Spatio temporal image correlation spectroscopy (STICS) theory, verification and application to protein velocity mapping in living CHO cells

¹ McGill University

^* To whom correspondence should be addressed. E-mail: paul.wiseman{at}mcgill.ca.

Submitted on October 20, 2004
Revised on November 30, 2004
Accepted on 7 February 2005

	Abstract

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from laser scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser scanning microscopy studies of -actinin/EGFP fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of µm/s from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of µm/s) and diffusion of interacting 5 integrin/ECFP and -actinin/EYFP within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within sub-regions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

Key Words: adhesion proteins, cell motility, flow analysis, fluctuation analysis, image correlation spectroscopy, vector mapping

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