Dual-color total internal reflection fluorescence cross-correlation spectroscopy

• 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.

Nyckelord

fluorescence spectroscopy

laser-induced fluorescence

correlation

high count-rate

microscopy

molecule

cell

biomolecules

kinetics

surface

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)