HALOmem, University of Halle, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
Chemphyschem. 2012 Apr 10;13(5):1221-31. doi: 10.1002/cphc.201100801. Epub 2012 Feb 16.
Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live-cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false-positive or overestimated cross-correlation amplitude arising from spectral cross-talk. Cross-talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross-talk in standard dcFCCS. The dependence of the cross-talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross-talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed-through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross-talk correction and its influence on experimental error are explored.
双色荧光相关光谱学 (dcFCCS) 可定量评估用不同荧光团标记的移动分子之间的相互作用。该技术广泛应用于重组和活细胞生物系统。dcFCCS 的一个主要缺点是由于光谱串扰而产生人为的假阳性或高估的交叉相关幅度的风险。通过快速交替激发可以减少或防止串扰，但该技术在标准商业设置中不易实现。设计了一种实验策略，该策略不需要专门的硬件和软件即可识别和纠正标准 dcFCCS 中的串扰。定量确认了串扰对粒子浓度和亮度的依赖性。此外，使用可快速访问的参数（即测量的（表观）荧光计数率和相关幅度）对串扰进行定量校正也很简单。仅需在校准测量中确定漏出比。最后，探讨了串扰校正的限制及其对实验误差的影响。
