In vivo confocal and two-photon (2P) microscopies have been a major driving force in deepening our understanding of cell dynamics by enabling the visualization of migration, distribution, morphology, and cell-cell interactions in three dimensions and at subcellular resolution. In this talk, I will first discuss the key features of confocal and 2P microscopes for in vivo imaging and introduce bio-applications in various mouse organs such as the small intestine, bone marrow, and lymph nodes.
Secondly, I will talk about 3P microscopy, which overcomes the depth limitation of confocal and 2P imaging. We applied 3P microscopy to visualize dynamic immune cell behavior in mouse lymph nodes for the first time. We determined safe laser parameters by monitoring immune cell motility in various imaging conditions to prevent photo-damage. Using the safe laser conditions, we were able to visualize blood vessels through the entire depth of mouse popliteal lymph node in vivo. Additionally, we could measure the motility of CD4+ and CD8+ T cells through the entire depth of T cell zone in vivo, allowing us to discover the depth dependence of CD4+ T cell motility in the T cell zone during lipopolysaccharide-induced inflammation.
Thus, in vivo 3P microscopy has the potential to uncover previously unknown cellular dynamics in deeper regions of many other organs beyond the depth limit of conventional confocal and 2P microscopies.
Talk will be in-person and virtual, see information below.