Volocity 6

Samples once mounted were positioned in the focus of the objectives, and data were collected by translation of the motorised stage using the yz driver. Laser power and camera integration times were set to provide sufficient contrast and intensity aiming to minimise total integration time while not compromising signal. The raw data were then passed to M-Squared Lasers proprietary deconvolution software to process for deconvolution and removal of Airy pattern in data. Richardson–Lucy deconvolution was processed with at least 100 iterations, and a final datafile is produced for each channel. These were then combined in Nikon Elements (version 5.02) to overlay channels and produce three-dimensional renders as shown in figures. In some instances, Volocity 6.3.1 (PerkinElmer) or ClearVolume (ImageJ plugin—https://imagej.net/ClearVolume) were used to produce 3D renders which was chosen only for convenience at the time.
Morphological measurements of lumen volume (Fig. 6) were performed using Volocity 6.3.1 (PerkinElmer) where an object is defined by use of the built-in “find-objects” method and selecting an intensity value to set the threshold for volumetric regions based on the channel used. An arbitrary volume of more than 150 µm³ was chosen from the dataset, and objects smaller than this were discarded.

To visualize DFC migration, embryos at a desired stage were manually dechorionated and mounted in 1% low-melting-point agarose. Time-lapse multiple-focal-plane (4D) microscopy was then performed at 25°C under an Olympus FV1000 multi-photon system using a Plan Apochromat 40×/1 W dipping objective or at 28°C with a Perkin Elmer Spinning Disk confocal microscope system using 20×/40×/60× (Silicon Oil) objectives. Movies were processed using Imaris software 7.1.0 (Bitplane AG) or Image-Pro Plus 6.0 software (Media Cybernetics) or Volocity 6.1.1 (Perkin Elmer). 3D reconstructions were processed with Volocity 6.1.1 (Perkin Elmer). Embryos were individually genotyped after observation when necessary.

Live-cell time lapse, live-cell imaging and fluorescence recovery after photobleaching (FRAP) experiments were performed with a Nikon Ti-E microscope equipped with an UltraVIEW VoX spinning disk confocal unit (PerkinElmer, UK), controlled by Volocity 6.3 software (PerkinElmer, UK), and equipped with a live-cell chamber (ACU control, Olympus) set at 37 °C with 5% CO₂ and 60% air humidity. Z-stacks were acquired with a ×60/1.49 NA CFI Apochromat TIRF oil immersion objective (voxel size, 0.12 × 0.12 × 0.3–1 µm; Nikon, Tokyo, Japan) or a 100x/1.49 NA CFI Apochromat TIRF oil immersion objective (voxel size, 0.071 × 0.071 × 0.5–1 µm; Nikon, Tokyo, Japan) and a cooled 14-bit CCD camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan, Cat.No.: C9100-50). Z-stack images were analyzed using Volocity 6.3 (PerkinElmer, UK) and Fiji. Mid Z-planes were assembled onto videos and annotated using Fiji^{98 (link)} (https://Fiji.nih.gov/ij/).

Fluorescent and bright-field images of the transgenic embryos were acquired using the LSM510 confocal laser-scanning microscope (Carl Zeiss Vision, Singapore). Projection of image stacks was made by the Zeiss image browser. Image analysis and signal quantification were performed using the Volocity 6.0 software (PerkinElmer, Singapore). Images were then imported into Adobe Photoshop for cropping, resizing, and orientation. Contrast and brightness were adjusted equally for all images of the same figure. For imaging the immune-stained cell culture plates, the Zeiss fluorescent microscope was used. For live imaging of transgenic lines, embryos were tricaine-anesthetized and embedded in low melting 1% agarose (A9414, Sigma-Aldrich, Milan, Italy). For post mortem imaging of stained embryos, samples were flat-mounted in 87% glycerol/PBS. Standard imaging was performed with a Leica M165 stereomicroscope, equipped with a Leica DFC480 digital camera. Confocal imaging was performed with a Leica SP5 spectral system. Image analysis and signal quantification were performed using the “Measurements” option of the Volocity 6.0 software (Perkin Elmer, Milan, Italy). Virtual qualitative localization of brain markers was performed using gene expression data available in the ViBE-Z (Virtual Brain Explorer for Zebrafish) database [35 (link)]. Final figures were assembled using Adobe Photoshop CS2.

The full length, truncated and mutated SPNS2 genes with N-terminal GFP fusion were cloned into pcDNA5/TO plasmid. The plasmid was transfected into HEK 293T cells for 24 h via lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Live cells were imaged with a Perkin Elmer Spinning Disk Microscope. 100×Olympus PlanApo Objective with Numerical Aperture of 1.4 was used. GFP was excited by the 488 nm laser and the fluorescence images were taken using EM-CCD camera (Hamamatsu). Images were analysed using Volocity 6.3.1 software (Perkin Elmer).

Bacteria were investigated for green fluorescent protein (GFP) expression using an Olympus BX-51 fluorescence microscope (Olympus, Hamburg, Germany). Analysis was performed using the F-View Soft Imaging System and the software cell^F at 1,000× magnification. Bacteria were concentrated after sampling by centrifugation and kept on ice until observation of aliquots using bright-field microscopy and the GFP fluorescence channel. To obtain three-dimensional pictures, an inverted confocal microscope (IX81, Olympus) was used at 400× magnification. Z-stacks were recorded and reconstructed with Volocity 6.0 software (Perkin Elmer).