Grewe Benjamin F, Helmchen Fritjof
Cold Spring Harb Protoc. 2014 Jun 2;2014(6):618-29. doi: 10.1101/pdb.prot081778.
Two-photon calcium imaging of neuronal populations allows optical measurements of spiking activity in living animals. However, laser-scanning microscopes with galvanometric scan mirrors are too slow to capture population activity on a millisecond timescale. This protocol describes a two-photon microscope that is based on two-dimensional laser scanning with acousto-optic deflectors (AODs), enabling high-speed in vivo recording of neuronal population activity at temporal resolutions of several hundred hertz. The detailed construction plan of the AOD-based microscope is accompanied by equally detailed optimization procedures. We also introduce a novel random-access pattern scanning (RAPS) technique for high-speed in vivo measurements of neuronal population activity. AOD-based RAPS can measure calcium transients in neocortical neuronal populations, revealing spike trains with near-millisecond precision. The current limitations of the AOD-based microscope are discussed, and we provide an outlook of its future applications.
对神经元群体进行双光子钙成像可实现对活体动物中神经元放电活动的光学测量。然而，配备振镜扫描镜的激光扫描显微镜速度过慢，无法在毫秒时间尺度上捕捉群体活动。本方案描述了一种基于声光偏转器（AOD）进行二维激光扫描的双光子显微镜，能够以数百赫兹的时间分辨率对神经元群体活动进行高速活体记录。基于AOD的显微镜详细构建计划还配有同样详细的优化程序。我们还介绍了一种用于神经元群体活动高速活体测量的新型随机访问模式扫描（RAPS）技术。基于AOD的RAPS可测量新皮质神经元群体中的钙瞬变，以近毫秒的精度揭示尖峰序列。文中讨论了基于AOD的显微镜当前的局限性，并展望了其未来应用。
