Imaris

Embryos subjected to whole-mount in situ hybridisation were cleared in serial incubations of glycerol (25, 50, 75 and 95%), the eyes and brains dissected and placed in a drop of glycerol, cover-slipped, and imaged with a 40X (0.8 NA) water-immersion lens using a Nikon E1000 microscope connected to a digital camera (Jenoptik) operated by Openlab (Improvision) software.
Cryosections were examined by confocal fluorescence microscopy (Leica Systems) using a 40X (1.2 NA) or 63X (1.4 NA) oil-immersion lens. Whole-mount immunostained embryos were imaged using a 25X (0.95 NA) water-immersion lens. All confocal images were processed using Volocity (Improvision) or Imaris software and all figures were composed with Adobe Photoshop and Adobe Illustrator.

Animals were anesthetized using 3-aminobenzoic acid ester (Tricaine), covered in 0.8% low-melting point agarose. Animals were then mounted laterally on their right side in glass-bottomed 35 mm petri dishes (Nichols et al., 2018 (link)). Images were acquired on a spinning disk confocal microscope custom built by 3i technology (Denver, CO) that contains: Zeiss Axio Observer Z1 Advanced Mariana Microscope, X-cite 120LED White Light LED System, filter cubes for GFP and mRFP, a motorized X, Y stage, piezo Z stage, 20X Air (0.50 NA), 63X (1.15NA), 40X (1.1NA) objectives, CSU-W1 T2 Spinning Disk Confocal Head (50 μm) with 1X camera adapter, and an iXon3 1Kx1K EMCCD camera, dichroic mirrors for 446, 515, 561, 405, 488, 561,640 excitation, laser stack with 405 nm, 445 nm, 488 nm, 561 nm, and 637 nm with laser stack FiberSwitcher, photomanipulation from vector high speed point scanner ablations at diffraction limited capacity, Ablate Photoablation System (532 nm pulsed laser, pulse energy 60J @ 200 HZ). Images in time-lapse microscopy were collected every 5 min for 24 h or from 24 to 72 h depending on the experiment. Adobe Illustrator, ImageJ, and IMARIS were used to process images. Only brightness and contrast were adjusted and enhanced for images represented in this study. All fluorescence quantifications were normalized to the background value of each image.

Repeated imaging of zebrafish was performed as described5 (link). Images were acquired on Zeiss LSM 780 NLO confocal and Leica M205 FA stereo-microscopes. ImageJ53 (link), Adobe Photoshop, Adobe Illustrator and Imaris were used for image processing and analysis. Maximum intensity projections of confocal scans of the fluorescent samples were uniformly adjusted for brightness and contrast. Scans of the bright field were stacked using “stack focuser” plugin and tile scans were stitched in Fiji54 (link).