Image cross-correlation spectroscopy: A new experimental biophysical approach to measurement of slow diffusion of fluorescent molecules

The technique of image cross-correlation spectroscopy has been applied to measurements of diffusion of fluorescent beads in polymer solution. The technique is based on measurement and analysis of temporal fluctuations of the intensity observed in fluorescence confocal microscope images. As with other fluctuation techniques, fluorescence fluctuations arise from stochastic concentration fluctuations about the equilibrium concentration. The dynamics of the fluctuations depend on the number of fluorescent molecules in the observation volume. This work presents the theory and illustrates how the technique can be applied to measurement of diffusion of fluorescent beads in solutions of various viscosities. Further we extend the concept of cross-correlation to studies on the cell surface to detect the relative distribution of molecules (receptors) on the cell surface, in which intensity fluctuations arising from samples containing two distinct types of labelled molecules are cross-correlated using two detectors with different wavelength sensitivities.