Dual-color fluorescence fluctuation spectroscopy in vitro and in vivo

Joachim D. Mueller; Mohac Tekmen; Lindsey Hillesheim; Weidong Yang; Yan Chen

doi:10.1117/12.537971

21 June 2004 Dual-color fluorescence fluctuation spectroscopy in vitro and in vivo

Joachim D. Mueller, Mohac Tekmen, Lindsey Hillesheim, Weidong Yang, Yan Chen

Proceedings Volume 5323, Multiphoton Microscopy in the Biomedical Sciences IV; (2004) https://doi.org/10.1117/12.537971
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

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.

Citation Download Citation

Joachim D. Mueller, Mohac Tekmen, Lindsey Hillesheim, Weidong Yang, and Yan Chen "Dual-color fluorescence fluctuation spectroscopy in vitro and in vivo", Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); https://doi.org/10.1117/12.537971

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KEYWORDS

Proteins

Fluorescence correlation spectroscopy

In vivo imaging

In vitro testing

Data modeling

Binary data

Luminescence

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