Inverted spinning disk confocal microscope

Images and time-lapse series were collected with Nikon inverted spinning disk confocal microscope equipped with Yokogawa CSU-X1 Spinning Disk, EMCCD camera, laser launch including 445 nm (to image Cerulean and CFP), 488 nm (to image GFP), 514 nm (to image Venus and YFP), 561 nm (to image mCherry), and 647 nm (to image Cy5), focus drift correction through the Perfect Focus System, and 60× Plan Apo 1.40 NA oil/0.13 mm WD, and controlled by the NIS-Elements software. For live-cell imaging experiments, cells were seeded in # 1.5 coverslip bottom dishes and incubated in a complete DMEM cell culture medium (described above) at 37°C with 5% CO₂ within a Tokai incubator on the microscope stage. Imaging experiments were performed at 60–80% confluency of stably transfected cells or 48–72 h after transient transfection, unless otherwise specified in the figure legend.

Cells were seeded onto a #1.5 coverslip-bottom tissue culture dish. For live-cell experiments, cells were maintained in complete DMEM cell culture medium at 37°C with 5% CO₂. Imaging experiments were performed at 50–80% confluency of stably-transfected cells or 48 h after transfection, unless otherwise indicated. All images were acquired using a Nikon inverted spinning disk confocal microscope equipped with a Yokogawa CSU-X1 Spinning Disk, EMCCD camera, 60x Plan Apo 1.40 NA oil / 0.13 mm WD, Tokai live-cell incubator, and 445 nm, 514, 561, and 647 nm laser lines. Focus drift was prevented through the Perfect Focus system.