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PURPOSE. The aims of this study were to use transscleral optical spectroscopy to analyze normal and tumor-infiltrated areas of enucleated human eyes, and to characterize the spectral properties of uveal melanomas in relation to various morphological features. METHODS. Nine consecutive eyes enucleated for uveal melanoma were examined by transscleral spectroscopy, using a fiber-optic probe that exerted a fixed pressure on the scleral surface. Spectroscopic measurements, covering the wavelength range of 400-1100 nm, were sequentially performed over the uveal melanoma and on the opposite (normal) side of each eye. The eyes were then processed for histological and immunohistochemical analyses. Comparisons between spectral and morphological parameters were performed by Spearman's rank correlation coefficient and unpaired t-test. RESULTS. The average reflection intensity obtained from the normal side of the eyes was higher than that from the tumors. The spectral imprint of hemoglobin was lower and that of water was considerably stronger when compared with the tumor side. The diffuse reflection spectra from the melanomas showed a strong correlation with the degree of tumor pigmentation (Spearman's rho = -0.87, P < 0.0001). A weaker correlation was observed between the amount of hemoglobin-related absorption and the density of intratumoral blood vessels (Spearman's rho = -0.25, P = 0.023). The mean diffuse reflection intensity obtained from the spindle cell melanomas was significantly higher than that from the mixed and epithelioid cell melanomas (P < 0.0001). CONCLUSIONS. Although future in vivo studies are required, these data suggest that transscleral optical spectroscopy is a feasible method for identification and morphological assessment of choroidal tumors. (Invest Ophthalmol Vis Sci. 2012;53:5379-5385) DOI: 10.1167/iovs.12-9840 (Less)