Intravital Imaging of T Cell-DC Interactions

Multiphoton imaging was done using a Zeiss LSM7 MP System equipped with a 20x/1.0NA water-immersion objective lens (Zeiss UK, Cambridge, UK) and a tunable Titanium: sapphire solid-state two-photon excitation source (Chamelon Ultra II; Coherent Laser Group, Glasgow, UK) and optical parametric oscillator (OPO; Coherent Laser Group). Popliteal LNs were excised 24 h after immunization, transferred into CO₂-independent media at room temperature and bound onto a plastic coverslip with veterinary glue (Vetbond, 3M, St. Paul, MN). Grease was used to attach the coverslip to the bottom of the imaging chamber, which was supplied with warmed (36.5°C) and gassed (95% O₂ and 5% CO₂) RPMI 1640 before and during the imaging. A laser output of 820 nm and OPO signal at 1,060 nm provided excitation of YFP CD11c- and DS-Red OTII T cells. Acquisition of the videos was done for 20–30 min with X-Y pixel resolution of 512 × 512 inches in 2 μm. Cellular 3D tracking was done using Volocity 6.1.1 (Perkin Elmer, Cambridge, UK). Mean velocity, displacement and meandering index was calculated for each object. Intersection of DsREd and YFP objects was used to determine interaction between T cells and DCs, respectively (28 (link), 30 (link)).