Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 μm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200-250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.

中文翻译：

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使用超快速基于AOD的随机访问双光子显微镜，对处于清醒状态的大脑内整个神经元的感觉诱发活动进行全面成像。

确定神经元如何转换突触输入并在动作电位（AP）触发输出中编码信息是理解树突整合，神经转换和编码所必需的。使用传统的基于检流计镜的激光扫描显微镜在体内对包括复杂的树突状乔木的3D体积的大脑进行成像的速度限制已阻碍了在单个神经元突触输入和体细胞AP的整个补充中完全捕获活动的荧光传感器。为了解决这个问题，我们开发了一种两光子显微镜，该显微镜通过使用无惯性声光偏转器（AOD）进行激光束定位来实现高速扫描，从而可以对数百至数千个点进行随机访问采样兴趣仅限于预定的神经元结构，避免浪费扫描周围的细胞外组织。该系统能够对完整和清醒的脊椎动物大脑中单个神经元的活动进行全面成像。在这里，我们展示了使用单细胞电穿孔标记的红色空间填充荧光团以确定树突状乔木形态，并用钙传感器jGCaMP7s或谷氨酸传感器iGluSnFR标记的白细胞非洲爪蟾t脑中的大脑皮质神经元成像。神经活动。使用离散的兴趣点扫描，我们获得了3 Hz的采样率，以2μm的分辨率对整个乔木进行饱和采样，获得了6 Hz的采样率，依次采样了包含树突状乔木和躯体的3个体积，以及200-250 Hz的扫描单个平面通过树突状乔木。