Analysis of Melanin Structure and Biochemical Composition in Conjunctival Melanocytic Lesions Using Pump–Probe Microscopy

PURPOSE: We analyze melanin structure and biochemical composition in conjunctival melanocytic lesions using pump-probe microscopy to assess the potential for this method to assist in melanoma diagnosis. METHODS: Pump–probe microscopy interrogates transient excited-state photodynamic properties of absorbing molecules, which yields highly specific molecular information with subcellular spatial resolution. This method is applied to analyze melanin in 39 unstained, thin biopsy specimens of melanocytic conjunctival lesions. Quantitative features of the biochemical composition and structure of melanin in histopathologic specimens are assessed using a geometric representation of principal component analysis (PCA) and principles of mathematical morphology. Diagnostic power is determined using a feature selection algorithm combined with cross validation. RESULTS: Conjunctival melanomas show higher biochemical heterogeneity and different overall biochemical composition than primary acquired melanosis of the conjunctiva (PAM) without severe atypia. The molecular signatures of PAMs with severe atypia more closely resemble melanomas than other types of PAMs. Pigment organization in the tissue becomes more disorganized as diagnosis of the lesions worsen, but nevi are more inconsistent biochemically and structurally than other lesions. Relatively high sensitivity (SE) and specificity (SP) is achieved for differentiating between various melanocytic lesions, particularly PAMs without severe atypia and melanomas (SE = 89%; SP = 87%). CONCLUSIONS: Pump–probe microscopy is a powerful tool that can identify quantitative, phenotypic differences between various types of conjunctival melanocytic lesions. TRANSLATIONAL RELEVANCE: This study further validates the use of pump–probe microscopy as a potential diagnostic aid for histopathologic evaluation of conjunctival melanocytic lesions.