Intravital multidimensional real-time imaging of the conjunctival immune system.

The conjunctiva, as a peripheral mucosal surface, is dependent on the migration of immune cells to facilitate an orchestrated immune response. So far, only limited data to visualize these dynamics directly have been obtained, mainly due to technical and experimental restrictions. To investigate migration on a cellular level, the following conditions need to be met: (1) intravital investigations need to be facilitated by suitable microscopic techniques; (2) tissues need to be investigated in three spatial dimensions and over time; (3) data need to contain detailed information about the tissue character. Whereas the use of confocal laser scanning microscopy allows high-resolution imaging of the superficial conjunctival immune system and enables the recording of rapid cellular migration, intravital two-photon microscopy further enables tracking of individual cells and characterization of cells and structures with unique optical features using autofluorescence detection, fluorescence lifetime measurements and second harmonic generation in deep tissue. Based on current results and experimental studies, two-photon microscopy has the potential for general use in basic research and clinical practice, and would greatly enhance possibilities for diagnosing and analyzing inflammatory processes of the ocular surface. In particular, inflammation in common diseases, such as allergy and dry eye, and its progress under treatment could be investigated in detail.