Pump-probe imaging differentiates melanoma from melanocytic nevi.

Melanoma diagnosis is clinically challenging: the accuracy of visual inspection by dermatologists is highly variable and heavily weighted toward false positives. Even the current gold standard of biopsy results in varying diagnoses among pathologists. We have developed a multiphoton technique (based on pump-probe spectroscopy) that directly determines the microscopic distribution of eumelanin and pheomelanin in pigmented lesions of human skin. Our initial results showed a marked difference in the chemical variety of melanin between nonmalignant nevi and melanoma, as well as a number of substantial architectural differences. We examined slices from 42 pigmented lesions and found that melanomas had an increased eumelanin content compared to nonmalignant nevi. When used as a diagnostic criterion, the ratio of eumelanin to pheomelanin captured all investigated melanomas but excluded three-quarters of dysplastic nevi and all benign dermal nevi. Additional evaluation of architectural and cytological features revealed by multiphoton imaging, including the maturation of melanocytes, presence of pigmented melanocytes in the dermis, number and location of melanocytic nests, and confluency of pigmented cells in the epidermis, further increased specificity, allowing rejection of more than half of the remaining false-positive results. We then adapted this multiphoton imaging technique to hematoxylin and eosin (H&E)-stained slides. By adding melanin chemical contrast to H&E-stained slides, pathologists will gain complementary information to increase the ease and accuracy of melanoma diagnosis.