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 co ntrast. The background and its associated noise can be made low enough to d etect single fluorescent molecules under ambient conditions. In this paper, different TIR geometries were compared for excitation and detection of sin gle rhodamine 6G (R6G) molecules at air-silica interfaces and single B-phyc oerythrin 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 photoblea ching (N-b) were optimum in different geometries. The greatest image contra st was obtained when using prism-TIR (SBR = 11.5), but the largest number o f detected signal photoelectrons was obtained by using through-objective TI R for R6G-air-silica ([N-b] = 10(4)). The results were discussed in terms o f the TIR field enhancements and the modified dipole emission pattern near a dielectric interface. The SBR;and total detected photons are important pa rameters for designing photon-limited experiments. Cytometry 30:224-231, 19 93 (C) 1999 Wiley-Liss, Inc.