Data_Sheet_1_Fast Optical Investigation of Cardiac Electrophysiology by Parallel Detection in Multiwell Plates.pdf

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells. MULTIPLE combines optical detection of action potentials using a red-shifted voltage-sensitive fluorescent dye (di-4-ANBDQPQ) with optical stimulation, employing optogenetic actuators, to ensure excitation of cardiomyocytes at constant rates. MULTIPLE was first characterized in terms of interwell uniformity of the illumination intensity and optical detection performance. Then, it was applied for probing action potential features in HL-1 cells (i.e., mouse atrial myocyte-like cells) stably expressing the blue light-activatable cation channel CheRiff. Under proper stimulation conditions, we were able to accurately measure action potential dynamics across a 24-well plate with variability across the whole plate of the order of 10%. The capability of MULTIPLE to detect action potential changes across a 24-well plate was demonstrated employing the selective Kv11.1 channel blocker (E-4031), in a dose titration experiment. Finally, action potential recordings were performed in spontaneous beating human induced pluripotent stem cell derived cardiomyocytes following pharmacological manipulation of their beating frequency. We believe that the simplicity of the presented optical scheme represents a valid complement to sophisticated and expensive state-of-the-art optical systems for high-throughput cardiac electrophysiological investigations.

当前用于快速表征心脏电生理学技术的手段，通常采用光学技术对置于多孔板中的单个细胞或细胞单层进行动作电位特征的操控与监测。通过在完全自动化的荧光显微镜下对每个孔位进行逐个分析，通常可以实现高速的探究能力。在此，我们介绍了一种替代性的、成本效益高的光学方法（MULTIPLE），该方法利用高功率LED阵列对培养板进行全局照明，并采用sCMOS传感器对多个孔位中发出的荧光进行并行检测。MULTIPLE结合了使用红移电压敏感荧光染料（di-4-ANBDQPQ）进行动作电位的光学检测和光学刺激，通过光遗传学驱动器确保心肌细胞以恒定的速率兴奋。MULTIPLE首先在照明强度的孔间均匀性和光学检测性能方面进行了表征。随后，它被应用于探测稳定表达蓝色光激活阳离子通道CheRiff的HL-1细胞（即小鼠心房肌样细胞）的动作电位特征。在适当的刺激条件下，我们能够精确测量整个24孔板上的动作电位动力学，整个板上的变异量级为10%。通过使用选择性Kv11.1通道阻断剂（E-4031）进行剂量滴定实验，证明了MULTIPLE在24孔板检测动作电位变化的能力。最后，在经过药物调节其节律频率后，对自发性搏动的人源诱导多能干细胞来源的心肌细胞进行了动作电位记录。我们认为，所提出的简单光学方案是对复杂且昂贵的先进光学系统进行高通量心脏电生理学研究的有效补充。