For our experiments, we used a Zeiss 880 microscope with a 20X 0.8 NA air lens at 0.8 zoom. The "tile" function of Zen was used to capture and stitch together two images, one including the forebrain, midbrain, and rostral part of the hindbrain and the other including the caudal hindbrain and anterior spinal cord. Step size was 2 microns. A brain usually comprised around 130-150 slices. Laser intensity and gain were calibrated such that the brightest neurons in the brain were saturated, because otherwise signal in the dimmest neurons was lost. (Note that saturated pixels exist in some portions of the reference brain as well-- it is best to attempt to match the staining of the reference brain as closely as possible.) 3-5 larvae were inspected before final settings were chosen, due to the variability in brightness of the GFP signal between brains. Ideally, the full range of each channel should be utilized. Once settings were determined, the same imaging settings were used for every brain in a staining batch. Images were saved in 8-bit, because they will be downsampled to 8-bit at a later step anyway.