Light quenching of fluorescence using time-delayed laser pulses as observed by frequency-domain fluorometry

We describe experimental observations of fluorescence quenching by time-delayed light pulses whose wavelength overlaps the emission spectrum of 4-(dimethylamino)-4′-cyanostilbene (DCS). The relative cross sections for light quenching were proportional to the amplitude of the emission spectra at the light quenching wavelength. The frequency-domain intensity and anisotropy decay measurements showed oscillations resulting from time-delayed light quenching. The amplitude of the oscillations depends upon the amount of light quenching. The frequency of the oscillations depends upon the delay between the excitation and quenching pulses. To the best of our knowledge, only a stepwise decrease in the intensity or anisotropy could produce such oscillations in the frequency-domain data. Light quenching of fluorescence is thus shown to provide a means to control the number and orientation of the excited fluorophores. The use of multiple light pulses for excitation and quenching can have far-reaching applications in the use of time-resolved fluorescence in physical chemistry and biophysics.