Dual-color fluorescence fluctuation spectroscopy in vitro and in vivo

J. D. Müller, M. Tekmen, L. Hillesheim, W. Yang, Y. Chen

Research output: Contribution to journalConference articlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)136-145
Number of pages10
JournalProceedings of SPIE - The International Society for Optical Engineering
StatePublished - 2004
EventProgress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV - San Jose, CA, United States
Duration: Jan 25 2004Jan 27 2004


  • Dual-color Photon Counting Histogram
  • Fluorescence Correlation Spectroscopy
  • Fluorescence Fluctuation Spectroscopy
  • Protein-protein interactions
  • Two-photon excitation


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