Lessons in fluctuation correlation spectroscopy.

Molecular diffusion and transport processes are fundamental in physical, chemical, and biological systems. Current approaches to measuring molecular transport in cells and tissues based on perturbation methods, e.g., fluorescence recovery after photobleaching, are invasive; single-point fluctuation correlation methods are local; and single-particle tracking requires the observation of isolated particles for relatively long periods of time. We discuss here the detection of molecular transport by exploiting spatiotemporal correlations measured among points at large distances (>1 μm). We illustrate the evolution of the conceptual framework that started with single-point fluorescence fluctuation analysis based on the transit of fluorescent molecules through a small volume of illumination. This idea has evolved to include the measurement of fluctuations at many locations in the sample using microscopy imaging methods. Image fluctuation analysis has become a rich and powerful technique that can be used to extract information about the spatial distribution of molecular concentration and transport in cells and tissues.