Sensitive detection of small shifts in fluorescence spectra

Abstract

Instrumentation is described for the sensitive detection of small shifts in the emission peaks of fluorescence spectra. The method is based on the modulation with time of the wavelength of the detected light at the emission maximum of the starting spectrum. Modulation of the wavelength is achieved by a novel use of a photoelastic light modulator and a specific arrangement of polarizing filters placed at the exit slit of the emission monochromator. A shift in the initial emission spectrum results in an alternating current in the detected light at double the fundamental frequency of the photoelastic modulator, which is detected with a phase-sensitive amplifier. The instrument is sensitive to shifts of 0.2 to 0.5 nm in the wavelength of maximal emission, verified with solutions of 5-dimethylaminonaphthalene-1-sulfonyl- dl-glutamic acid and reduced β-nicotinamide adenine dinucleotide by following fluorescence shifts resulting from alterations in solvent polarity. Also, the fluorescence of quinine (at micromolar levels) is detectable in the presence of a tenfold higher emission intensity of 9-aminoacridine, although the emission maxima of the two compounds are separated by less than 10 nm. The results of these experiments suggest applications of the technique to problems of biological interest which require sensitive detection of minute changes in fluorescence spectra, changes which are due to a shift in the emission spectrum of the chromophore studied or to the occurrence of a new emitting species which has a slightly shifted fluorescence spectrum.