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Proceedings Paper

Dual-color fluorescence fluctuation spectroscopy in vitro and in vivo
Author(s): Joachim D. Mueller; Mohac Tekmen; Lindsey Hillesheim; Weidong Yang; Yan Chen
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Paper Abstract

The combination of dual-color fluorescence correlation spectroscopy (FCS) and two-photon excitation is a powerful tool for probing protein-protein interactions. The submicron resolution and single molecule sensitivity of the technique make it attractive for in vivo applications. However, the strong spectral cross talk between the two emission channels of most fluorescent dye mixtures provides a challenge for the analysis of dual-color FCS experiments. We describe a new technique, dual-color photon counting histogram (PCH) analysis that overcomes some of the challenges associated with spectral cross talk. Dual-color PCH is an extension of regular PCH that simultaneously analyses the photon counts of two detection channels. We demonstrate that dual color PCH quantitatively resolves protein mixtures in vitro. We also apply dual-color PCH to study proteins in biological cells. The fluorescent proteins ECFP and EYFP, which are commonly used for dual-color studies in cells, have significant spectral cross talk. We will discuss the resolvability of these fluorescent proteins and present data that successfully resolve the protein mixtures in vitro and in vivo. Our results show that dual color PCH is a promising technique for the characterization of protein-protein interactions in intact cells.

Paper Details

Date Published: 21 June 2004
PDF: 10 pages
Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); doi: 10.1117/12.537971
Show Author Affiliations
Joachim D. Mueller, Univ. of Minnesota (United States)
Mohac Tekmen, Univ. of Minnesota (United States)
Lindsey Hillesheim, Univ. of Minnesota (United States)
Weidong Yang, Univ. of Minnesota (United States)
Yan Chen, Univ. of Minnesota (United States)

Published in SPIE Proceedings Vol. 5323:
Multiphoton Microscopy in the Biomedical Sciences IV
Ammasi Periasamy; Peter T. C. So, Editor(s)

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