Fv31s sw software

ZART T cells were cultured in complete RPMI without phenol red supplemented with 2% heat-inactivated fetal bovine serum, 50 U mL⁻¹ penicillin, 50 μg mL⁻¹ streptomycin, 1 mM sodium pyruvate, 10 mM HEPES and 50 μM 2-mercaptoethanol and maintained at 37°C. Cells were placed in a petri dish above the TEMPO device. In vitro two-photon imaging of calcium signals was performed with an upright microscope (FVMPE-RS, Olympus), a 25×/1.05 numerical aperture, water-dipping objective and using FV31S-SW software (Olympus). Excitation was provided by an Insight DeepSee dual laser (Spectra-Physics) tuned at 880 nm and image acquisition was performed using a resonant scanner (0.067 μs/pixel, 10 integrations). The following filters were used for fluorescence detection CFP:483/32; FRET: 542/27. Photoactivation was performed with the TEMPO device using a 5s pulse of blue-LED illumination. Time-lapse movies were processed and analyzed using Fiji.²⁷ (link) Calcium was calculated after cell tracking (surface tracking mode, Imaris 7.4.2 software) by dividing FRET signals by CFP signals in individual cells.

At room temperature, diluted RBC solution (approximately 0.025% hematocrit) was injected into a homemade cell chamber. RBC fluorescence imaging was performed on an Olympus FV3000RS confocal microscope with an IX83 body combined with an FV31-HSU Hybrid Scanning Unit (Tokyo, Japan). Calcium mobilization can then be imaged with laser illumination (Ex 490/Em 525), where 15% power (maximum power: 50 mW) was used in the laser diode. Bright-field imaging was performed simultaneously on the same microscope. Images were illuminated using the silver-coated resonance scanning mirrors at 10 fps and 512 × 512 frame size, controlled with Olympus CellSens Dimension software. Light signals were collected via an Olympus 40 × /0.95 UPLXAPO (Air) objective. Two channels inside the InGaAsP high-sensitivity detector with quantum efficiency > 45% were used to concurrently acquire images of both bright-field light and fluorescent light. The acquisition was performed with averaging every two frames. No significant photobleaching effect was observed during the time interval used in the experiments. All images were then collected with the Olympus FV31S-SW software.

The eGFP fluorophore was excited at 488 nm and emission was detected at 490–540 nm. The RFP fluorophore was excited at 561 nm and emission was detected at 560–620 nm. The images were taken by a laser scanning confocal microscope FV3000 (Olympus) and processed with FV31S‐SW software (Olympus).