Multiphoton Laser Scanning Microscopy on Non-Melanoma Skin Cancer: Morphologic Features for Future Non-Invasive Diagnostics

This study describes the morphologic features of human non-melanoma skin cancer obtained using multiphoton laser scanning microscopy (MPLSM) on freshly excised specimens from 14 patients. Optical sectioning parallel to the tissue surface was performed, resulting in en face autofluorescence images of the epidermis and upper dermis, reaching tissue depths of 135 μm. The microscopy was carried out ex vivo using a femtosecond pulsed laser at 780 nm and a × 40/0.8 objective. The autofluorescence was detected in the range of 450–530 nm. Traditional histopathological criteria such as bowenoid dysplasia, multinucleated cells, or hyperkeratosis in squamous cell carcinoma in situ (SCCIS) (five specimens), and peripheral palisading of tumor cells in superficial basal cell carcinoma (SBCC) (six specimens) were clearly discerned. The morphologic features differed significantly between these lesions and perilesional skin. However, characteristic tumor aggregates were found in only one of the three investigated nodular basal cell carcinomas (NBCCs) due to limited imaging depth. In addition, speckled perinuclear fluorescence was observed in both lesions and normal perilesional skin. In conclusion, MPLSM could potentially be applied for non-invasive diagnostics of SCCIS and SBCC, whereas the ability to characterize NBCC is unclear at this point.