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

Paul W. Wiseman; Jeffrey A. Squier; Kent R. Wilson

doi:10.1117/12.384193

2 May 2000 Dynamic image correlation spectroscopy (ICS) and two-color image cross-correlation spectroscopy (ICCS): concepts and application

Paul W. Wiseman, Jeffrey A. Squier, Kent R. Wilson

Author Affiliations +

Proceedings Volume 3919, Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII; (2000) https://doi.org/10.1117/12.384193
Event: BiOS 2000 The International Symposium on Biomedical Optics, 2000, San Jose, CA, United States

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.

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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, (2 May 2000); https://doi.org/10.1117/12.384193

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