Wp. Ambrose et al., Single-molecule detection with total internal reflection excitation: Comparing signal-to-background and total signals in different geometries, CYTOMETRY, 36(3), 1999, pp. 224-231
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.