Intravital Microscopy With an Airy Beam Light Sheet Microscope Improves Temporal Resolution and Reduces Surgical...

Intravital microscopy has emerged as a powerful imaging tool, which allows the visualization and precise understanding of rapid physiological processes at sites of inflammation in vivo, such as vascular permeability and leukocyte migration. Leukocyte interactions with the vascular endothelium can be characterized in the living organism in the murine cremaster muscle. Here, we present a microscopy technique using an Airy Beam Light Sheet microscope that has significant advantages over our previously used confocal microscopy systems. In comparison, the light sheet microscope offers near isotropic optical resolution and faster acquisition speed, while imaging a larger field of view. With less invasive surgery we can significantly reduce side effects such as bleeding, muscle twitching, and surgical inflammation. However, the increased acquisition speed requires exceptional tissue stability to avoid imaging artefacts. Since respiratory motion is transmitted to the tissue under investigation, we have developed a relocation algorithm that removes motion artefacts from our intravital microscopy images. Using these techniques, we are now able to obtain more detailed 3D time-lapse images of the cremaster vascular microcirculation, which allow us to observe the process of leukocyte emigration into the surrounding tissue with increased temporal resolution in comparison to our previous confocal approach.