EFFECT OF CONCENTRATION QUENCHING ON FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING MEASUREMENTS
EFFECT OF CONCENTRATION QUENCHING ON FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING MEASUREMENTS
复制标题
DOI：
10.1016/s0006-3495(95)80397-8
复制
发表时间：
1995-05-01
影响因子：
3.4
通讯作者：
TILTON, RD
中科院分区：
文献类型：
--
作者：
ROBESON, JL;TILTON, RD
光漂白后的荧光恢复（FRAP）数据的标准分析是有效的，只有当未光漂白的荧光团的量子产率是独立的浓度，但在二维系统中的紧密分子包装可能会导致显着的荧光浓度淬灭。使用全内反射荧光，我们定量的表面浓度依赖的相对量子产率的荧光素异硫氰酸酯标记的蛋白质吸附到聚合物表面进行测量的荧光恢复后，图案光漂白。FITC标记的核糖核酸酶A的吸附层显示出显著的浓度淬灭，因此标准FRAP分析方法是不可接受的。我们提出了一个扩展的FRAP分析程序，占扩散荧光团的浓度淬灭的影响系统中的量子产率的变化。扩展的分析表明，如果浓度猝灭条件占上风，可能会有显着的误差，从FRAP测量通过使用标准程序获得的输运参数。
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.