Department of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA.
J Phys Chem B. 2010 Jan 28;114(3):1336-49. doi: 10.1021/jp906648q.
Quantitative analysis of the staining of cell membranes with the cationic amphiphile, octadecyl rhodamine B (R18), is confounded by probe aggregation and changes to the probes' absorption cross section and emission quantum yield. In this paper, flow cytometry, quantum-dot-based fluorescence calibration beads, and FRET were used to examine real-time transfer of R18 from water to two limiting models of the cellular plasma membrane, namely, a single-component disordered membrane, dioleoyl-L-alpha-phosphatidylcholine (DOPC), and a ternary mixture of DOPC, cholesterol, and sphingomyelin (DSC) membranes, reconstituted on spherical and monodisperse glass beads (lipobeads). The quenching of R18 was analyzed as the probe concentration was raised from 0 to 10 mol % in membranes. The data show a > 2-fold enhancement in the quenching level of the probes that were reconstituted in DSC relative to DOPC membranes at the highest concentration of R18. We have parametrized the propagation of concentration-dependent quenching as a function of real-time binding of R18 to lipobeads. In this way, phenomenological kinetics of serum-albumin-mediated transfer of R18 from the aqueous phase to DOPC and DSC membranes could be evaluated under optimal conditions where the critical aggregation concentration (CAC) of the probe is defined as 14 nM. The mass action kinetics of association of R18 with DOPC and DSC lipobeads are shown to be similar. However, the saturable capacity for accepting exogenous probes is found to be 37% higher in DOPC relative to that for DSC membranes. The difference is comparable to the disparity in the average molecular areas of DOPC and DSC membranes. Finally, this analysis shows little difference in the spectral overlap integrals of the emission spectrum of a fluorescein derivative donor and the absorption spectrum of either monomeric or simulated spectrum of dimeric R18. This approach represents a first step toward a nanoscale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells.
阳离子两亲性十八烷基罗丹明 B（R18）与细胞膜的染色定量分析受到探针聚集以及探针吸收截面和发射量子产率变化的干扰。在本文中，我们使用流式细胞术、基于量子点的荧光校准珠和 FRET 来实时研究 R18 从水中向两种细胞质膜的极限模型的转移，即单组分无序膜、二油酰基-L-α-磷脂酰胆碱（DOPC）和 DOPC、胆固醇和神经鞘磷脂（DSC）的三元混合物膜，在球形和单分散玻璃珠（脂质珠）上重建。当探针浓度从 0 增加到 10 mol%时，分析了 R18 的猝灭情况。数据表明，在最高 R18 浓度下，与 DOPC 膜相比，DSC 膜中探针的猝灭水平提高了 2 倍以上。我们已经将浓度依赖性猝灭的传播参数化，作为 R18 与脂质珠实时结合的函数。通过这种方式，可以在探针的临界聚集浓度 (CAC) 定义为 14 nM 的最佳条件下，评估血清白蛋白介导的 R18 从水相向 DOPC 和 DSC 膜的转移的经验动力学。R18 与 DOPC 和 DSC 脂质珠的缔合的质量作用动力学是相似的。然而，在 DOPC 中，接受外源性探针的饱和容量比 DSC 膜高 37%。这种差异与 DOPC 和 DSC 膜的平均分子面积的差异相当。最后，该分析表明，荧光素衍生物供体的发射光谱的光谱重叠积分与单体或模拟的二聚体 R18 的吸收光谱之间几乎没有差异。这种方法代表了通过分析活细胞中独特受体表达部位之间的差异局部 FRET，对活细胞中膜异质性进行纳米级探测的第一步。
