

N2 - 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.

AB - 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.

KW - Circularly polarized light

KW - CORNU-depolarizer

KW - Diagonal-polarization

KW - Homo-FRET

KW - Orientation factor for FRET

KW - Polarizationally structured light

UR - http://www.scopus.com/inward/record.url?scp=84949659920&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84949659920&partnerID=8YFLogxK

U2 - 10.1016/j.bbamcr.2015.12.003

DO - 10.1016/j.bbamcr.2015.12.003

M3 - Article

C2 - 26657258

AN - SCOPUS:84949659920

VL - 1863

SP - 322

EP - 334

JO - Biochimica et Biophysica Acta - Molecular Cell Research

JF - Biochimica et Biophysica Acta - Molecular Cell Research

SN - 0167-4889

IS - 2

ER -