Pump-probe imaging of pigmented cutaneous melanoma primary lesions gives insight into metastatic potential.

Metastatic melanoma is associated with a poor prognosis, but no method reliably predicts which melanomas of a given stage will ultimately metastasize and which will not. While sentinel lymph node biopsy (SLNB) has emerged as the most powerful predictor of metastatic disease, the majority of people dying from metastatic melanoma still have a negative SLNB. Here we analyze pump-probe microscopy images of thin biopsy slides of primary melanomas to assess their metastatic potential. Pump-probe microscopy reveals detailed chemical information of melanin with subcellular spatial resolution. Quantification of the molecular signatures without reference standards is achieved using a geometrical representation of principal component analysis. Melanin structure is analyzed in unison with the chemical information by applying principles of mathematical morphology. Results show that melanin in metastatic primary lesions has lower chemical diversity than non-metastatic primary lesions, and contains two distinct phenotypes that are indicative of aggressive disease. Further, the mathematical morphology analysis reveals melanin in metastatic primary lesions has a distinct "dusty" quality. Finally, a statistical analysis shows that the combination of the chemical information with spatial structures predicts metastatic potential with much better sensitivity than SLNB and high specificity, suggesting pump-probe microscopy can be an important tool to help predict the metastatic potential of melanomas.

Ultrafast transient absorption of eumelanin suspensions: the role of inverse Raman scattering.

An ultrafast investigation is carried out on synthetic eumelanin suspended either in water or in DMSO-methanol. Upon photoexcitation by visible femtosecond pulses, the transient absorption (TA) dynamics of the suspensions are probed in a broad visible spectral range, showing clear nonlinearities. The latter arise from pump-probe interactions that induce the inverse Raman scattering (IRS) effect. We show how eumelanin TA dynamics are modified in proximity of the solvent Stokes and anti-Stokes scattering peaks, demonstrating that IRS affects the sign of TA but not the relaxation times. We compare the results obtained in both suspensions, unveiling the role of the surrounding environment. Eventually, the intrinsic response of synthetic eumelanin to ultrafast photoexcitation is evaluated.

In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature.

We performed epi-mode pump-probe imaging of melanin in excised human pigmented lesions and both hemoglobin and melanin in live xenograft mouse melanoma models to depths greater than 100 µm. Eumelanin and pheomelanin images, which have been previously demonstrated to differentiate melanoma from benign lesions, were acquired at the dermal-epidermal junction with cellular resolution and modest optical powers (down to 15 mW). We imaged dermal microvasculature with the same wavelengths, allowing simultaneous acquisition of melanin, hemoglobin and multiphoton autofluorescence images. Molecular pump-probe imaging of melanocytes, skin structure and microvessels allows comprehensive, non-invasive characterization of pigmented lesions.