Weidemann Thomas, Schwille Petra
Biophysics/BIOTEC, Technische Universität Dresden, Dresden, Germany.
Methods Enzymol. 2013;518:43-70. doi: 10.1016/B978-0-12-388422-0.00003-0.
Fluorescence correlation spectroscopy evaluates local signal fluctuations arising from stochastic movements of fluorescent particles in solution. The measured fluctuating signal is correlated in time and analyzed with appropriate model functions containing the parameters that describe the underlying molecular behavior. The dual-color extension, fluorescence cross-correlation spectroscopy (FCCS) allows for a comparison between spectrally well-separated channels to extract codiffusion events that reflect interactions between differently labeled molecules. In addition to solution measurements, FCCS can be applied with subcellular resolution and is therefore a very promising approach for a quantitative biochemical assessment of molecular networks in living cells. To derive thermodynamic and kinetic reaction parameters, the influence of a number of other factors like background noise, illumination intensity profiles, photophysical processes, and cross talk between the channels have to be treated. Here, we provide a roadmap to derive binding reaction data with dual-color FCCS using continuous wave laser excitation, as it is now accessible with many state-of-the-art confocal microscopes.
荧光相关光谱法用于评估溶液中荧光粒子随机运动引起的局部信号波动。所测量的波动信号会随时间进行关联，并使用包含描述潜在分子行为参数的适当模型函数进行分析。双色扩展技术，即荧光交叉相关光谱法（FCCS），允许对光谱上充分分离的通道进行比较，以提取反映不同标记分子之间相互作用的共扩散事件。除了溶液测量外，FCCS还可应用于亚细胞分辨率，因此对于定量生化评估活细胞中的分子网络是一种非常有前景的方法。为了推导热力学和动力学反应参数，必须考虑许多其他因素的影响，如背景噪声、照明强度分布、光物理过程以及通道之间的串扰。在这里，我们提供了一个路线图，用于使用连续波激光激发通过双色FCCS推导结合反应数据，因为现在许多先进的共聚焦显微镜都可以实现这一点。
