Xiao Sheng, Cunningham William J, Kondabolu Krishnakanth, Lowet Eric, Moya Maria V, Mount Rebecca, Ravasio Cara, Economo Michael N, Han Xue, Mertz Jerome
Department of Biomedical Engineering, Boston University, Boston MA 02215.
Neurophotonics Center, Boston University, Boston MA, 02215.
bioRxiv. 2023 Jul 21:2023.07.21.548930. doi: 10.1101/2023.07.21.548930.
Voltage imaging with cellular specificity has been made possible by the tremendous advances in genetically encoded voltage indicators (GEVIs). However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines signal-to-noise ratio (SNR) and induces crosstalk between cells, making reliable imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating, while also maximizing signal detection efficiency. The resulting benefits in SNR and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different GEVI classes.
基因编码电压指示器（GEVIs）的巨大进展使具有细胞特异性的电压成像成为可能。然而，电压成像所需的千赫兹速率会导致信号微弱。此外，离焦荧光和组织散射产生的背景既会降低信噪比（SNR），又会诱导细胞间的串扰，使得在密集标记组织中进行可靠成像极具挑战性。我们描述了一种显微镜，它结合了靶向照明和共聚焦门控的独特优势，同时还能最大化信号检测效率。通过实验和理论对由此在降低SNR和串扰方面带来的益处进行了量化。我们的显微镜为在大尺度和穿透深度上实现高保真电压成像提供了一种通用解决方案，我们在广泛的成像条件和不同的GEVI类别中都展示了这一点。
