Robeson J L, Tilton R D
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890, USA.
Biophys J. 1995 May;68(5):2145-55. doi: 10.1016/S0006-3495(95)80397-8.
Standard analysis of fluorescence recovery after photobleaching (FRAP) data is valid only if the quantum yield of unphotobleached fluorophores is independent of concentration, yet close molecular packing in two-dimensional systems may lead to significant fluorescence concentration quenching. Using total internal reflection fluorescence, we quantified the surface concentration dependence of the relative quantum yield of fluorescein isothiocyanate-labeled proteins adsorbed to polymeric surfaces before performing measurements of fluorescence recovery after pattern photobleaching. Adsorbed layers of FITC-labeled ribonuclease A displayed significant concentration quenching, and thus the standard FRAP analysis method was unacceptable. We present an extended FRAP analysis procedure that accounts for the changing quantum yield of diffusing fluorophores in systems that are influenced by concentration quenching. The extended analysis shows that if concentration quenching conditions prevail, there may be significant error in the transport parameters obtained from FRAP measurements by using the standard procedures.
只有在未漂白荧光团的量子产率与浓度无关时，光漂白后荧光恢复（FRAP）数据的标准分析才有效，但二维系统中紧密的分子堆积可能会导致显著的荧光浓度猝灭。利用全内反射荧光，我们在进行图案光漂白后的荧光恢复测量之前，量化了吸附在聚合物表面的异硫氰酸荧光素标记蛋白的相对量子产率对表面浓度的依赖性。异硫氰酸荧光素标记的核糖核酸酶A吸附层表现出显著的浓度猝灭，因此标准的FRAP分析方法是不可接受的。我们提出了一种扩展的FRAP分析程序，该程序考虑了在受浓度猝灭影响的系统中扩散荧光团不断变化的量子产率。扩展分析表明，如果浓度猝灭条件普遍存在，那么使用标准程序从FRAP测量中获得的传输参数可能会有显著误差。
