In vivo imaging and optical manipulation of the small intestinal mucosa using 2-photon microscopy and femtosecond laser nano surgery

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Novel methods for intravital 2-photon microscopy based on tissue auto-fluorescence have successfully been established in the first funding period. In a newly developed mouse model, we discovered several previously unknown processes of epithelial cell biology and mucosal immunology. For the first time, the dynamics of intraepithelial lymphocytes was observed and their kinetics quantified. Integration of a UV manipulation laser and development of an online dosimetry made it possible to cause highly localized tissue damage and examine the in vivo tissue responses. In the present renewal, the non-linear imaging range will be shifted to the infrared, endogenous and exogenous fluorophores will be excited quasi-simultaneously at multiple wavelengths, the focus quality will be improved by adaptive optics, and laser nano-surgery controlled by integrated bubble interferometry will be established. Using these new imaging techniques, mechanisms of lymphocyte migration and antigen processing in the gut epithelium will be studied. Controlled laser manipulation will provide new insight into mechanisms involved in tissue damage and repair. Alterations of tissue dynamics in inflammatory disease will be investigated by establishing an inflammation model for the small intestinal mucosa, directly observing the invasion of bacteria, and tracking immune cells and tissue repair after local damage.