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Dual-color total internal reflection fluorescence cross-correlation spectroscopy.

Marcel M Leutenegger, Hans H Blom, Jerker J Widengren, Christian C Eggeling, Michael M Gösch, Rainer A RA Leitgeb, and Theo T Lasser

J Biomed Opt 11(4):040502 (2006) PMID 16965125

We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three-fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.

DOI: 10.1117/1.2221714
Version: za2963e q8za5 q8zb5 q8zc3 q8zd8 q8zec q8zf6 q8zg2

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