Peng Luxin, Zou Peng
College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871, China.
PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing, 100871, China.
Chem Biomed Imaging. 2023 Jul 20;1(5):448-460. doi: 10.1021/cbmi.3c00054. eCollection 2023 Aug 28.
Membrane potential and its fluctuation are fundamental biophysical phenomena essential to cellular activities and functions. Compared to traditional electrode-based techniques, the optical recording via developed genetically encoded voltage indicators (GEVIs) offers a combination of noninvasiveness, high spatial resolution, and increased measurement throughput. However, its application is limited by the insufficient acquisition rate and time accuracy of the camera. Here we design and apply a stroboscopic illumination scheme to boost the temporal resolution of voltage imaging, while simultaneously eliminating the artifacts caused by nonsynchronized exposure in the rolling-shutter mode. We demonstrate that commonly used GEVIs are compatible with stroboscopic voltage imaging (SVI), and our SVI scheme offers a 5-fold faster acquisition frame rate than that of conventional continuous illumination. The GEVIs tested maintain high sensitivities in the SVI mode, supporting faithful reports of intracellular depolarization waveform and intercellular gap junction-mediated depolarization coupling in human embryonic kidney 293T (HEK 293T) cell populations. SVI allows resolving the action potential (AP) waveform with less distortion and mapping action potential initiation and propagation dynamics in cultured neurons in kilohertz, beyond the restriction from the camera in the field of view.
膜电位及其波动是细胞活动和功能所必需的基本生物物理现象。与传统的基于电极的技术相比，通过开发的基因编码电压指示剂（GEVIs）进行光学记录具有非侵入性、高空间分辨率和更高的测量通量等优点。然而，其应用受到相机采集速率和时间精度不足的限制。在此，我们设计并应用了一种频闪照明方案来提高电压成像的时间分辨率，同时消除滚动快门模式下非同步曝光所产生的伪影。我们证明常用的GEVIs与频闪电压成像（SVI）兼容，并且我们的SVI方案提供的采集帧率比传统连续照明快5倍。所测试的GEVIs在SVI模式下保持高灵敏度，支持对人胚肾293T（HEK 293T）细胞群体中细胞内去极化波形和细胞间缝隙连接介导的去极化耦合进行可靠报告。SVI能够以较小的失真解析动作电位（AP）波形，并以千赫兹级映射培养神经元中动作电位的起始和传播动态，突破了视野内相机的限制。
