Csu x1 spinning disk

For imaging, 35 mm glass bottom dishes with 20 mm well (Cellvis, #D35-20-1.5-N) were used. Glass was first treated with 10 μg/mL of fibronectin in PBS for 30 min in 37oC. 293 T UAS-dGFP cell line was plated on dish and allowed to adhere onto the plate. For mCherry tagged Gal4LOVSK22 tracking experiment, 2500 ng of pLZA144 encoding Gal4LOVSK22-VP64-mCherry was transfected to 293 T UAS-dGFP cells with the FuGENE HD Transfection Reagent following the protocol in manual and kept in dark first. 4 h after transfection, cells were either switched to blue light illumination or kept in dark for another 20 h. Then cell media was aspirated and cells was fixed in 4% PFA in PBS for 15 min in room temperature, followed by PBS washing for three times. Cells were then stained with 2 μg/mL DAPI for 15 min in room temperature, followed by PBS wash for three times and kept in 4oC before imaging. Imaging was done using Nikon Eclipse Ti microscope with a Prior linear motorized stage, a Yokogawa CSU-X1 spinning disk, an Agilent laser line module containing 405, 488, 561 and 650 nm lasers, an iXon DU897 EMCCD camera, and a 40X oil immersion objective lens. Several images of fixed cells in the 405, 488 and 561 channels were collected.

Imaging experiments were conducted on a Nikon Eclipse Ti confocal microscope equipped with a Yokogawa CSU-X1 spinning disk, a Prior Proscan III motorized stage, an Agilent MLC 400B laser launch, and a cooled iXon DU897 EMCCD camera. An environmental chamber was used to maintain cells at 37°C and 5% CO₂ during imaging. In microscopy experiments using optogenetic stimuli, an X-cite XLED1 light source linked to a Polygon400 Mightex Systems digital micromirror device was used to stimulate cells with 500-ms pulses of 450 nm blue light every 1 min, which we define as continuous blue light stimulation. All images were collected using a 20× air, 40× air, or 60× oil objective. Time-lapse images were acquired every 1–3 min.