Drift-Diffusion Analysis of Neutrophil Migration during Inflammation Resolution in a Zebrafish Model
Inflammatory neutrophils exhibit restricted migration away from the site of tissue injury. 3 dpf embryos from transgenic zebrafish expressing Kaede in neutrophils were subjected to tailfin transection under anaesthesia using a sterile scalpel. The embryos were recovered for 4 hours. At four hours after injury the embryo was mounted in 0.5% low melting point agarose for imaging on a Laser Confocal System (Perkin Elmer Inc). The PhotoKinesis device was then used to photoconvert all neutrophils present within the tip of the tailfin. Photoconversion was carried out according to the methods described (120 cycles of 40% 405 nm laser energy), and time-lapse videomicroscopy was performed using a TE2000 fluorescent inverted microscope (Nikon). (a) Composite images of DIC overlaid with the red and green fluorescence channels showing a representative zebrafish tail before (above) and after (below) photoconversion. (b) A montage of DIC images overlaid with the red fluorescence channel at then timepoints indicated after tailfin injury. The redistribution of photoconverted cells can be clearly seen over time. (c) For each neutrophil in six individual fish, the distance from the wound was calculated using algorithms within Volocity and plotted against time.