Background Optical mapping of transmembrane voltage and intracellular calcium is a powerful tool for investigating cardiac physiology and pathophysiology. However, simultaneous dual mapping of two fluorescent probes remains technically challenging. We introduce a novel, easy-to-use approach that requires a path splitter, single camera and excitation light to simultaneously acquire voltage and calcium signals from whole heart preparations, which can be applied to other physiological models - including neurons and isolated cardiomyocytes. Results Complementary probes were selected that could be excited with a single wavelength light source. Langendorff-perfused hearts (rat, swine) were stained and imaged using a sCMOS camera outfitted with an optical path splitter to simultaneously acquire two emission fields at high spatial and temporal resolution. Voltage (RH237) and calcium (Rhod2) signals were acquired concurrently on a single sensor, resulting in two 384 × 256 images at 814 frames per second. At this frame rate, the signal-to-noise ratio was 47 (RH237) and 85 (Rhod2). Imaging experiments were performed on small rodent hearts, as well as larger pig hearts with sufficient optical signals. In separate experiments, each dye was used independently to assess crosstalk and demonstrate signal specificity. Additionally, the effect of ryanodine on myocardial calcium transients was validated - with no measurable effect on the amplitude of optical action potentials. To demonstrate spatial resolution, ventricular tachycardia was induced - resulting in the novel finding that spatially discordant calcium alternans can be present in different regions of the heart, even when electrical alternans remain concordant. The described system excels in providing a wide field of view and high spatiotemporal resolution for a variety of cardiac preparations. Conclusions We report the first multiparametric mapping system that simultaneously acquires calcium and voltage signals from cardiac preparations, using a path splitter, single camera and excitation light. This approach eliminates the need for multiple cameras, excitation light patterning or frame interleaving. These features can aid in the adoption of dual mapping technology by the broader cardiovascular research community, and decrease the barrier of entry into panoramic heart imaging, as it reduces the number of required cameras.

中文翻译：

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灯光、摄像头、路径分离器：一种使用单个摄像头真正同时进行心脏双光学映射的新方法。

背景跨膜电压和细胞内钙的光学映射是研究心脏生理学和病理生理学的有力工具。然而，两个荧光探针的同时双重映射在技术上仍然具有挑战性。我们介绍了一种新的、易于使用的方法，该方法需要路径分离器、单相机和激发光来同时从整个心脏制剂中获取电压和钙信号，这可以应用于其他生理模型 - 包括神经元和分离的心肌细胞。结果选择了可以用单波长光源激发的互补探针。Langendorff 灌注心脏（大鼠、猪）使用配备光路分离器的 sCMOS 相机进行染色和成像，以同时以高空间和时间分辨率获取两个发射场。在单个传感器上同时采集电压 (RH237) 和钙 (Rhod2) 信号，从而以每秒 814 帧的速度生成两张 384 × 256 图像。在此帧速率下，信噪比为 47 (RH237) 和 85 (Rhod2)。在小型啮齿动物心脏以及具有足够光信号的较大猪心脏上进行了成像实验。在单独的实验中，每种染料都独立用于评估串扰并证明信号特异性。此外，证实了兰尼定对心肌钙瞬变的影响——对光学动作电位的幅度没有可测量的影响。为了证明空间分辨率，诱发室性心动过速 - 导致新发现空间不一致的钙交替存在于心脏的不同区域，即使电交替保持一致。所描述的系统擅长为各种心脏准备提供宽视野和高时空分辨率。结论 我们报告了第一个使用路径分离器、单相机和激发光同时从心脏准备中获取钙和电压信号的多参数映射系统。这种方法消除了对多个相机、激发光图案化或帧交错的需要。这些功能可以帮助更广泛的心血管研究界采用双映射技术，并降低进入全景心脏成像的障碍，因为它减少了所需的摄像机数量。所描述的系统擅长为各种心脏准备提供宽视野和高时空分辨率。结论 我们报告了第一个使用路径分离器、单相机和激发光同时从心脏准备中获取钙和电压信号的多参数映射系统。这种方法消除了对多个相机、激发光图案化或帧交错的需要。这些功能可以帮助更广泛的心血管研究界采用双映射技术，并降低进入全景心脏成像的障碍，因为它减少了所需的摄像机数量。所描述的系统擅长为各种心脏准备提供宽视野和高时空分辨率。结论 我们报告了第一个使用路径分离器、单相机和激发光同时从心脏准备中获取钙和电压信号的多参数映射系统。这种方法消除了对多个相机、激发光图案化或帧交错的需要。这些功能可以帮助更广泛的心血管研究界采用双映射技术，并降低进入全景心脏成像的障碍，因为它减少了所需的摄像机数量。单相机和激发光。这种方法消除了对多个相机、激发光图案化或帧交错的需要。这些功能可以帮助更广泛的心血管研究界采用双映射技术，并降低进入全景心脏成像的障碍，因为它减少了所需的摄像机数量。单相机和激发光。这种方法消除了对多个相机、激发光图案化或帧交错的需要。这些功能可以帮助更广泛的心血管研究界采用双映射技术，并降低进入全景心脏成像的障碍，因为它减少了所需的摄像机数量。