Ff500 646 di01

Freshly prepared samples were placed on the microscope setup, and movies of typically 300 frames with a frame rate of 10 Hz were recorded. We used a home build setup arranged around an inverted microscope (IX70, Olympus) with a 1.45 NA oil immersion objective (PLAPON 60×0TIRFM, Olympus). Samples were illuminated using a 633 nm HeNe laser (circularly polarized light, 1.4 kW/cm²) in oblique illumination mode to excite the samples slightly above the glass-cell interface, minimizing in this way any potential artifacts associated with the proximity of the cell membrane to the glass substrate. The fluorescence emission of the ATTO-647N was separated from the excitation light using a dichroic mirror (Semrock, FF500/646-Di01). A 660 nm long pass filter (Semrock, BLP01-635R-25) then selectively allowed the fluorescent emitted light to be detected by an EMCCD (Hamamatsu) camera. Temperature was maintained at 37°C with 5% CO₂ by a custom made incubator built around the microscope stage. In experiments where cells were stimulated with CCL21 and/or ICAM-1, movies of 30 s were recorded before, and at one-minute intervals after stimulation up to 10 minutes.

Single QD655 tracking was performed using a custom setup built around an inverted microscope (IX70, Olympus) equipped with a 1.49 numerical aperture oil immersion objective (Apon ×60 total internal reflection fluorescence, Olympus). Samples were excited using a 488-nm laser (Sapphire 488–150CW CDRH) in oblique illumination configuration. A dichroic mirror (Semrock, FF500/646-Di01) was used to direct the laser light onto the sample while allowing transmission of the QD fluorescence emission, which was directed onto a CMOS camera (Hamamatsu, ORCA-Flash 4.0) after further long-pass filtering (Semrock, BLP01–635R-25). A custom-made incubator built around the microscope allowed the samples to be maintained at 37 °C with 5% CO₂ during the measurements. During a typical experiment, movies of 1000 frames were recorded every 60 s at a frame rate of 62 Hz.