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

摘要 活性敏感的荧光指示剂和显微镜已被证明是测量神经元 活动，但大多数形式的细胞显微镜只能产生大脑表面附近的神经元图像， 通常仅在去除覆盖脑表面的组织如骨之后。新皮层中的许多神经元 是细胞显微镜无法企及的在这里，我们建议优化3-光子（3 P）激发荧光 成像，细胞显微镜的一种形式，使更深入的成像到大脑。我们的目标是获得 从果蝇、老鼠、猫和猕猴的深层细胞中提取细胞信息。 优化3 P显微镜后，我们将方便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，详细的信息，我们的显微镜， 用神经科学家能理解的术语来描述实验过程。此外，我们还将与 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， 许多神经科学实验室使用3 P激发来深入大脑成像， 准备工作，重复了许多天，几周或几个月。 为了实现我们的广泛目标，我们将追求五个具体目标： 具体目标1：进一步优化快速3 P激发。 具体目标2：记录来自完整、行为正常的果蝇中枢运动回路的钙信号 黑腹菌 具体目标3：研究小鼠新皮层的感觉信息加工和小鼠的空间记忆 海马体。 具体目标4：研究猫和猕猴皮层深层的视觉信息处理。 具体目标5：分享方法并促进商业快速3 P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.