Optogenetic stimulation combined with optical mapping of membrane potential (Vm) and calcium transients (CaT) is a powerful electrophysiological tool. We developed a novel experimental platform in which tissue is stimulated optogenetically while Vm and CaT are imaged simultaneously. The Vm indicator is an organic dye, while the CaT indicator is genetically encoded. We used cardiac spheroids containing cardiomyocytes and fibroblasts differentiated from human induced pluripotent stem cells as model tissue. The spheroids were genetically encoded with an optogenetic actuator, CheRiff, and the calcium indicator jRCaMP1b. The Vm indicator was the organic dye RH237. CheRiff was excited using blue light (450 nm), and both RH237 and jRCaMP1b were excited using a single band of green light (either 525–575 nm or 558–575 nm). Fluorescence emission was split and imaged by two cameras (CaT: 595–665 nm; Vm: >700 nm). The spheroids were successfully stimulated optogenetically and Vm and CaT were recorded simultaneously without cross-talk using both excitation light bands. The 525–575 nm band produced higher signal-to-noise ratios than the 558–575 nm band, but caused a slight increase in tissue excitability because of CheRiff activation. The optogenetic actuator and CaT indicator are genetically encoded and can be expressed in engineered tissue constructs. In contrast, the Vm indicator is an organic dye that can stain any tissue. This system is well-suited for studying coupling between engineered tissue grafts and host tissue because the two tissue types can be stimulated independently, and tissue activation can be unambiguously attributed to either graft or host.
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