Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries.

Excitation of fluorescence with total internal reflection (TIR) excitation yields very low background scattered light and good signal-to-background contrast. The background and its associated noise can be made low enough to detect single fluorescent molecules under ambient conditions. In this paper, different TIR geometries were compared for excitation and detection of single rhodamine 6G (R6G) molecules at air-silica interfaces and single B-phycoerythrin proteins at water-silica interfaces. Through-objective, objective-coverslip, and prism-based TIR geometries were investigated. The signal-to-background ratio (SBR) and the number of photons detected before photobleaching (Nb) were optimum in different geometries. The greatest image contrast was obtained when using prism-TIR (SBR = 11.5), but the largest number of detected signal photoelectrons was obtained by using through-objective TIR for R6G-air-silica ( = 10(4)). The results were discussed in terms of the TIR field enhancements and the modified dipole emission pattern near a dielectric interface. The SBR and total detected photons are important parameters for designing photon-limited experiments.