Airyscan lsm880

Retinas were imaged and processed using a Zeiss confocal laser-scanning microscope with Airy Scan (LSM 880) with Zen software (Carl Zeiss, Thornwood, NY). Confocal microscopy was performed with set parameters for laser power, photomultiplier gain, and offset, with a pinhole of diameter of 1 Airey unit for all samples with a × 40 oil immersion objective. Images of Z-stacks (10–15 optical sections with 0.5–1 μm step size) were acquired and maximum intensity projections of the Z-series were processed in Zen software. All images were compiled for publication in Adobe Photoshop CC 2015 software.

A Zeiss Airyscan LSM 880 confocal microscope was used to image VGluT2/TdTom⁺ or BDA/TdTom⁺ neuronal processes apposing SPNs (ChAT⁺ neurons) in thoracic IML. For each rostral (∼T1 – T6/7) and caudal (∼T6/7 – T13) region, 14–20 ROIs containing the IML, with at least 90 μm of inter-section distance, were selected to obtain a representative rostro-caudal distribution of IML regions and SPNs per mouse (n = 4). Each IML area was imaged (7–14 Z-stack images through a focusing range of 3.6–12.8 μm) at 63× magnification. Laser settings were optimized and replicated within each animal, for consistency between images. Left and right IML SPNs were selected and imaged based on visibility of at least one SPN soma per ROI. Z-stack images were then imported into Imaris software for 3D reconstruction and quantitative analysis using the “Surface” tool. Synaptic boutons (VGluT2) less than 1 μm from SPN somas were quantified and considered doubled labeled if there was overlap with TdTom⁺ surfaces. Similarly, doubled labeled contacts of BDA/TdTom⁺ were considered as synaptic boutons when less than 1 μm from SPN soma.

All images were acquired under minimally saturating conditions, unless otherwise noted. LSM780 and LSM710 confocal microscopes (Zeiss, Jena, Germany) were used to collect image stacks of neuron cell bodies and proximal dendrites using a 63× oil objective and 2× digital zoom. Unless indicated otherwise, laser intensities and digital gain for quantitative analyses were set using signals from a representative control neuron, and then the same settings were used for the analyses of all other experimental conditions (Kv2.1-KD, proNRG2 variants, etc.). The image stacks were then projected in Z using the maximum intensity method. For the images collected in Figure 1A, laser intensity and digital gain were set for each individual coverslip and used for all images collected from that coverslip. The Z-projected superresolution images of proNRG2 puncta under Kv2.1 knockdown conditions shown in Figure 1G were acquired on an Airyscan LSM880 (Zeiss), with the laser intensity and digital gain for proNRG2 and Kv2.1 set using a representative neuron from a control coverslip. The representative images of proNRG2/VAP-transfected HeLa cells shown in Figure 3B,D were acquired as single planes, and laser settings were optimized for each image.

Samples were imaged on a Zeiss Airyscan LSM 880 confocal microscope with a C-Apochromat 40x/1.2 W Korr M27 water immersion objective or a Plan-Apochromat 63x/1.4 OIL DIC M27 objective. For super-resolution imaging, an Airyscan detector was used⁷⁹ . Volocity (version 6.3.1, PerkinElmer) and Zen (Zeiss) software were used to produce maximum intensity projections and 3D opacity renderings. Image analysis was performed on optical sections. For signal intensity profiles along the apical-basal axis and across tricellular junctions, the arrow tool in the Zen software was used. Anterior and posterior embryo widths measurements were made using the line tool in Volocity.
For F-actin signal intensity profiles across the apical surface of epiblast or ectoderm cells, Fiji’s freehand line tool with a width of ‘3’ was used^{80 (link)}. Because the size of the apical domain was different for each cell measured, distances were expressed as percentages, with 100% representing the total distance across the apical domain. To account for depth-dependent signal attenuation, F-actin signal intensity at the apical domain was normalised by mean F-actin intensity in the nucleus of the cell measured. In each experiment, for each genotype, three embryos were used for measurements and five cells were analysed per embryo. The LOWES method was used to fit a line to the data.