(A) In fluorescence correlation spectroscopy, a confocal laser illumination spot is used to monitor the intensity fluctuation due to fluorescence molecules diffusing in and out of the focus. The auto-correlation function (ACF) of the resulting time trace is used to compute the particle number (N, from the y-intercept) and residence time (τd, from the half-point of the ACF decay) of the fluorescent molecule in the laser focus, which is related to the diffusion coefficient by w2 = 4Dτd. In this example, a GFP-labeled protein on a supported lipid bilayer is excited by a blue (488 nm) laser. (B) In total internal relfection fluorescence microscopy (TIRFM) single molecule tracking, the displacement of the particle between each frame are reconstructed into particle trajectories (example trajectory shown in inset). The step size distribution may be used to calculate the diffusion coefficient and the relative population of multiple diffusing species, following the step size equation P(r) (Equations 5 and 6 for single and two species, respectively). The fit to a single species step size distribution is shown. (C) Left: theoretical binding curves for a dimerization reaction for a wide range of dissociation constants, right: simulated FCS data using experimentally determined diffusion coefficients for monomers and dimers for the same dissociation constants.
