Non-linear laser imaging of skin lesions

We investigated different kinds of human cutaneous ex-vivo skin samples by combined two photon intrinsic fluorescence (TPE), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), spectral lifetime imaging (SLIM), and multispectral two photon emission detection (MTPE). Morphological and spectroscopic differences were found between healthy and pathological skin samples, including tumors. In particular, we examined tissue samples from normal and pathological scar tissue (keloid), and skin tumors, including basal cell carcinoma (BCC) and malignant melanoma (MM). By using combined TPE-SHG microscopy we investigated morphological features of different skin regions, as BCC, tumor stroma, healthy dermis, fibroblastic proliferation, and keloids. A score, based on the SHG to autofluorescence aging index of dermis (SAAID), was assigned to characterize each region. We found that both BCC and surrounding dermis have a negative SAAID value, tumor stroma has a positive SAAID value, whereas fibroblastic proliferation and keloids have a SAAID value close to the unit. Further comparative analysis of healthy skin and neoplastic samples was performed using FLIM, SLIM, and MTPE. In particular, BCC showed a blue-shifted fluorescence emission, a higher fluorescence response at 800 nm excitation wavelength and a slightly longer mean fluorescence lifetime. MM showed an emission spectrum similar to the corresponding healthy skin emission spectrum, and a mean fluorescence lifetime distribution shifted towards shorter values. Finally, the use of aminolevulinic acid as a contrast agent has been demonstrated to increase the constrast in BCC border detection. The results obtained represent further support for in-vivo non-invasive imaging of diseased skin.