Evaluation of spectral correction techniques for fluorescence measurements on pigmented lesions in vivo

Recently, the use of optical spectroscopy for non-invasive diagnosis of malignant melanoma has been suggested. The reliability of such optical measurements can be seriously compromised by spatial variations in the optical properties of the tissue that are not related to malignancy. In the present paper we report a novel approach to fluorescence spectroscopy which allows for elimination of spatial variations in the optical properties of the tissue investigated. To test this concept we performed fluorescence and color measurements on moles and unpigmented control skin in human volunteers before and after topical application of δ-aminolevulinic acid (ALA). Two types of fluorescence data analysis were investigated; a single ratio technique based on the ratio of the red to the yellow fluorescence (660-750 nm to 550-600 nm) at 405 nm excitation and a double-ratio technique, the red-to-yellow ratio at 405 nm excitation divided by the red-to-yellow ratio at 435 nm excitation. The two excitation wavelengths were selected to be located close to the maximum and at some distance from the Soret excitation band of the porphyrins. The single ratio showed a significant correlation between fluorescence and color. The double ratio was independent of the color of the lesion. These findings indicate that the double-ratio technique is suitable for in-vivo detection of local differences in concentration of fluorescent tumor-localizing drugs in pigmented lesions. This enables in-vivo studies of the pharmacokinetics of tumor-localizing agents in pigmented lesions and may significantly contribute to the development of a non-invasive diagnostic tool for malignant melanoma.