Cfi plan fluor 40

Microscopy slides were imaged using an inverted fluorescence microscope (Nikon Ti-2 Eclipse with Crest X-light V2 spinning disk module (disk unit 60 μm), Nikon Europe BV, Amsterdam, Netherlands) with a CFI Plan Fluor 40× oil immersion objective (CFI Plan Fluor 40×/1.30 W.D. 0.24, Nikon Europe BV). Brightfield and fluorescence images were recorded by an Andor Zyla 4.2 Plus USB3 camera in Widefield and Spinning Disk Confocal mode using LED light excitation. HPTS was imaged with excitation at 395 nm and 470 nm and emission at 515 nm using appropriate exciter, emitter, dichroic filter cubes. Liss Rhod PE was imaged with excitation at 550 nm and emission at 595 nm and DY-647P1 with excitation at 640 nm and emission at 698 nm using appropriate exciter, emitter, dichroic filter cubes. Z-Stacks were recorded in confocal mode from top to bottom (below the slide surface) with a step size of 1 μm and 72 steps. Time series were recorded as indicated.

Differentiated beating hiPSC-CMs were dissociated by Accutase and replated in Matrigel (BD Bioscience) pre-coated 25 mm round cover glass (Warner Scientific Inc.). Cells were recovered for 3-4 days. For Fura-2 AM imaging, cells were loaded with 10 µM Fura-2 AM (stock solution of Fura-2 AM was pre-dissolved in 20% Pluronic F-127 solution in DMSO) in Tyrode’s solution (140 mM NaCl, 1 mM MgCl₂, 5.4 mM KCl, 1.8 mM CaCl₂, 10 mM glucose, and 10 mM Hepes pH = 7.4 with NaOH at RT) for 30 min at room temperature. To pace cells, we used a custom-made LabView script to generate pulse waveforms with a stimulus isolation unit (Warner Scientific Inc. SIU-102) that generates pulse signals to stimulate hiPSC-CMs in the imaging chamber (Warner Scientific Inc. RC-21BRFS). Cell samples were activated with Lamda-4 light source (Sutter) with fast switching between 340 and 380 nm wavelength, and the emission signals were recorded with high-frame rate EMCCD (Andor iXon Ultra897) that was connected to a reverse fluorescence microscope (Nikon. Ti-S) with a 40X oil immersed objective (CFI Plan Fluor 40 × , NA 0.75). Signaling was captured in fast frame rate video mode (512 × 512 frame) at a speed of 50 fps. For data analysis, a custom-made script with Mat-lab was used. Transient amplitude was expressed as R340/R380.