We performed multiphoton fluorescence (MF) and second-harmonic generation (SHG) imaging on human basal cell carcinoma samples. In the dermis, basal cell carcinomas can be identified by masses of autofluorescent cells with relatively large nuclei and marked peripheral palisading. In the normal dermis, SHG from dermal collagen contributes largely to the multiphoton signal. However, within the cancer stroma, SHG signals diminish and are replaced by autofluorescent signals, indicating that normal collagen structures responsible for SHG have been altered. To better delineate the cancer cells and cancer stroma from the normal dermis, a quantitative MF to SHG index is developed. We demonstrate that this index can be used to differentiate cancer cells and adjacent cancer stroma from the normal dermis. Our work shows that MF and SHG imaging can be an alternative for Mohs' surgery in the real-time guidance of the secure removal of basal cell carcinoma.

We performed multiphoton imaging on fungi of medical significance. Fungal hyphae and spores of Aspergillus flavus, Micosporum gypseum, Micosoprum canis, Trichophyton rubrum, and Trichophyton tonsurans were found to be strongly autofluorescent but generate less prominent second harmonic signal. The cell wall and septum of fungal hyphae can be easily identified by autofluorescence imaging. We found that fungi of various species have distinct autofluorescence characteristics. Our result shows that the combination of multiphoton imaging and spectral analysis can be used to visualize and identify fungal species. This approach may be developed into an effective diagnostic tool for fungal identification.

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