Plan fluor

The samples were visualized with a custom-built fluorescence microscope shown in Figure 2a. A blue LED (C503B-BCS-CV0Z0461, Durham, NC, USA) with a peak wavelength of 470 nm was used as an excitation source (power P_out = 265 μW) and focused with a 10× objective lens (Plan Fluor, NA 0.30, Nikon, Tokyo, Japan) on the sample placed on a piezoactuator stage (Tritor 102SG, PiezoSystemJena, Jena, Germany). The QD emission was collected with a 100× oil-immersion objective lens (Plan Fluor, NA 1.30, Nikon, Japan, Tokyo) from the substrate side and split between a complementary metal oxide semiconductor (CMOS) camera (Andor Zyla 4.2, sCMOS, Oxford Instruments, Oxford, UK) with an acquisition rate of 10 Hz and a single-photon counting module (SPCM-ARQH-15, Excelitas Technologies, Wiesbaden, Germany) with an integration time of 10 ms. The excitation light was blocked with a band-pass filter (ET542lp, Chroma, Bellows Falls, VT, USA) while the emission was integrated over the area of a slit (50 μm × 1 mm, S50RD, Thorlabs, Newton, NJ, USA), aligned parallel to the film edge. The acquired photon counts were averaged with a sliding bin size of 250 ms.

Cell movement was monitored using an inverted Nikon‐Ti wide‐field microscope (Nikon, Japan) and an incubation chamber (Life Imaging Services, Switzerland). The medium was maintained at a controlled temperature of 37 °C and CO₂ concentration of 5%. Images were collected with a 20×, 0.45 NA long‐distance objective (Plan Fluor, Nikon, Japan). Time‐lapse experiments were set to routinely collect images, in different spatial positions of the sample, in the BF channel with a time resolution of 20 min.
ZO‐1, tricellulin, EDU‐stained nuclei, and Hoechst or DAPI stained nuclei distribution were acquired in immunostained samples using a 60×, 1.4 NA oil immersion objective (Plan Fluor, Nikon, Japan), and the FITC, Cy5, TRITC, and DAPI filter respectively. Samples were images with an inverted Nikon‐Ti spinning disk confocal microscope (Nikon, Japan) equipped with an Andor DU‐888 camera (Oxford Instruments, UK) and a pE‐100 LED illumination system (CoolLED Ltd., Andover, UK).

Images for confocal laser scanning light microscopy (CLSM) were obtained using the 4 × (Plan Apo, N.A. = 0.2, Nikon), 20 × (Plan Fluor, N.A. = 0.5, Nikon), and 40 × (Plan Fluor, N.A. = 0.75, Nikon) objectives. The 4 × objective was used to visualize the entire striatum in a single frame in CLSM, whereas the 20 × and 40 × objectives were used to identify and analyze interneurons and MSNs with sufficient resolution, respectively. The size of each frame was 1024 × 1024 pixels, and images of optical slices were acquired from the section surface to the bottom at the preset optimal step size (one-third of full width at half maximum of z airy disk) and stored as stacked files for each frame using the three single laser beams alternately at each z-position of the stage to collect images of different fluorescence signals. The intensity of the signal in each pixel was recorded at 8 bits for each channel.

Ovulation was induced in females by injection of LHRH (Salmon, 4013835, Bachem AG, Bubendorf BL, Switzerland) dissolved in Holtfreter’s solution according to the method of Berger et al. [23 ], and the eggs were artificially inseminated with sperm from the male according to the method of Ohtani et al. [24 (link)]. For microscopic observation of gonads (with Nikon Eclipse 80i/Plan Fluor, DS-US/DS-Ri1 camera), gonads were cut out and fixed with Nawashin fixing solution (27% formalin). Paraffin sections (10 μm) were double-stained with hematoxylin and eosin.

Cultured cells were subjected to Live/Dead staining (consisting of calcein/ethidium; ThermoScientific, Waltham, MA, USA) to check their viability after 72 h in culture on the cryosection dECM hydrogels. Cells were fixed with 4% PFA (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and washed with 1X PBS 3 times.
For F-actin fibroblast were fixed in 4% PFA (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and permeabilized with 0.1% triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min. Subsequently, the samples were incubated with Phalloidin-iFluor 488 Reagent (ab176753, Abcam, Cambridge, UK) at a dilution of 1:750 for 45 min at room temperature. The samples were then washed 3 times with PBS1x and incubated with a 1:1000 solution of DAPI (NucBlue, ThermoScientific, Waltham, MA, USA). The samples were then washed with PBS 3 times and transferred with the seeded surfaces facing down onto a coverslip using Fluoromount-G mounting medium (ThermoScientific, Waltham, MA, USA).
For fluorescence imaging acquisition, a Nikon D-Eclipse Ci confocal microscope was used in conjunction with a ×10 Plan Fluor (Nikon) for Live/Dead staining-(epifluorescence mode) and ×20 Plan Apo immersion oil objective (Nikon) for F-actin staining (z-stack confocal mode).

Whole organ imaging was performed with a custom LSM [33 (link)]. The light sheet was generated in digital scanning mode using a galvanometric mirror (6220H, Cambridge Technology, Bedford, MA, USA); confocal detection was achieved by synchronizing the galvo scanner with the line read-out of the sCMOS camera (Orca Flash4.0, Hamamatsu Photonics, Shizuoka, Japan). The laser light was provided by a diode laser (Cobolt, HÜBNER Photonics GmbH, Germany), and an acousto-optic tunable filter (AOTFnC-400.650-TN, AA Opto-Electronic, France) was used to adjust laser intensity. The excitation objective was a 10×, 0.3 NA Plan Fluor from Nikon, while the detection objective was a 10×, 0.6 NA Plan Apochromat from Olympus. The whole brain sample was recorded using a cuvette containing 40% TDE/PBS. The cuvette was mounted on a motorized x-, y-, z-, -stage (M-122.2DD and M-116.DG, Physik Instrumente, Karlsruhe, Germany), which allowed free 3D motion and rotation. Stacks were acquired with a z-step of approximately 3 µm and a xy resolution resulting from the setup configuration of 0.65 µm, with a field of view of 1.3 mm × 1.3 mm. The microscope was controlled via custom-written LabVIEW code (National Instruments, Austin, TX, USA), which coordinated the galvo scanners, the rolling shutter, and the stack acquisition.