Light quenching and depolarization of fluorescence observed with laser pulses. A new experimental opportunity in time-resolved fluorescence spectroscopy

We report the first time-resolved studies of quenching of fluorescence by light, i.e. "light quenching". The dye 4-(dicyanomethylene)-2-methyl-6-(p-dimethamino)-4H-pyrane (DCM) was excited in the anti-Stokes region from 560-600 nm. At high illumination power the intensifies of DCM were sub-linear with incident power. The extent of light quenching was proportional to the emission spectrum at the incident wavelength, as expected for light-stimulated decay from the excited state. The frequency-domain intensity decays indicated the effect was not due to heating or other photochemical effects. Importantly, the decay time was unchanged, as expected for light quenching with a single pulsed laser beam, while the time-zero anisotropy was decreased due to orientation-dependent quenching of the excited state population. Light quenching of fluorescence provides a new method to control the excited state population and orientation of fluorophores, and offers new experimental opportunities for biophysical applications of time-resolved fluorescence.