Large-scale voltage imaging in the brain using targeted illumination
Large-scale voltage imaging in the brain using targeted illumination
复制标题
DOI：
10.1101/2021.04.05.438451
复制
发表时间：
2021-04
期刊：
影响因子：
--
通讯作者：
Sheng Xiao;E. Lowet;H. Gritton;Pierre Fabris;Yangyang Wang;J. Sherman;Rebecca A. Mount;Hua-an Tseng;H. Man;J. Mertz;Xue Han
中科院分区：
文献类型：
--
作者：
Sheng Xiao;E. Lowet;H. Gritton;Pierre Fabris;Yangyang Wang;J. Sherman;Rebecca A. Mount;Hua-an Tseng;H. Man;J. Mertz;Xue Han
遗传编码的电压指标的最新进展，使光学成像的动作电位和阈下膜电压动态从单个神经元在哺乳动物大脑。为了进行高速电压成像，宽视野显微镜仍然是一个重要的工具，用于记录活动，从许多神经元同时在一个大的解剖区域。然而，光学切片的缺乏使得宽视场显微镜更容易受到背景信号污染，并且到目前为止，使用完全遗传编码的电压指示器的电压成像仍然限于在有限的视场内同时对几个细胞进行采样。在这里，我们展示了一种策略，大规模的电压成像，使用完全遗传编码的电压指标SomArchon和有针对性的照明。我们实现了一个简单的，低成本的数字化的照明设备为基础的有针对性的照明策略，以限制照明感兴趣的细胞，并系统地量化了这种显微镜设计的改进理论和实验与SomArchon表达神经元在单层细胞培养物和清醒的小鼠的大脑。我们发现，与宽场照明相比，靶向照明增加了SomArchon信号对比度，并减少了大脑中的背景交叉污染。这种改进允许降低照明强度，从而减少荧光光漂白和延长成像持续时间。当与高速大面积sCMOS相机结合时，我们在几分钟内同时对大脑中数十个尖峰神经元进行常规成像。因此，本文所述的具有集成靶向照明系统的宽视场显微镜设计为行为动物中的大型神经元群体的电压成像分析提供了一种简单的解决方案。
Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold membrane voltage dynamics from single neurons in the mammalian brain. To perform high speed voltage imaging, widefield microscopy remains an essential tool for recording activity from many neurons simultaneously over a large anatomical area. However, the lack of optical sectioning makes widefield microscopy more prone to background signal contamination, and thus far voltage imaging using fully genetically encoded voltage indicators remains limited to simultaneous sampling of a few cells over a restricted field-of-view. We here demonstrate a strategy for large scale voltage imaging using the fully genetically encoded voltage indicator SomArchon and targeted illumination. We implemented a simple, low-cost digital micromirror device based targeted illumination strategy to restrict illumination to the cells of interest, and systematically quantified the improvement of this microscopy design theoretically and experimentally with SomArchon expressing neurons in single layer cell cultures and in the brains of awake mice. We found that targeted illumination, in comparison to widefield illumination, increased SomArchon signal contrast and reduced background cross-contamination in the brain. Such improvement permitted the reduction of illumination intensity, and thus reduced fluorescence photobleaching and prolonged imaging duration. When coupled with a high-speed, large area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in the brain. Thus, the widefield microscopy design with an integrated targeted illumination system described here offers a simple solution for voltage imaging analysis of large neuron populations in behaving animals.