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On cellular or tissue level, electrophysiology studies during light stimulation with shaped light can potentially enable the design of effective stimulation strategies towards future cardiac therapies. In this work, we explore the use of optogenetics in probing the electrophysiology of a functional cardiac syncytium. A holographic technique with the use of a spatial light modulator allows beam shaping and precise spatiotemporal control of the origin of action potential in a cardiac-like cell line monolayer. Depolarization was performed by using a blue laser (488 nm) incident to a reflective ferroelectric liquid crystal on silicon spatial light modulator (FLCOS-SLM) which generates multiple foci on to cardiomyocyte-like cells (HL1 line) expressing the light-gated protein Channelrhodopsin-2 (ChR2). The calcium-sensitive fluorophore Cal-630 was used to visualize the cellular electrical activity and thus can be combined with ChR2 as an all-optical method to stimulate and visualize electrophysiology of cardiac-like cells in a non-contact manner. We generated single or multiple foci each with spot size of ~5 µm for optogenetic stimulation. The probability of triggering an action potential, which we refer to as excitation probability, is highly dependent on the laser intensity. The threshold intensity for a single beamlet, where approximately 50% of the laser pulses successfully trigger an action potential, is ~4 µW/μm² (n=5 regions, 5 cells analyzed per region, performed in triplicates) at frequency of 1 Hz and pulse duration of 10 ms. We also demonstrate spatial summation of membrane potential in HL1-ChR2. Generated multiple foci can render the excited cells as individual inputs to elicit action potential in a syncytium. We demonstrate the dependence of the excitation probability on the foci gap distance. Two foci, each with sub-threshold intensity of 2 µW/μm² were generated. At spacing of 50 µm, the median excitation probability increases compared to single spot stimulation with the same intensity.
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