Plan apochromat 63 1.40 oil dic objective

The structures were printed using the Nanoscribe Photonic Professional GT+ system (Nanoscribe, Karlsruhe, Germany). This system makes use of the 2PP fabrication technique to reach a lateral resolution < 200 nm and an axial resolution < 700 nm [28 (link)]. The system employs a 780 nm Ti-Sapphire laser with a 150 fs pulse durations at 80 MHz repetition rate [26 (link)], and can print either by scanning the laser over the field of view (FOV) of the objective lens with galvo mirrors or by moving the sample using a piezoelectric stage. The structures were printed on a 30 mm Ø borosilicate glass coverslips using the IP-L 780 polyacrylate photoresist (Nanoscribe, Karlsruhe, Germany) and a Plan-APOCHROMAT 63×/1.40 Oil DIC objective (Carl Zeiss, Oberkochen, Germany). In order to avoid stitching errors, the microstructures were arranged in microarrays corresponding to a square with a side length below 140 µm, or a hexagon with a side length below 100 µm. The sizes were selected so that the microarrays could be inscribed in the maximum effective writing field for this microscope objective, which is a circle with a diameter of 200 µm. Several microarrays were printed on the same substrate. Individual micro arrays were printed using the galvo mirrors to scan the laser beam, after which the stage was displaced to a new area on the substrate using the piezoelectric motors.

Imaging was performed on a Zeiss spinning disk confocal fluorescence microscope equipped with a Zeiss Plan Apochromat 63× /1.40 Oil/DIC objective. Samples were in 5% CO₂ atmosphere at 37 °C during imaging and were illuminated with laser light alternating between 488 nm and 639 nm, exciting the cell membrane stain and the Atto 647N dye (labeling the ceria nanoparticles), respectively. Image sequences were captured with an electron multiplier charge-coupled device camera (Evolve 512, Photometrics, USA). Several planes of the cells were imaged with a spacing of 250 nm and a detection time of 100 ms per confocal section.

Undifferentiated podocytes were plated on glass cover slips coated with 0.25 μg/cm² laminin 521 (CORNING) and allowed to differentiate for 7 days. They were then fixed in 4% PFA before being permeabilized with 0.1% Triton. After blocking with 3% bovine serum albumin in PBS, cells were immunostained with the respective antibodies and stained with phalloidin. Immunofluorescence images were obtained using a Zeiss LSM780 laser scanning confocal microscope with the Zeiss Plan Apochromat 63 × 1.40 Oil DIC objective. All imaging parameters were maintained constant throughout image acquisition of all samples. Quantification of FA complex was done using ImageJ software, as described previously^{49 (link)}. Briefly, cell contour was traced using phalloidin-stained images with the ‘Freehand Line’ function and cell area was measured. After shifting to images with vinculin staining, the ‘Clear Outside’ function was used to erase the area outside the cell region. The number of particles between 1 and 8 μm² was counted. FAs were classified into three groups: small (1–2 μm²), medium (2–6 μm²), and large (6–8 μm²). The proportion of the number of FAs in each group relative to total FAs was calculated.