Csu w1 scanner unit

3D confocal imaging was performed using a spinning disk CSU-W1 scanner unit (Yokogawa) and a DMi6 inverted microscope, equipped with a 100× NA 1.40 objective (Leica Microsystems). Excitation was done with solid state lasers (100 mW 488 nm, 50 mW 560 nm, 100 mW 642 nm) and images were captured on an Ixon Ultra EMCCD camera (Andor). Z-stacks with 100 nm step size were recorded using a piezo stage insert (LUDL Electronic Products, 96A900). The setup was operated using MicroManager^{46 (link)}. Edge localization precision and reference shape measurements were performed with the 50 mW 560 nm laser, where laser power output was varied between 1–50% and image stacks were acquired with 50 ms exposure per slice (144 μm × 144 μm each).
Instantaneous structured illumination microscopy (iSIM) was performed using a VisiTech iSIM scan head (VisiTech International), an Optospin fast filter wheel (Cairn), and an IX73 microscope, equipped with a 60 × NA 1.3 silicone oil objective (Olympus) and a Hamamatsu Flash 4.0 v2 camera. For live cell imaging a WSKM-F1 stagetop incubator (Tokai Hit) was used. Images were acquired using MetaMorph software (Molecular Devices).

Prior to live imaging, culture inserts containing the organoid slices were transferred to a six-well glass bottom plate (Cellvis) and fresh cortical culture medium was added. GFP-positive cells were live imaged for 48 hours on a spinning disk wide microscope equipped with a Yokogawa CSU-W1 scanner unit. The microscope was equipped with a high working distance (WD 6.9–8.2, 414 mm) 20× Plan Fluor ELWD NA 0.45 dry objective (Nikon), and a Prime95B SCMOS camera. Z-stacks of 80–100 µm range were taken with a step size of 4–5 µm at intervals of 15 minutes. Temperature and CO₂ levels were controlled with a stage top incubator (Tokai Hit). Raw images were processed with NIS-Elements and FIJI ImageJ.

Mouse embryonic brains were dissected out of the skull, fixed in 4% Pfa for 2 h, and 80 µm-thick slices were prepared with a Leica VT1200S vibratome in PBS. Slices were boiled in citrate sodium buffer (10 mM, pH6) for 20 min and cooled down at room temperature (antigen retrieval). Slices were then blocked in PBS-Triton X100 0.3%-Donkey serum 2% at room temperature for 2 h, incubated with primary antibody overnight at 4 °C in blocking solution, washed in PBS-Tween 0.05%, and incubated with secondary antibody overnight at 4 °C in blocking solution before final wash and mounting in aquapolymount. Imaging was performed on a fully motorized spinning disk wide microscope driven by Metamorph software (Molecular Devices) and equipped with a Yokogawa CSU-W1 scanner unit to increase the field of view and improve the resolution deep in the sample. Image analysis, modifications of brightness and contrast were carried out with Fiji. Statistical analysis was carried out with Prism. Figures were assembled in Affinity Designer.