Bx61wi fluorescence microscope

Most wide field images were acquired on a Zeiss Axio Imager Z2 (Zeiss, Germany) with a 10× objective (effective NA 0.45), a colibri LED 7 and Zen blue software (Zeiss, Germany), using a multi‐band bandpass filter (Zeiss, Germany) with the filter excitation wavelengths 370–400 nm, 450–488 nm, 540–570 nm, 614–647 nm, 720–750 nm and the filter emission wavelengths 412–438 nm, 501–527 nm, 582–601 nm, 662–700 nm, 770–800 nm for DAPI, Alexa 647, and GFP and a single‐band bandpass filter (TxRed‐4040C‐ZHE‐ZERO, Semrock) with the excitation wavelengths 540–552 nm and emission wavelengths 590–4095 nm for mCherry. LED‐module 385 nm was used for DAPI, LED‐module 630 nm for Alexa 647, LED‐module 475 nm for GFP, and LED‐module 567 nm for mCherry. Some images were taken using an Olympus BX61WI fluorescence microscope (Olympus, Japan) with a 10× (effective NA 0.40) objective and a Volocity 4.1.0 software (Quorum Technologies) or a Zeiss LSM700 confocal microscope (BioVis facility, Uppsala University). Brightness and contrast were adjusted in the Fiji software (Schindelin et al. 2012 (link)), equally for the whole image and without obscuring any data.
Coordinates were identified based on the Paxinos and Franklin atlas (Paxinos and Franklin 2012 ). Abbreviations and brain region identification followed the Allen brain atlas data portal (http://atlas.brain‐map.org). Cell counts were done manually with the Fiji counter plugin and the cell diameter was measured manually in Fiji. For cortical layer counts DAPI was used to distinguish the layers. For calculating cell distance to cortex surface, two points were marked at the cortex surface, then a line was interpolated from these points for each coronal image, and cell dorsoventral (DV) coordinates were calculated as the shortest Euclidean distance between each cell and the interpolated line. For the visualization of cell distance to cortex surface, the traced cells were divided in three different groups; by the injection site (on brain sections with starter cells), anterior (>0.5 mm anterior of the most anterior starter cell) and posterior (> 0.1 mm posterior of the most posterior starter cell). The probability density function of cells was calculated by performing a bidimensional gaussian kernel density estimate on the cell coordinates (ML × AP or AP × DV) interpolated 100 points on each axis.
In the Chrna2Cre‐EGFP mouse, there were seven Chrna2/GFP and mCherry double‐positive cells, of which at least six were classified as starter cells based on their location in cortical layer 5 by the injection site and based on their extensive dendritic staining compared to the surrounding GFP labelled Chrna2+ cells, indicating that they had a higher GFP expression after being transfected with the GFP‐expressing helper virus. One Chrna2/EGFP and mCherry double‐positive cell was classified as a likely traced cell based on its location more than 1 mm away from the injection site.

Wide field images were taken using an Olympus BX61WI fluorescence microscope (Olympus, Japan) with a Volocity software (Quorum Technologies) or a Zeiss Axio Imager Z2 (Zeiss, Germany) with a colibri LED 7 and Zen blue software (Zeiss, Germany). Some images were taken using a Zeiss LSM700 confocal microscope (BioVis facility, Uppsala University). Brightness and contrast were adjusted in ImageJ, equally for the whole image and without obscuring any data. Images of brainstem were stitched using the Pairwise Stitching in ImageJ (ImageJ, RRID:SCR_003070) (Preibisch et al., 2009 (link)). Cell counts were done manually with ImageJ's Cell counter plugin (Schneider et al., 2012 (link)). For estimation of the total number of traced cells in the spinal cord, cells were identified and counted using an ImageJ macro based on signal intensity and size. A selection of automatically counted images was re‐counted manually, and the macro was found to identify 50–80% of cells.
Brain regions were identified based on the Allen Brain atlas and The Mouse Brain in Stereotaxic Coordinates (MBSC) (Franklin & Paxinos, 2008 ; Lein et al., 2007 (link)). Abbreviations followed the MBSC.

Mice were anesthetized with ketamine/xylazine and immobilized on a custom-built stage. Laser-scanning microscopy images were collected using a BX61WI fluorescence microscope (Olympus) and a 20× 0.95NA water immersion objective (Olympus) and dedicated single-beam TriM Scope II (LaVision Biotec) controlled by IMSpector software. The microscope was outfitted with a Chameleon Vision II Ti:Sapphire laser (Coherent) with pulse precompensation. For 4D analysis of cell migration, stacks of 13–17 optical sections with 3-µm z spacing were acquired every 20 or 30 s for 30 min with the laser tuned to a wavelength of 845–875 nm. Pharmacological antagonists AMD3100 (Tocris Bioscience), 4F-benzoyl-TN14003 (a gift from H. Tamamura, Tokyo Medical and Dental University, Tokyo, Japan), and anti–VCAM-1 (clone M/K2; Bioexpress Inc.), anti-α4 (clone PS/2; LygoCyte Pharma Inc.), and anti-CXCL12 (clone 79014; R&D Systems) blocking antibodies were injected i.v. 1–5 min before imaging. Videos in which significant tissue drifting was detected were excluded from analyses. A few videos showed small tissue drifting, which was computationally corrected with Imaris software tool “Correct Drift” (Bitplane).
The axis ratio of parenchymal GFP⁺ cells was calculated by measuring the distance between the two furthest GFP⁺ points on the x and y planes of individual cells after collapse of the z plane using Imaris. Parenchymal GFP⁺ cells that were distinguishable from neighboring cells were selected for analysis to avoid miscalculation of the axis ratio of a single GFP⁺ cell.