Characterizing 3D trajectories of immune cells in response to bacterial toxin secretion apparatus in live larval zebrafish

Immune responses involve complex chemical and physical dynamics. Innate immune cells are the first responders at a variety of disturbances such as infected cells, tumors, and wounds. They also maintain microbial homeostasis, especially in the gut, where they show tolerance towards commensal microbes while preserving strong reactions to pathogens. Physical characterization of immune responses is typically limited to total cell count, but in-vitro studies show cellular motility and morphology are also altered upon stimulation. How these physical behaviors are manifested inside a living host remains unclear. We examined innate immune cells - neutrophils and macrophages - in larval zebrafish, a model vertebrate. Using light sheet fluorescence microscopy to obtain 3D images over several hours, we tracked immune cells in wound assays and in the presence of an opportunistic pathogen. Characterizing speeds, measures of random-walk motion, and cellular morphology, we were able to identify a sub-population of cells that perform directed motion towards stimuli while most cells show random or largely stationary trajectories. In addition, imaging reporters of a bacterial toxin secretion mechanism allows measurements of timescales connecting bacterial activity and immune cell reorganization.