Plan apo vc 100 1.4 na oil immersion objective

For imaging of unconjugated HEK cells to study ZAP70-mRuby2 recruitment to plasma membrane, cells were seeded directly into the imaging dish and transfected as described above. Prior to imaging, cells were washed gently in DPBS once and 1 ml of imaging medium was added to cover the cells in the dish. All live-cell imaging experiments in this study used a Nikon Ti inverted microscope in a thermally-controlled enclosure (OKOLabs), equipped with a CSU-X1 spinning-disc confocal head (Yokogawa). Laser lines used to excite samples were: 405 nm (100 mW), 488 nm (60 mW), 561 nm (50 mW) and 640 nm (100 mW), controlled by an acousto-optic tunable filter (Andor). Fluorescence emission was collected through filters for mTagBFP (460±15 nm), eGFP (525±25 nm), mRuby2 (607±18 nm) and IFP2.0 (708±38 nm). All images were collected using a Plan Apo VC 100× 1.4 NA oil-immersion objective (Nikon) onto an iXon Ultra EM-CCD camera (Andor) with a calculated pixel size of 85 nm. Stage movement was controlled by a Prior motorized stage with a Piezo Z-drive. The entire microscope system was controlled by μManager software that was used to create multi-channel, multi-time point image data-sets and at multiple positions when required. The built-in perfect-focus unit of the microscope was used to correct for axial focus drift due to fluctuations in temperature.