Bene László, Gogolák Péter, Ungvári Tamás, Bagdány Miklós, Nagy István, Damjanovich László
Department of Surgery, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Department of Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Biochim Biophys Acta. 2016 Feb;1863(2):322-34. doi: 10.1016/j.bbamcr.2015.12.003. Epub 2015 Dec 3.
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.
通过使用偏振光和非偏振光进行激发，已证明了荧光共振能量转移（FRET）在异质和同质FRET系统中对供体光选取向分布的敏感性。在传统流式细胞仪中实现的双发射偏振方案中，通过单激光激发并应用针对MHC I和MHC II细胞表面受体的荧光团偶联单克隆抗体，同时测量了FRET以及供体和受体各向异性。基于科尔努原理的晶体去偏振器、用于圆偏振的四分之一波片以及用作双偏振激发的对角偏振器的平行分束器实现了原始偏振光的去偏振。基于强度的FRET效率和受体去偏振的同时分析明确表明，光的去偏振可能会增加FRET，增加的量取决于受体与供体的浓度比。受体去偏振对FRET比供体超偏振甚至比基于强度的FRET效率更敏感。它可以用作监测供体-受体对动力学变化的灵敏工具。对于异质FRET获得的FRET增强和受体去偏振增加的基本观察结果，与去偏振激发下同质FRET增强的类似观察结果相似。就FRET的取向因子而言，在宏观各向同性取向分布（如细胞表面结合荧光团的分布）条件下，去偏振时的FRET增强表明存在供体和受体的局部取向失配，这在偏振情况下会阻止最佳FRET，而激发去偏振可能会消除这种失配。还提出了一种用于去偏振激发的荧光各向异性理论。
