TIRFM was performed as previously described (14 (link)). Briefly, 22×22mm coverslips were acid-washed and coated with 0.2mg/mL gelatin (Corning, #2850–22). For all imaging, a 60x Nikon 1.49 NA TIRFM DIC objective (Nikon) was used combined with an additional 1.8x tube lens (yielding a final magnification of 108x), mounted on a Ti-Eclipse inverted microscope with Perfect Focus System (Nikon). TIRFM illumination was achieved using an Andor “Diskovery Platform/borealis widefield illuminator”. Time-lapse series were acquired at a penetration depth of 80 nm and a frame rate of 1Hz using a PCO-Edge 5.5 sCMOS camera. During imaging, cells were maintained at 37°C in DMEM/F12 supplemented with 10% fetal bovine serum.
Confocal images were acquired with a similar setup as described above, using a 50 μm pinhole disk and an Andor Zyla 4.2 sCMOS camera.
Both microscopes have a custom-built full body environmental chamber with temperature control and CO2 stage incubator operated by Bold Line controller and OKO-Touch with SmartBox for data logging, and Molecular Devices MetaMorph software.
Confocal images were acquired with a similar setup as described above, using a 50 μm pinhole disk and an Andor Zyla 4.2 sCMOS camera.
Both microscopes have a custom-built full body environmental chamber with temperature control and CO2 stage incubator operated by Bold Line controller and OKO-Touch with SmartBox for data logging, and Molecular Devices MetaMorph software.
