Oral mucosa is a soft tissue lining the inside of the mouth, protecting the oral cavity from microbiological insults. The mucosal immune system is composed of diverse types of cells that defend against a wide range of pathogens. The pathophysiology of various oral mucosal diseases has been studied mostly by ex vivo histological analysis of harvested specimens. However, to analyze dynamic cellular processes in the oral mucosa, longitudinal in vivo observation of the oral mucosa in a single mouse during pathogenesis is a highly desirable and efficient approach. Herein, by utilizing micro GRIN lens-based rotatory side-view confocal endomicroscopy, we demonstrated non-invasive longitudinal cellular-level in vivo imaging of the oral mucosa, visualizing fluorescently labeled cells including various immune cells, pericytes, nerve cells, and lymphatic and vascular endothelial cells. With rotational and sliding movement of the side-view endomicroscope on the oral mucosa, we successfully achieved a multi-color wide-area cellular-level visualization in a noninvasive manner. By using a transgenic mouse expressing photoconvertible protein, Kaede, we achieved longitudinal repetitive imaging of the same microscopic area in the buccal mucosa of a single mouse for up to 10 days. Finally, we performed longitudinal intravital visualization of the oral mucosa in a DNFB-derived oral contact allergy mouse model, which revealed highly dynamic spatiotemporal changes of CSF1R or LysM expressing immune cells such as monocytes, macrophages, and granulocytes in response to allergic challenge for one week. This technique can be a useful tool to investigate the complex pathophysiology of oral mucosal diseases.
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