Characterizing trajectories of innate immune cells in larval zebrafish.

It is well established from in vitro studies of immune cells that stimulation by a wide range of potential signals leads to motility and morphology changes. How these physical behaviors manifest inside a living animal remains unclear due to limitations of conventional imaging and analysis approaches. Here, we establish a quantitative framework for imaging and tracking neutrophil and macrophage dynamics in larval zebrafish, spanning a large fraction of the animal for multi-hour timescales with few-minute temporal resolution. We focus especially on the gut, examining innate immune responses to different preparations of the intestinal microbiome. Using light sheet fluorescence microscopy and trajectory analysis of hundreds of individual cells, we characterize speeds, directional persistence measures, and cellular morphology to reveal distinct population behaviors. Individual immune cells exhibit stable motility phenotypes, favoring predominantly motile or non-motile states rather than frequent transitions between them. Gut architecture constrains migration patterns as demonstrated by preferential anterior-posterior movement and a high probability of cells remaining in the vicinity of the gut throughout the imaging duration. Macrophages display significantly reduced sphericity during motile periods compared to non-motile periods, providing a morphological signature that may enable inference of dynamic behavior from static snapshots. Surprisingly, migration patterns remain consistent across diverse microbial conditions - germ-free, conventionally reared, and colonized by two strains of a zebrafish-native