Images were captured on a Zeiss Axio Observer.Z1 Motorised Inverted Widefield Microscope fitted with a Hamamatsu Flash 4.0 Camera using Plan-Apochromat 20×/0.8 M27 air objective with Colibri.2 LED illumination. Data acquisition was controlled by Zeiss Zen Blue 2012 software configured at a bit depth of 16-bit and binning mode 2×2.
Whole tissue tiled preview scans were obtained using an EC Plan-Neofluar 10×/0.3 Ph1 air objective with phase contrast. For Reg3B analysis, a 20 point array was chosen at random with point focusing achieved with the Plan-Apochromat 20×/0.8 M27 air objective using EGFP 488 filter as the reference channel for local surface focusing.