T cell activation monitoring on lipid bilayers

For T cell stimulation, the CD3ζ-EGFP expressing Jurkat cells were allowed to attach to the lipid bilayers at 37 °C for 2 min prior to imaging. For stimulation by antibody coating as a control experiment, anti-CD3ε-antibody-coated surfaces were used rather than the lipid bilayers. The surfaces were prepared by adsorbing 1 μg/mL anti-CD3ε antibody solution overnight at 4°C onto a coverslip^{30 (link)}.
Cells were imaged with a custom-built TIRF and HILO (highly inclined and laminated optical sheet) microscope setup^{17 (link), 33 (link)} based on an inverted microscope (IX-81, Olympus, Japan) equipped with an infinity-corrected objective (PlanApo 100× NA 1.45 oil TIRFM, Olympus, Japan). A beam from a solid-state laser (488 nm and 20 mW; Sapphire 488-20-OPS; Coherent, Japan) was used for fluorescence illumination. Optical filters (custom-order, Olympus) included a dichroic mirror (DM488) and emission filters (Em 495-545 for EGFP, Em 569-624 for Qdot 585, and Em 650-705 for Qdot 655). Images were captured with three electron-multiplying charge-coupled device (EMCCD) cameras (C9100-13, Hamamatsu Photonics, Japan) controlled by AQUACOSMOS software (Hamamatsu Photonics). Specimens were observed at 37 °C using a temperature control system with a stage top incubator and an objective heater (IBC-IU2-TOP/-CB/-LH, MI-IBC-IU2, Tokai Hit, Japan).