Csu w1 spinning disk

Polyacrylamide gels (0.69 and 4.07 kPa shear modulus, 40 μm thickness) were prepared on glass coverslips with embedded 40 nm fluorescent beads (Thermo Scientific, TransFluoSpheres 633/720) and the surfaces were coated with neutravidin as described^{71 (link)}. TGT was immobilized on the surface and the Cy3 signal of TGT was checked to confirm the immobilization. cRGDfK or MUPA-LDVPAAK ligand was conjugated on the immobilization strand of TGT to prevent force-induced rupture. Cells were seeded on the gels for 1 h. The bead images were taken before and after cell removal with the addition of 0.1% SDS. Bead displacements were determined using particle image velocimetry, and the corresponding contractile energy was estimated with Fourier transform traction cytometry, using ImageJ plugins^{72 (link)}. The cell area was determined by manually drawing an ROI in the DIC channel. Imaging was performed on Nikon Eclipse Ti2 microscopes equipped with Perfect Focus, CSU-W1 spinning disk, and Hamamatsu Orca-flash 4.0 v3 camera. Illumination was provided by optical fiber (Oz Optics) coupled solid-state lasers: 561 nm (200 mW) for Cy3 and, 655 nm (100 mW) for fluorescent beads from Spectral Applied Research. Emission was collected via single-bandpass emission filter (605/52 nm) and a long-pass LP647 nm filter for far-red from Chroma.