Spinning disk confocal and tirf combined system

Imaging was performed on an inverted microscope (Nikon TiE,Tokyo, Japan) equipped with a Yokogawa spinning disk confocal and TIRF combined system (Spectral Diskovery, Ontario, Canada), a Nikon 100× Plan Apo 1.49 NA oil immersion objective, and four laser lines (405, 488, 561, and 640 nm), a Hamamatsu Flash 4.0, and μManager software to run the microscope and capture the images. Confocal images were captured using an Andor iXon electron-multiplying charge-coupled device camera. For TIRF imaging, a polarizing filter was placed in the excitation laser path to polarize the light perpendicular to the plane of incidence. The angle of illumination was controlled with either a standard Nikon TIRF motorized positioner or a mirror moved by a motorized actuator (CMA-25CCCL; Newport). Data collection was performed at 37°C. Before imaging, cells were pelleted, washed, and resuspended with the imaging buffer. The experiments were performed two times with different T cell donors. Each experiment consisted of each SLB condition with each CAR construct in triplicate.