Recent clinical studies on human skin indicated that in vivo multi-harmonic generation microscopy (HGM) can achieve sub-micron resolution for histopathological analysis with a high penetration depth and leave no energy or photodamages in the interacted tissues. It is thus highly desired to apply HGM for in vivo mucosa histopathological diagnosis. In this paper, the first in vivo optical virtual biopsy of human oral mucosa by using epi-HGM is demonstrated. We modified an upright microscope to rotate the angle of objective for in vivo observation. Our clinical study reveals the capability of HGM to in vivo image cell distributions in human oral mucosa, including epithelium and lamina propria with a high penetration depth greater than 280 μm and a high spatial resolution better than 500 nm. We also found that the third-harmonic-generation (THG) contrast on nucleus depends strongly on its thicknesses, in agreement with a numerical simulation. Besides, 4% acetic acid was found to be able to enhance the THG contrast of nucleus in oral mucosa, while such enhancement was found to decay due to the metabolic clearance of the contrast enhancer by the oral mucosa. Our clinical study indicated that, the combined epi-THG and epi-second-harmonic-generation (SHG) microscopy is a promising imaging tool for in vivo noninvasive optical virtual biopsy and disease diagnosis in human mucosa.
Keywords: (180.5810) Scanning microscopy; (180.6900) Three-dimensional microscopy; (190.4160) Multiharmonic generation.