Dissemination of 3-photon imaging for chronic cellular imaging across species

摘要 活动敏感的荧光指示器和显微镜被证明是测量神经元的有价值的工具 活动，但大多数形式的细胞显微镜只能产生图像的神经元附近的大脑表面和 一般情况下，只有在移除了覆盖在大脑表面的组织，如骨骼之后。新皮质内有许多神经元 都是细胞显微镜所不能及的。在这里，我们提出了优化三光子(3P)激发荧光 成像是细胞显微镜的一种形式，可以对大脑进行更深层次的成像。我们的目标是获得 苍蝇、老鼠、猫和猕猴体内深层细胞的细胞信息。 在优化3P显微镜后，我们将促进我们的显微镜和其他程序的复制 神经科学实验室。我们将提供开放获取的有关我们的显微镜和 神经学家可以理解的术语中的实验程序。此外，我们将与 一个或多个显微镜制造商，以确保我们的显微镜设计的商业可用性，使 许多神经科学实验室使用3P激发以更小的侵入性深入大脑进行成像 准备工作，反复进行几天、几周或几个月。 为了达到我们的总体目标，我们将实现五个具体目标： 的具体目标1：进一步优化快速3P激发。 特定目标2：记录完整、行为正常的果蝇中枢运动回路的钙信号 黑猩猩。 特定目标3：研究小鼠新皮质的感觉信息处理和小鼠的空间记忆 海马体。 的具体目标4：研究猫和猕猴皮质深层的视觉信息处理。 的具体目标5：分享方法并促进商用快速3P显微镜的可用性。

ABSTRACT Activity-sensitive fluorescent indicators and microscopy have proven valuable tools for measuring neuronal activity, but most forms of cellular microscopy can produce images of neurons only near the brain surface and generally only after removal of tissues overlying the brain surface, such as bone. Many neurons in neocortex are out of reach of cellular microscopy. Here we propose to optimize 3-photon (3P) excitation fluorescence imaging, a form of cellular microscopy, to enable deeper imaging into the brain. Our aims are to acquire cellular information from deep cells in flies, mice, cats and macaques. After optimizing 3P microscopy, we will facilitate the duplication of our microscope and procedures in other neuroscience laboratories. We will provide open-access, detailed information on our microscope and experimental procedures in terminology that’s comprehensible to neuroscientists. In addition, we will work with one or more microscope manufacturers to ensure commercial availability of our microscope design, enabling many neuroscience laboratories to use 3P excitation to image deep into the brain with less invasive preparations, repeatedly over many days, weeks or months. To achieve our broad aims, we will pursue five specific aims:  Specific aim 1: Further optimize fast 3P excitation.  Specific aim 2: Record calcium signals from central motor circuits in intact, behaving Drosophila melanogaster.  Specific aim 3: Study sensory information processing in mouse neocortex and spatial memory in mouse hippocampus.  Specific aim 4: Study visual information processing in deep layers of cat and macaque cortex.  Specific aim 5: Share methods and facilitate availability of a commercial fast 3P microscope.