It is generally accepted that oral cancer arises in the presence of oral precancerous lesions. However, the clinical courses of these lesions are quite unpredictable, and a fundamental enigma remains that when and how these lesions turn to malignant growth. Characterization of these potentially malignant lesions is thus important and could serve as early indicators of this neoplastic transformation process, potentially facilitates the treatment outcome and improves the survival rate. Higher harmonic generation microscope (HGM), providing images with a <500nm lateral resolution at a 300μm penetration depth without leaving photodamages in the tissues, was used for this purpose. Oral cavity biopsies were obtained from 18 patients with clinical suspected oral precancerous lesions scheduled for surgical biopsy. HGM images were compared with histological images to determine the results. By visualization of subtle cellular and morphological changes, the preliminary result of this HGM image discloses excellent consistency with traditional histolopathology studies, without the need for fixation, sectioning and staining. More specifically speaking, the keratin thickness was found to be increased comparing with normal adjacent controls. In some cases, variations in cell size, nuclear size and increased nuclear/cytoplasmic ratio, and increased size of nucleoli were identified, indicating different stages of malignant transformation. These results together indicated that HGM provides the capability to characterize features of oral precancerous lesions as well as oral cancer progression, and holds the greatest potential as an ideal tool for clinical screening and surveillance of suspicious oral lesions.
Third-harmonic generation (THG) microscopy has been reported to provide intrinsic contrast in elastic fibers, cytoplasmic membrane, nucleus, actin filaments, lipid bodies, hemoglobin, and melanin in human skin. For advanced molecular imaging, exogenous contrast agents are developed for a higher structural or molecular specificity. We demonstrate the potential of the commonly adopted tattoo dye as a THG contrast agent for in vivo optical biopsy of human skin. Spectroscopy and microscopy experiments were performed on cultured cells with tattoo dyes, in tattooed mouse skin, and in tattooed human skin to demonstrate the THG enhancement effect. Compared with other absorbing dyes or nanoparticles used as exogenous THG contrast agents, tattoo dyes are widely adopted in human skin so that future clinical biocompatibility evaluation is relatively achievable. Combined with the demonstrated THG enhancement effect, tattoo dyes show their promise for future clinical imaging applications.