Prairieview software

CD4SP cells were enriched from 1MO and 12MO thymi and stained with CMTPX CellTracker Red or 2 μM Indo1AM dyes (Life Technologies), prior to incubation for ≥1 h on pCX‐EGFP thymic slices. Slices were imaged (Lancaster et al., 2019 ) every 15 s, through a depth of 40 μm, at 5‐μm intervals for durations of 15 min, using an Ultima IV microscope (Bruker) with a 20× water immersion objective (NA 1.0) and PrairieView software (v.5.4, Bruker). The sample was illuminated with two MaiTai titanium:sapphire lasers (Newport). Migratory cell paths were tracked, and mean cell velocities and path straightness calculated using Imaris (v9, Bitplane). Cell densities were quantified in manually demarcated cortical and medullary regions at the first time point for each dataset.

In vivo imaging of the L4 DRG in awake mice was conducted more than 5–7 days post-surgery, as previously described (Chen et al., 2019 (link)). The mouse was vertebrae-fixed under the two-photon microscope. In brief, mice were placed inside a 2.9-cm-diameter transparent plastic cylinder securely affixed to a heavy metal base to minimize motion artifacts. Prior to imaging, the mice were habituated for at least 30 min.
For Ca²⁺ imaging of afferent sensory somata in the DRG, Thy1.2-GCaMP6s line 3 mice were used. The in vivo Ca²⁺ imaging experiments were carried out using a Bruker two-photon system equipped with a DeepSee Ti:sapphire laser (Spectra-Physics) tuned to 920 nm. Images were collected at frame rates of 2 Hz, with a resolution of 512 × 512 pixels, using a 25× objective (NA, 1.05) immersed in artificial cerebrospinal fluid, along with a 1× digital zoom. Image acquisition was performed using Bruker PrairieView software. To prevent tissue damage, the laser power reaching the DRG was restricted to ≤ 20 mW. Additionally, to avoid bleaching of fluorescent signals during imaging, we imposed a time limit of less than 10 min for each imaging session. Simultaneously, video recording of hindlimb movement was conducted, and only imaging sessions without hindlimb movement were included for spontaneous activity analysis.

Animals were anesthetized with 3–5% Isoflurane in oxygen and received an intravenous injection of 100 µL Alexa 680-Dextran. Animals were placed inside the recording platform on a heating blanket. Anesthesia was continued with 1.5% Isoflurane in oxygen. Images were obtained using an Ultima two-photon laser scanning microscopy system from Bruker Fluorescence Microscopy equipped with an Ultra II femtosecond Ti:Sapphire laser (Coherent) coupled to an Optical Parametric Oscillator (Chameleon Compact OPO, Coherent) tuned to 1240 nm. Alexa Fluor 680 was imaged using a GaAsP detector (H7422P-40, Hamamatsu). We used a ×4 objective (XLFluor4x/340, NA = 0.28, Olympus) to obtain low-resolution images of the exposure. A ×20 water-immersion objective (XLUMPlanFLNXW, NA = 1.0, Olympus) was used for high-resolution imaging. The microscope was operated using PrairieView software (Bruker).