Advances in Image Correlation Spectroscopy for Measurements in Heterogeneous Cell Environments

Abstract

Image correlation spectroscopy (ICS) methods provide a spatial regime based approach for measurements of membrane associated protein-protein interactions and macromolecular transport properties using fluorescence microscopy images of living cells as input. These approaches are based on space and time correlation analysis of fluctuations in fluorescence intensity within images recorded as a time series on a laser scanning or TIRF microscope. We recently introduced spatio-temporal image correlation spectroscopy (STICS) which measures vectors of protein flux in cells based on the calculation of a spatial correlation function as a function of time from an image time series. Here we will describe extensions of (ICS) that are suited for measurements in the heterogeneous cell environment. We will introduce a two color extension, spatio-temporal image cross-correlation spectroscopy (STICCS) with a bivariate fitting method that accounts for directional confinement in the cell. We will illustrate the method with transport maps of the adhesion related macromolecules alpha5, alpha6 and alphaL integrins with paxillin, and actin within, or associated with the basal membrane in adherent cells plated on extracellular matrix components laminin, collagen or fibronectin. Using the method we detect transient flow patterns as well as anisotropic diffusion that are correlated with adhesion fluxing activity in specific regions of the cell. Finally we will also highlight recent advances we have made with an extension of reciprocal (k-) space ICS (kICS) that can be used to measure molecular confinement in membrane domains from analysis of the correlation functions in both k-space and time.