Wavelength-selective light quenching of biochemical fluorophores

The use of light quenching to selectively eliminate the emission of biochemical fluorophores based on the emission wavelength is described. To demonstrate the possibility of wavelength-selective light quenching, a mixture of two fluorophores, 4-(dimethylamino)-4′-cyanostillene (DCS) and Prodan, emitting at different wavelengths was examined first. The emission spectrum and intensity decay were altered by the 570-nm quenching pulse due to selective quenching of the longer wavelength emission of DCS. Quenching of the solvent-sensitive fluorophore partially bound to human serum albumin and partially in the aqueous phase was then examined. Light quenching with a long wavelength (570 nm) time-delayed pulse selectively quenched the Prodan fluorophore in the aqueous phase while in the presence of the Prodan bound to human serum albumin, which emitted at shorter wavelengths, was not quenched. Using one-beam short wavelength excitation and quenching, a selective quenching of the blue-shifted emission of ribonuclease T₁ in the presence of the red-shifted emission of the tryptophan residue in adrenocorticotropic hormone was observed. In both systems wavelength-selective light quenching was demonstrated by a shift in the emission spectra, and by changes in the intensity decay consistent with preferential quenching of one species. Light quenching is instantly reversible by blocking or defocusing of the quenching beam. It can occur for inaccessible residues or in viscous solvents, and thus can be of wide applicability for resolving the complex emission of biological macromolecules.