Two-photon image correlation spectroscopy and image cross-correlation spectroscopy

Paul W. Wiseman; Jeffrey A. Squier

doi:10.1117/12.424565

24 April 2001 Two-photon image correlation spectroscopy and image cross-correlation spectroscopy

Paul W. Wiseman, Jeffrey A. Squier

Proceedings Volume 4262, Multiphoton Microscopy in the Biomedical Sciences; (2001) https://doi.org/10.1117/12.424565
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

Image correlation spectroscopy (ICS) was developed as an imaging analog of fluorescence correlation spectroscopy (FCS) optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. Ics was first implemented on a confocal laser scanning microscope (CLSM) and entails spatial autocorrelation analysis of fluorescence fluctuations within an image sampled from an area of the cell. Spatial and temporal autocorrelation analysis of image time series enables measurement of both the molecular dynamics and aggregation state of the imaged molecules. The parallel nature inherent in the collection of multiple fluctuations in an imaging scheme improves the signal to noise ratio of the correlation analysis, which enhances dynamic measurements for slowly moving species in membrane systems. We outline our development of two-photon ICS and describe recent applications of the method for measurements of flow, diffusion and aggregation behavior of green fluorescent protein/integrin receptor constructs in living cells. We also describe the use of two-photon excitation to perform two-color image cross-correlation spectroscopy to measure the dynamics and colocalization of non-identical species labeled with different fluorophores.

Citation Download Citation

Paul W. Wiseman and Jeffrey A. Squier "Two-photon image correlation spectroscopy and image cross-correlation spectroscopy", Proc. SPIE 4262, Multiphoton Microscopy in the Biomedical Sciences, (24 April 2001); https://doi.org/10.1117/12.424565

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