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Proceedings Article

Dynamic image correlation spectroscopy (ICS) and two-color image cross-correlation spectroscopy (ICCS): concepts and application

[+] Author Affiliations
Paul W. Wiseman

Univ. of California/San Diego (Canada)

Jeffrey A. Squier, Kent R. Wilson

Univ. of California/San Diego (USA)

Proc. SPIE 3919, Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII, 14 (May 2, 2000); doi:10.1117/12.384193
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From Conference Volume 3919

  • Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII
  • Jose-Angel Conchello; Carol J. Cogswell; Andrew G. Tescher; Tony Wilson
  • San Jose, CA | January 22, 2000

abstract

The interaction of macromolecules in space and time are known to be important for the regulation of many biochemical reactions. Image correlation spectroscopy (ICS) was recently introduced as an imaging analog of fluorescence correlation spectroscopy optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. We present two novel developments of dynamic ICS that will greatly enhance our abilities to measure molecular interactions as a function of time and space in living cells. We illustrate the use of a rapid scan two-photon microscope system to collect image series at high time resolution (30 frames/s) for dynamic ICS analysis. Secondly, we demonstrate the implementation of two-color image cross-correlation spectroscopy (ICCS) with a CLSM using multiple wavelength excitation, and with two-photon excitation of samples containing two different fluorescent species. Cross-correlation analysis allows the degree of co- localization of two different fluorophores to be measured directly. By performing two-color ICCS, we can monitor the interactions of non-identical labeled macromolecules as a function of time and space. We describe the experimental setup for both methods and illustrate the application for measurements of the diffusion coefficients of singly and doubly labeled fluorescent microspheres in aqueous solutions.

© (2000) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
Citation

Paul W. Wiseman ; Jeffrey A. Squier and Kent R. Wilson
"Dynamic image correlation spectroscopy (ICS) and two-color image cross-correlation spectroscopy (ICCS): concepts and application", Proc. SPIE 3919, Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII, 14 (May 2, 2000); doi:10.1117/12.384193; http://dx.doi.org/10.1117/12.384193


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