Air objective

Bright-field reflectivity spectra at normal incidence were collected using a 20× air objective (NIKON, NA = 0.45), directed to a fiber-coupled spectrometer and normalized with reflection from a standard dielectric-coated silver mirror.

Cell tracking on the 2D and “2.5D” surfaces was performed with the TrackMate plugin in ImageJ. T cells were imaged with differential interference contrast microscopy at 10 s intervals using a ×20 air objective (Nikon Instruments, Japan) and tracking was later performed manually, one cell at a time, at each interval. Cells were maintained at 37 °C in 5% CO₂ (Tokai Hit, Japan) for the duration of the live-cell imaging session. Images were captured at resolutions of 512 × 512 or 1024 × 1024 pixels. For phenotype perturbations, T cells were treated with either 70 nM Taxol or 10 µM Nocodazole.

Repopulated L-Scaffolds and L-Hydrogels were fixed in 4% formaldehyde for 45 min at room temperature and stored in PBS with 0.05% sodium azide (Sigma-Aldrich) at 4 °C until further processing. This included permeabilization in 0.1% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and staining with 1X Phalloidin-iFluor 555 (Abcam, Cambridge, UK) for F-actin staining and DAPI (nuclei staining) prepared in 1% BSA in PBS for 1 h at room temperature. The L-Scaffolds and L-Hydrogels were immersed in Ce3D (prepared as previously described [21 (link)]) in chambers built on top of slides using iSpacers (SunJin Lab) and glass coverslips. They were imaged in a resonant scanner A1RHD confocal microscope (Nikon) controlled with the NIS Elements AR software (Nikon) using a 10× air objective (Nikon) and laser excitation. Images were corrected for brightness and prepared for publication in NIS Elements AR Analysis software (Nikon).

All experiments were conducted 16–28 hr after seeding the cells on the sample. Then the cells were observed on an inverted Nikon Ti-E2 microscope with an Orca Flash 4.0 sCMOS camera (Hamamatsu), a temperature control system set at 37°C, a humidifier, and a CO ₂ controller. For the opto-experiments on cell doublets and singlets, a Nikon ×60 oil objective was used and for the opto-experiments on tissues a Nikon ×40 air objective was used. The E-cadherin and vinculin staining images were taken with an Eclipse Ti inverted confocal microscope (Nikon France Instruments, Champigny sur Marne, France), equipped with sCMOS prime camera (Photometrics), a ×60 objective, and a CSU X1 spinning disk (Yokogawa, Roper Scientific, Lisses, France). MetaMorph software was used for controlling the microscope (Universal Imaging Corporation, Roper Scientific, Lisses, France). Unless otherwise stated, all photoactivations were done with one pulse per min for $10\min$ , and each pulse had a duration of $200\mathrm{ms}$ , a power density of $0.9\mathrm{mW}{\mathrm{mm}}^{-2}$ , and a wavelength of $470\mathrm{nm}$ . The power density was measured with a power meter right after the objective by shining light on a surface of a given size and dividing the measured power by this size. Photoactivation regions were aligned with respect to the micropattern to ensure reproducibility.