We combined a non-linear microscope with a sensitive prism-based spectrograph and employed it for the imaging of the auto fluorescence of skin tissues. The system has a sub-micron spatial resolution and a spectral resolution of better than 5 nm. The spectral images contain signals arising from two-photon excited fluorescence (TPEF) of endogenous fluorophores in the skin and from second harmonic generation (SHG) produced by the collagen fibers, which have non-centrosymmetric structure. Non-linear microscopy has the potential to image deep into optically thick specimens because it uses near-infrared (NIR) laser excitation. In addition, the phototoxicity of the technique is comparatively low. Here, the technique is used for the spectral imaging of unstained skin tissue sections. We were able to image weak cellular autofluorescence as well as strong collagen SHG. The images were analyzed by spectral unmixing and the results exhibit a clear spectral signature for the different skin layers.

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doi.org/10.1117/12.660069, hdl.handle.net/1765/57188
Laser Florence 2004: A Window on the Laser Medicine World
Department of Radiation Oncology

Palero, J., Latouche, G., de Bruijn, R., Gerritsen, H., & Sterenborg, D. (2005). Non-liniear microscopy and spectroscopy of skin tissues. Presented at the Laser Florence 2004: A Window on the Laser Medicine World. doi:10.1117/12.660069