Comparison of 90° and Epi-Geometry for Two-Photon Excitation in Capillaries

Abstract

Two geometries of excitation/emission optics are compared for two-photon excitation of time-resolved fluorescence in capillaries. The test fluorophore is the enzymatic tag β-naphthylamine (BNA). Two 0.25 NA (numerical aperture) microscope objectives are used in the 90° geometry: This configuration has a sensitivity of 1.9 × 10¹⁰ count M⁻¹ min⁻¹ and a detection limit of 2.5 nM [signal-to-noise (S/N) = 3]. The epi-geometry uses single objectives of 0.25, 0.50, or 1.25 NA. This second configuration has a sensitivity of 7.6 × 10¹⁰ count M⁻¹ min⁻¹ and a detection limit of 1.7 nM (S/N = 3) for the 1.25 NA objective. The similar performance of the two configurations is in stark contrast to the factor of 46 enhancement predicted by geometric optics for epi-excitation. The discrepancy is attributed to large increases in collected background counts, which make detection of the signal difficult.