Advances in Image Correlation Spectroscopy: Measuring Number Densities, Aggregation States, and Dynamics of Fluorescently Labeled Macromolecules in Cells

A brief historical outline of fluorescence fluctuation correlation techniques is presented, followed by an in-depth review of the theory and development of image correlation techniques, including: image correlation spectroscopy (ICS), temporal ICS (TICS), image cross-correlation spectroscopy (ICCS), spatiotemporal ICS (STICS), k-space ICS (kICS), raster ICS (RICS), and particle ICS (PICS). These techniques can be applied to analyze image series acquired on commercially available laser scanning or total internal reflection fluorescence microscopes, and are used to determine the number density, aggregation state, diffusion coefficient, velocity, and interaction fraction of fluorescently labeled molecules or particles. A comprehensive review of the application of ICS techniques to a number of systems, including cell adhesion, membrane receptor aggregation and dynamics, virus particle fusion, and fluorophore photophysics, is presented.