Signal discrimination between fluorescent proteins in live cells by long-wavelength optical modulation

Fluorescent proteins (FPs) have revolutionized molecular and cellular biology; yet, discrimination over cellular autofluorescence, spectral deconvolution, or detection at low concentrations remain challenging problems in many biological applications. By optically depopulating a photoinduced dark state with orange secondary laser coexcitation, the higher-energy green AcGFP fluorescence is dynamically increased. Modulating this secondary laser then modulates the higher-energy, collected fluorescence, enabling its selective detection by removing heterogeneous background from other FPs. Order-of-magnitude reduction in obscuring fluorophore background emission has been achieved in both fixed and live cells. This long-wavelength modulation expands the dimensionality to discriminate FP emitters based on dark state lifetimes and enables signal of interest to be recovered by removing heterogeneous background emitter signals. Thus, AcGFP is not only useful for extracting weak signals from systems plagued by high background, but it is a springboard for further FP optimization and utilization for improving sensitivity and selectivity in biological fluorescence imaging.