Single molecule detection on surfaces

Monitoring biological relevant reactions on the single molecule level based on fluorescence spectroscopy techniques has become one of the most promising approaches for understanding a variety of phenomena in biophysics, biochemistry and life science. By applying techniques of fluorescence spectroscopy to labeled biomolecules a manifold of important parameters becomes accessible. For example, molecular dynamics, energy transfer, and ligand-receptor reactions can be monitored at the molecular level. This huge application field was and still is a major drive for innovative optical methods as it opens the door for new quantitative insights of molecular interactions on a truly micro- and nano-scopic scale. This thesis contributes new single molecule detection (SMD) concepts, correlation analysis and optical correlation spectroscopy to study fluorophores or labeled biomolecules close to a surface. The search beyond the classical confocal volume towards improved confinement was a key objective. In a first approach, fluorescence correlation spectroscopy (FCS) using near field light sources to achieve highly confined observation volumes for detecting and measuring fluorophores up to micromolar concentration was investigated. In a second approach, FCS and fluorescence intensity distribution analysis (FIDA) based on dual-color total internal reflection fluorescence (TIRF) microscopy was conceived to achieve a common observation volume for dual-color fluorescence measurements. This resulted in two novel fluorescence fluctuation spectroscopy instruments providing observation volumes of less than 100al. The first instrument generates a near field observation volume around and inside nano-apertures in an opaque metal film. Back-illumination of such an aperture results in a highly confined excitation field at the distal aperture exit. This instrument was characterized with FCS and observation volumes as small as 30al were measured. The second instrument confines the observation volume with total internal reflection (TIR) at a glass-water interface. Today, the last-generation instrument provides a dual-color ps pulsed excitation and time-resolved detection for coincidence analysis and time-correlated single photon counting. It was characterized with FCS and FIDA and observation volumes of 70al to 100al were achieved. Moreover, the presence of the interface favors emission into the optically denser medium, such that nearly 60% of the emitted fluorescence can be collected. This very efficient light collection resulted in a two- to three-fold stronger fluorescence signal and led to a high signal to background ratio, which makes this instrument particularly suitable for SMD studies on surfaces. In parallel to these experimental investigations, a theoretical analysis of the total SMD process including an analysis of optical focus fields, molecule-interface interactions, as well as the collection and detection efficiency was performed. This analysis was used as a guide