A1r mp system

Confocal micrographs are acquired by an inverted microscope Eclipse Ti equipped with a confocal A1R-MP system (Nikon), using an Argon ion laser (excitation wavelength, λ = 488 nm). The sample emission is collected by a 60× (oil immersion NA = 1.40, Nikon) objectives and the fluorescence signal is detected by a spectral detection unit equipped with a multi-anode photomultiplier (Nikon).
The AFM characterization of the nanopatterned surfaces is carried out by “peak force” imaging mode in air using a Bruker Dimension Icon system equipped with a Nanoscope V controller. The used silicon tip (nominal radius of curvature of 2 nm) is mounted on silicon nitride cantilever with 0.4 N/m nominal spring constant. SEM is performed with a Nova NanoSEM 450 system (FEI), using an acceleration voltage around 8 kV and an aperture size of 30 mm.

After completing flow measurements, immunocytochemistry experiments were performed in order to assess the morphology and growth of renal tubule cells within the device. Staining was carried out directly in the chip, rinsing with PBS, fixing cells grown on the PC membrane by a solution of 4% paraformaldehyde in PBS for 20 min, and washing 3 times with PBS for 5 min each. Cell membranes were permeabilized by incubation with 0.1% (v/v) Triton-X100 in PBS for 10 min, followed by incubation in 1% BSA in PBS for 30 min to reduce nonspecific background staining. For investigating cell morphology, the device was incubated for 40 min with phalloidin-FITC/TRITC (25 µg/mL), washed with PBS and stained with DAPI (3 µg/mL) for 10 min. To visualize the markers AQP2, Na⁺K⁺ATPase, before phalloidin and DAPI staining, chips were incubated for 2 h in the primary antibody (anti-AQP2 1:50 in BSA; anti- Na⁺K⁺ATPase 1∶50 in BSA), washed with PBS and incubated with the secondary antibody (anti goat-FITC 1∶100 for AQP2 staining; anti mouse–FITC 1∶400 for Na⁺K⁺ATPase staining) for 1 h. Finally, the membrane, with cell attached, was taken apart from the device and visualized by inverted microscopy Eclipse Ti equipped by confocal A1 R MP system (Nikon, Melville, NY). Experiments were repeated three times on different devices.

The mice with window chambers were anesthetized by inhaling 1.0-3.0% isoflurane in oxygen flow through a Matrx VMS small animal anesthesia machine (Midmark, Dayton, OH, USA). The window chamber was fixed on a warm plate (Thermo Plate) using a custom-made holder and then fastened to the microscope stage. Intravital imaging was obtained with the large-field imaging function on a motorized stage using an A1R MP+ System (Nikon, Tokyo, Japan). The images were captured using the 20× objective (N.A. 0.75, Nikon). A confocal laser scanning microscope was used to simultaneously image the CFP-B16 cells (405 nm excitation, 400-500 nm emission), and CXCR6-GFP cells (488 nm excitation, 500-550 nm emission).

An inverted microscope, Eclipse Ti (Nikon), equipped with a confocal A1R‐MP system (Nikon) was used for the microscopic optical measurements. For the characterization of the light backscattered by the CDD material, a polarized Ar ion laser (wavelength, λ = 488 nm) was used as the light source, while the backscattered signal was collected by a 10 × objective (numerical aperture, N.A. = 0.25) and measured by a photomultiplier. At the beginning of the measurement, the objective is positioned in order to have the focal plane at the surface of the sample (air/sample interface), and this objective position is fixed throughout the overall measurement. Upon sublimation from the sample, the light intensity that is backscattered by the air/sample interface and measured by the detector of the confocal microscope is to decrease because of the increasing spatial gap between the air/sample interface and the fixed focal plane. The sublimated thickness was estimated by the focal depth of the confocal systems: Δz = 2λ/(N.A.)².^[17] The bright field imaging time‐lapse was collected by a DS‐Ri1 color charged‐coupled‐device (CCD) camera (Nikon) in cross‐polarization mode. Analysis was performed by the ImageJ software. QR‐codes were imaged by using either a smartphone camera or a CCD camera (Leica) coupled to a long working‐distance optical system (MVL7000, Thorlabs).