Studying Slow Membrane Dynamics with Continuous Wave Scanning Fluorescence Correlation Spectroscopy

Here we discuss the application of scanning fluorescence correlation spectroscopy (SFCS) using continuous wave excitation to analyze membrane dynamics. The high count rate per molecule enables the study of very slow diffusion in model and cell membranes, as well as the application of two-foci fluorescence cross-correlation spectroscopy for parameter-free determination of diffusion constants. The combination with dual-color fluorescence cross-correlation spectroscopy with continuous or pulsed interleaved excitation allows binding studies on membranes. Reduction of photobleaching, higher reproducibility, and stability compared to traditional FCS on membranes, and the simple implementation in a commercial microscopy setup make SFCS a valuable addition to the pool of fluorescence fluctuation techniques.