Aquacosmos software

Cells were imaged using an inverted microscope (IX83, Olympus, Japan) equipped with custom-built TIRF and HILO microscope setup^{37 (link),38 (link)}. The microscope is equipped with an infinity-corrected objective (PlanApo 100x NA 1.45 oil TIRFM, Olympus) and two solid-state lasers (Sapphire 488-20 & Compass 561-50, Coherent, Japan) for the fluorescent illumination. The microscope optical filters were custom-ordered (Olympus) to include a dichroic mirror (DM488) and emission filters (Em 495-545 for EGFP, Em569-624 for TagRFP-T). The Jurkat T cells were imaged at 37 °C using temperature control system with a stage top incubator and an objective heater (IBC-IU2-YOP/-CB/-LH, MI-IBC-IU2, Tokai Hit, Japan). The dual-colour images were simultaneously captured at an approximately 80 nm/pixel magnification with two electron-multiplying charge-coupled device (EMCCD) cameras (C9100-13, Hamamatsu Photonics, Japan) controlled by AQUACOSMOS software (Hamamatsu Photonics) at a frame rate of 1 frame/s. The magnification difference, shift and rotation between the two colour images were corrected using ImageConverter (Olympus, Japan) based on Bicubic interpolation with two colour images of a 10-μm square lattice (Olympus) captured at the same time.

Freshly isolated islets of Langerhans were loaded with 5 µM Fura-2 AM for at least 1 h at room temperature. Calcium recordings in islets were obtained by imaging intracellular calcium under an inverted epifluorescence microscope (Zeiss, Axiovert 200). Images were acquired every 2 s with an extended Hamamatsu Digital Camera C4742-95 (Hamamatsu Photonics, Barcelona, Spain) using a dual filter wheel (Sutter Instrument CO, CA, USA) equipped with 340 nm and 380 nm, 10 nm bandpass filters (Omega optics, Madrid, Spain). Data was acquired using Aquacosmos software from Hamamatsu (Hamamatsu Photonics, Barcelona, Spain). Fluorescence changes are expressed as the ratio of fluorescence at 340 nm and 380 nm (F340/F380). Results were plotted and analyzed using commercially available software (Sigmaplot, Jandel Scientific).

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).

A quantity of 0.4 μM dynein was added to a 5 mg/ml BSA precoated flow chamber (Matsunami Glass, Osaka, Japan) and adsorbed onto the bottom for 5 min. A total of 10.5 mg/ml TMR-labeled tubulin and 7.64 mg/ml white tubulin were polymerized at a ratio of 1:40 and stabilized in 50 μM Taxol (Sigma)/BRB80 buffer (40 mM PIPES, pH 7.2, 0.5 mM MgSO₄, and 0.5 mM EGTA). The MT solution was diluted to 0.5 μM in ATP buffer (1 mM ATP and 100 μM Taxol) with oxygen scavenger (225 μg/ml glucose, 216 μg/ml glucose oxidase, 36 μg/ml catalase, and 1% 2-mercaptoethanol) and introduced into the flow chamber. Following confirmation of MT gliding by cytoplasmic dynein (0.5 μM), recombinant proteins (p80; 0.4 μM, Δ1-56 aa; 0.4 μM, p.G33W; 0.4 μM, p.S535 L; 0.4 μM, p.L540R; and 0.4 μM, BRB80) were added. MT gliding was observed via conventional inverted fluorescence microscopy (Olympus IX71, Tokyo, Japan) with an oil-immersion objective lens (UPlanSAPO, 100X, NA = 1.4, Olympus, Tokyo, Japan) and an EMCCD camera (ImagEM, Hamamatsu Photonics, Hamamatsu, Japan). Captured images were analyzed with AquaCosmos software (Hamamatsu Photonics, Hamamatsu, Japan). All experiments were performed at 37 °C with a stage incubator (Tokai-Hit, Shizuoka, Japan).

Assays were performed with a μ-Slide Chemotaxis^3D (ibidi GmbH) according to the manufacturer’s instructions. Cells were conjugated with collagen I gel and applied to the observation area in the presence or absence of 100 ng/ml CXCL12 in one side of the reservoir. The chamber was placed in a CO₂-incubator (Tokai hit) on the stage of a fluorescence microscope (ECLIPSE Ti; Nikon). Transmission images of cells were obtained every 2 min over 8 hrs using a digital CCD camera (ORCA-R2; Hamamatsu Photonics), and analyzed using a particle tracking tool of AQUACOSMOS software (Hamamatsu Photonics), a filtering tool of ImageJ (NIH) and a Chemotaxis and Migration tool (ibidi GmbH). Cells which moved directionally toward CXCL12 were evaluated by the position in the 90-degree area facing CXCL12 at the end of the experiment. The extent of chemotaxis was expressed as the percentage of directionally moved cells which moved over a distance equal to the average cell size (A20, ≥15 μm; splenocytes, ≥10 μm). When pharmacological inhibitors were used, they were applied to both sides of the reservoir.