Dry 20 0.75 na objective

The 1 × 10⁴ tfRFP-B16 cells were cultured overnight in a chambered coverslip with 8 wells (ibidi, Germany). Then, 5 × 10⁵ bone marrow cells from ROSA^mT/mG mice were added with 50 μg anti-tfRFP IgG alone, or 50 μg anti-tfRFP IgG in combination with 50 μL normal mouse serum. After 4 hours, the cultured cells were imaged with a spinning disk inverted confocal microscope (PerkinElmer) with a dry 20×/0.75 NA objective (Olympus).

Before performing long-term intravital imaging of the liver, mice needed to be assembled with a drawer-type abdominal window (DAW) 31 . The mice that successfully assembled DAW were immobilized in custom-made boxes, exposing the imaging window. Then, intravital imaging was performed by a spinning disk inverted confocal microscope (PerkinElmer) with a dry 20×/0.75 NA objective (Olympus). Intravital imaging of liver metastases was performed on days 2/3/4/5/7 after tumor inoculation. During imaging, mice were maintained under anesthesia with 1.0% isoflurane in oxygen flow at 0.6 L/min controlled by a small animal anesthesia machine (RWD, Shenzhen, China) and maintained at a constant temperature of 37 °C.

Intravital imaging was performed using an UltraVIEW VoX Spinning Disk inverted Confocal Microscope (PerkinElmer, USA) with a dry 20×/0.75 NA objective (Olympus, Japan) 1 day after activated CTLs were transferred into tumor-bearing mice. The mice were first anesthetized with a mixture of 10 mg/kg xylazine and 100 mg/kg ketamine hydrochloride. Next, the liver or the spleen was exposed by surgery, and the mouse was placed in a box maintained at a constant temperature of 37℃. The liver or the spleen of the mouse was attached to a slide and moistened with pre-warmed PBS. Mice were then anesthetized with 0.5-1.0% isoflurane in oxygen flow at 0.6 L/min controlled by a small animal anesthesia machine (RWD, China). The fluorescent signals of CFP and FRET were imaged using excitation at 440 nm, and the emissions were recorded at 450-500 nm (CFP) and 510-560 nm (FRET). The fluorescent signal of tdTomato (OT-I CTLs) was imaged using excitation at 561 nm, and its emission was recorded at 580-650 nm. Data were collected with velocity software and videos were processed further with Imaris (version 7.6.1, Bitplane) and the Fiji software. Trajectories of individual OT-I CTLs were plotted following the alignment of their starting positions by MATLAB 50 (link).