Micam02 imaging system

hiPSC-CMs were loaded with a voltage-sensitive dye (FluoVolt™ Membrane Potential Kit, F10488, ThermoFisher SCIENTIFIC) for 30 min at RT. The excitation and emission wavelengths were 522 and 535 nm, respectively. Then, 10 µM blebbistatin (Wako, 027-17043), an excitation-contraction uncoupler, was applied to avert motion artifacts. All experiments were performed at 37°C under aerial conditions. Optical action potential (OAP) imaging was acquired at a sampling rate of 5 or 10 ms per frame using the MiCAM02 imaging system (Brainvision, Tokyo, Japan) equipped with a high-speed CMOS camera, alongside field-of-view and spatial resolution, which were 5.76 × 4.8 mm and 30 × 30 μm, respectively. OAP parameters including average CL, d (−F)/dt_max, and APD, were calculated using OriginPro 8.6J software (LightStone, Tokyo, Japan).

Cells were prepared similar to that for membrane potential measurement and treated with the membrane potential kit (F10488; Thermo Fisher Scientific). Optical mapping was performed using the MiCAM02 imaging system (BrainVision, Tokyo, Japan) combined with MyoPacer EP (IonOptix, Westwood, MA, United States), as reported previously (Nakanishi et al., 2019 (link)). Briefly, after loading the cells, the cultures were electrically stimulated using a bipolar electrode. For aligned pattern, pacing was performed parallel or perpendicular to the direction of arranged nanofibers, using the same pacing positions for random and flat patterns. The pacing rate ranged from 0.5 Hz to 2 Hz in a gradient. The whole procedure was performed at 37°C under atmospheric conditions. Optical imaging was performed using the BV_Ana software (BrainVision, Tokyo, Japan).