Large-scale deep tissue voltage imaging with targeted illumination confocal microscopy.
Large-scale deep tissue voltage imaging with targeted illumination confocal microscopy.
复制标题
使用靶向照明共聚焦显微镜进行大规模深层组织电压成像。
DOI：
10.1101/2023.07.21.548930
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Mertz,Jerome
中科院分区：
文献类型：
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作者：
Xiao,Sheng;Cunningham,WilliamJ;Kondabolu,Krishnakanth;Lowet,Eric;Moya,MariaV;Mount,Rebecca;Ravasio,Cara;Economo,MichaelN;Han,Xue;Mertz,Jerome
基因编码电压指示器的进步使得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破坏了信噪比，又引起细胞之间的串扰，使得在密集标记的组织中进行可靠的体内成像极具挑战性。我们描述了一种显微镜，它结合了靶向照明和共焦门控的独特优势，同时还最大限度地提高了信号检测效率。由此产生的信噪比和串扰减少方面的好处通过实验和理论上进行了量化。我们的显微镜提供了一种多功能解决方案，可实现大尺度和穿透深度的高保真体内电压成像，我们在各种成像条件和不同的基因编码电压指示剂类别中进行了演示。
Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. 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 the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo 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 signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes.