Intensity measurements in scattering media

We describe a new method which allows quantitative measurements of fluorescence intensity in highly scattering media. The measurement principle is based on observing the emission from both the fluorophore of interest with a nanosecond decay time and of a reference fluorophore which displays a much longer microsecond lifetime. The reference fluorophore is placed on rather than in the sample to mimic a sensing device with the long lifetime reference held against the skin. The amplitude modulation of the emission is observed using the standard method of frequency-domain (FD) fluorometry. At an intermediate modulation frequency, the modulation is equivalent to the fractional intensity of the nanosecond fluorophore. The method was tested in 0.5% intralipid, which is more highly scattering than skin. Quantitative intensity measurements were obtained for various concentrations of fluorescein in intralipid, and of the pH sensor 6-carboxyfluorescein (6-CF). Low frequency modulation measurements provide a general method for quantitative measurements in the presence of factors which preclude direct intensity measurements.