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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.

Original publication

DOI

10.1117/1.2221714

Type

Journal article

Journal

J Biomed Opt

Publication Date

07/2006

Volume

11

Keywords

DNA, Equipment Design, Equipment Failure Analysis, Image Enhancement, Microscopy, Fluorescence, Multiphoton, Reproducibility of Results, Sensitivity and Specificity, Spectrometry, Fluorescence