Orca flash 4 v3 camera

Confocal images of intracranial lymphatics were acquired using a Nikon Ti2 inverted microscope with Yokogawa CSU-W1 spinning disk confocal, Hamamatsu Orca Flash 4 v3 camera with the following Nikon objectives: 4X Air 0.2 N.A., 10X Air 0.45 N.A., 20X water immersion 0.95 N.A., and 25X silicone immersion 1.05 NA, 40X water immersion 1.15 NA, 60X water immersion 1.20 N.A. Stereo microscope pictures were taken using a Leica M205 microscope using MultiFocus focus stacking. The large size of the juvenile and adult zebrafish head often required tile acquisitions that were later stitched using Nikon Elements software. Approximately five fish were imaged for each data point per experiment. All images were compared, and the images selected for presentation were representative of all the images collected, including vascular and lymphatic complexity. Most of the fish in this study were imaged at juvenile stages of development before visual determination of male or female sexual development is possible, however when adult fish were imaged both male and female fish were selected for analysis.