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Biophys. J. BioFAST: First Published July 21, 2006. doi:10.1529/biophysj.106.082768
© 2006 by the Biophysical Society.

A more recent version of this article appeared on October 15, 2006.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

k-Space Image Correlation Spectroscopy (kICS): A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics

David L. Kolin 1, David Ronis 1 and Paul W. Wiseman 1*

1 McGill University

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

Submitted on February 13, 2006
Revised on April 1, 2006
Accepted on 21 June 2006


  Abstract
We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope (TIRFM). In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or "blinking." Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities which agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of {alpha}5-integrin/eGFP constructs in a transfected CHO cell imaged on a TIRFM using CCD area detection.

Key Words: fluorescence correlation spectroscopy, light scattering theory, membrane dynamics, photobleaching, reciprocal space, temporal image correlation spectroscopy




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Copyright © 2006 by the Biophysical Society.