M 116 dg

Whole organ imaging was performed with a custom LSM [33 (link)]. The light sheet was generated in digital scanning mode using a galvanometric mirror (6220H, Cambridge Technology, Bedford, MA, USA); confocal detection was achieved by synchronizing the galvo scanner with the line read-out of the sCMOS camera (Orca Flash4.0, Hamamatsu Photonics, Shizuoka, Japan). The laser light was provided by a diode laser (Cobolt, HÜBNER Photonics GmbH, Germany), and an acousto-optic tunable filter (AOTFnC-400.650-TN, AA Opto-Electronic, France) was used to adjust laser intensity. The excitation objective was a 10×, 0.3 NA Plan Fluor from Nikon, while the detection objective was a 10×, 0.6 NA Plan Apochromat from Olympus. The whole brain sample was recorded using a cuvette containing 40% TDE/PBS. The cuvette was mounted on a motorized x-, y-, z-, -stage (M-122.2DD and M-116.DG, Physik Instrumente, Karlsruhe, Germany), which allowed free 3D motion and rotation. Stacks were acquired with a z-step of approximately 3 µm and a xy resolution resulting from the setup configuration of 0.65 µm, with a field of view of 1.3 mm × 1.3 mm. The microscope was controlled via custom-written LabVIEW code (National Instruments, Austin, TX, USA), which coordinated the galvo scanners, the rolling shutter, and the stack acquisition.

We generated two stable zebrafish transgenic lines using a Tol2 construct with elavl3 promoter that drives the expression of the genetically encoded calcium indicator GCaMP6s in all neurons (Freeman et al., 2014 (link)). Tg(elavl3:GCaMP6s) line showed a cytoplasmic expression of the transgene whereas line Tg(elavl3:H2B-GCaMP6s) expressed GCaMP6s in fusion with histone H2B and consequently showed nuclear localization. Larvae were kept according to standard procedures (Westerfield, 1995 ). Each 4-5dpf Tg(elavl3:H2B-GCaMP6s) larva, of unknown sex, used in the experiments was transferred into a reaction tube containing 1.5 % low gelling temperature agarose (A9414, Sigma) in fish water (150 mg Instant Ocean, 6.9 mg NaH 2PO 4, 12.5 mg Na 2HPO 4 per 1 l of dH 2O), kept at 38 °C. The zebrafish larva was then drawn with a syringe into a glass capillary (O.D. 1.5 mm); after gel polymerization, the agarose cylinder containing the larva was gently extruded until the head protruded from the glass (Figure S1b). The glass capillary was then mounted onto an x−, y−, z−, Θ-stage (M-122.2DD and M-116.DG, Physik Instrumente, Germany) and immediately immersed into the sample chamber containing fish water. The fish water was kept at a constant temperature of 28.5 °C.

Specimens were imaged using a custom-made confocal light sheet microscope (CLSM) described in Silvestri29 (link). The light sheet was generated by scanning the excitation beam with a galvanometric mirror (6220H, Cambridge Technology, MA) and confocality was achieved by synchronizing the galvo scanner with the line read-out of the sCMOS camera (Orca Flash4.0, Hamamatsu Photonics, Japan). Five different cw wavelengths were available (MLDs and DPSSs, Cobolt, Sweden) for fluorescence excitation and an acoustooptic tunable filter (AOTFnC-400.650-TN, AA Opto-Electronic, France) was used to regulate laser power. The excitation light was focused with a long working distance, low magnification objective (10x 0.3NA WD 17.5 mm, Nikon, Japan) and fluorescence was collected on a perpendicular axis with a specialized objective for high refractive index immersion and a correction collar for refractive indices ranging from 1.41 to 1.52 (XLSLPLN25XGMP, 25x 1.0NA, WD 8mm Olympus, Japan). The samples were mounted on a motorized x-, y-, z-, θ-stage (M-122.2DD and M-116.DG, Physik Instrumente, Germany) which allowed free 3D motion plus rotation in a custom-made chamber filled with 63% TDE/PBS. The microscope was controlled via custom written LabVIEW code (National Instruments) which coordinated the galvo scanners, the rolling shutter and the stack acquisition.