Triplet Shelving in Fluorescein and Its Derivatives Provides Delayed, Background-Free Fluorescence Detection.

Fluorescence from the xanthene dyes rose bengal, erythrosine B, eosin Y, and fluorescein is modulated by reversibly optically populating and depopulating their long-lived triplet states through coillumination with a second, long-wavelength laser. Here, we show that repumping the S1 state from the triplet generates strong, nanosecond-lived optically activated delayed fluorescence (OADF), microseconds to milliseconds after primary pulsed excitation. This time-delayed emission upon long-wavelength illumination generates fluorescence after triplet shelving and is a major contribution to fluorescence enhancement/modulation. The time-delayed and background-free OADF component is further increased using a >1 μs burst continuous wave excitation scheme to increase the steady-state triplet populations, yielding strong OADF even from strongly emissive fluorescein. Because emission is delayed long after the high-energy primary excitation, yellow-orange fluorescence is readily observed on zero background. As OADF generation depends on the triplet quantum yields and the reverse intersystem crossing rates, we directly probe the usually difficult-to-measure photophysics, create new zero-background detection schemes, and increase OADF through tailored excitation schemes, all improving sensitivity. The excellent match between experiments and simulations demonstrates the promise of these studies for OADF characterization, while enabling us to determine that OADF (in contrast to ground-state recovery and re-excitation) is the major component of fluorescence enhancement for xanthenes studied with triplet quantum yields exceeding 0.1.