Art and artifacts of fluorescence correlation spectroscopy

摘要

Fluorescence correlation spectroscopy (FCS) is an important technique for studying analyte molecules on a single molecule level in solution. The core molecular characteristic that is addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for studying molecular binding interactions or conformational changes of macromolecules. We present a thorough theoretical analysis of the FCS technique, paying special attention to the various frequently occurring technical artifacts. Particularly, we consider the influence of refractive index mismatch, cover-slide thickness, fluorescence anisotropy, optical adjustment, and optical saturation on the measured autocorrelation curve (ACF). The impact of these factors on the apparently determined diffusion coefficient is quantitatively evaluated. Extensive experimental results are presented demonstrating the theoretically predicted effects and dependencies.