Quantification of the hematoporphyrin derivative by fluorescence measurement using dual-wavelength excitation and dual-wavelength detection

A theory is presented describing a method for quantitative measurement of fluorophores in vivo. The method employs a combination of two-wavelength excitation and two-wavelength detection. A dimensionless fluorescence ratio is defined. The theory predicts that this ratio is a function of the concentration of the fluorophore only and is independent of optical properties, independent of geometrical parameters, and independent of autofluorescence. Experiments using a O.1-10-microg/mL hematoporphyrin derivative in a tissue phantom with the optical properties of the dermis show good agreement with theory.