Optically Activated Delayed Fluorescence.

We harness the photophysics of few-atom silver nanoclusters to create the first fluorophores capable of optically activated delayed fluorescence (OADF). In analogy with thermally activated delayed fluorescence, often resulting from oxygen- or collision-activated reverse intersystem crossing from triplet levels, this optically controllable/reactivated visible emission occurs with the same 2.2 ns fluorescence lifetime as that produced with primary excitation alone but is excited with near-infrared light from either of two distinct, long-lived photopopulated dark states. In addition to faster ground-state recovery under long-wavelength co-illumination, this "repumped" visible fluorescence occurs many microsceconds after visible excitation and only when gated by secondary near-IR excitation of ∼1-100 µs-lived dark excited states. By deciphering the Ag nanocluster photophysics, we demonstrate that OADF improves upon previous optical modulation schemes for near-complete background rejection in fluorescence detection. Likely extensible to other fluorophores with photopopulatable excited dark states, OADF holds potential for drastically improving fluorescence signal recovery from high backgrounds.

Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery.

Excitation of few-atom Ag cluster fluorescence produces significant steady-state dark state populations that can be dynamically optically depopulated with long wavelength coillumination. Modulating this secondary illumination dynamically repopulates the ground state, thereby directly modulating nanodot fluorescence without modulating background. Both fast and slow modulation enable unmodulated background to be quantitatively removed in fluorescence correlation spectroscopy (FCS) through simple correlation-based averaging. Such modulated dual-laser FCS enables recovery of pure Ag nanodot fluorescence correlations even in the presence of strong, spectrally overlapping background emission. Fluorescence recovery is linear with Fourier amplitude of the modulated fluorescence, providing a complementary approach to background-free quantitation of modulatable emitter concentration in high background environments. Using the expanding range of modulatable fluorophores, such methodologies should facilitate biologically relevant studies in both complex autofluorescent environments and multiplexed assays.