Single Molecule Detection at Surfaces: Dual-Color Fluorescence Fluctuation Spectroscopy with Total Internal Reflection Excitation

摘要

Fluorescence fluctuation spectroscopy based on an evanescent field excitation scheme leads to an alternative concept for single molecule detection with interesting experimental features. This concept is described and analyzed regarding the total fluorescence process including excitation, fluorescence emission and collection close to a dielectric interface. The realized experimental scheme showed superior performance of this concept with substantially enhanced molecular brightness when compared to the classical confocal setup based on water immersion objectives. Selected applications in life sciences including dual-color surface fluorescence correlation spectroscopy, enzyme kinetics and receptor-ligand binding are underlining the interest of this experimental approach.