Axio observer z1 advanced mariana microscope

Embryos were manually dechorionated at 48 hpf and anesthetized with 3-amino-benzoic acid ester (Tricaine). Anesthetized embryos were immersed in 0.8% low-melting point agarose and mounted on their right side in glass-bottomed 35 mm Petri dishes^{48 (link)}. A spinning disk confocal microscope from 3i technology^© was utilized and is equipped with a Zeiss Axio Observer Z1 Advanced Mariana Microscope with X-cite 120LED White Light LED System and filter cubes for GFP and mRFP, a motorized X,Y stage, piezo Z stage, 20X Air (0.50 NA) objective with 2 mm working distance, 63× (1.15 NA) water objective with 0.66 mm working distance, 40× (1.1 NA) water objective with 0.62 mm working distance, CSU-W1 T2 Spinning Disk Confocal Head (50 μM) with 1× camera adapter, and/or iXon3 1Kx1K EMCCD camera, dichroic mirrors for 446, 515, 561, 405, 488, 561, 640 excitation, laser stack containing 405 nm, 445 nm, 488 nm, 561 nm, and 637 nm with laser stack FiberSwitcher that has 250 μs switch time, photomanipulation with vector^© high speed point scanner ablations at diffraction limited capacity, Ablate!TM^© Photoablation System (532 nm pulsed laser, pulse energy 60J @ 200 HZ). All images of DRG were taken in the trunk of the animal. Time lapse images were taken every 5 min for 24 h starting at 48 hpf. Adobe Illustrator and ImageJ were used to process images and enhance image brightness and contrast.

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.