A multi-plane 3-photon microscope for volume imaging in NHP cortex

项目摘要 显微镜的最新进展使体积成像成为可能--几乎同时对电路中的许多神经元进行成像-- 通过双光子(2P)激发，对相对简单行为背后的电路产生了新的见解。卷 成像技术和2P激发主要局限于小动物，如啮齿动物，包括对 小鼠皮质。在非人类灵长类动物和人类中，小鼠的皮质与新皮质不同，因此不太可能对 小鼠的皮质将足以理解皮质在人类中的作用。 在R34的应用中，我们建议建立一个多平面三光子(3P)荧光显微镜来进行体积 在猕猴身上进行成像。与2P激发不同，3P激发支持深入猕猴皮质的成像。在未来的R01 应用，我们将使用这台3P显微镜对猕猴初级视皮层进行大量成像，研究如何 运动信息集中在4B层的神经元上，并将3P显微镜与连续切片电子配对 显微镜研究颜色信息的处理，特别是在4C层β中。 我们将追求四个具体目标： -具体目标1：建造和测试多平面3P显微镜 -特定目标2：测量猕猴1-4层中约50,000个神经元的视觉调谐。 -特定目标3：通过猕猴皮质的第5层和第6层扩展多平面3P成像。 -具体目标4：免费提供硬件设计、软件和协议。

PROJECT ABSTRACT Recent advances in microscopy permit volume imaging - the near-simultaneous imaging of many neurons in a circuit - with 2-photon (2P) excitation, yielding new insights into the circuits underlying relatively simple behaviors. Volume imaging techniques and 2P excitation have been limited mainly to small animals such as rodents, including studies of mouse cortex. Mouse cortex differs from neocortex in non-human primates and humans and it is unlikely that studies of mouse cortex will be sufficient to understand the role of cortex in humans. In this R34 application, we propose to build a multi-plane 3-photon (3P) fluorescence microscope to perform volume imaging in macaques. Unlike 2P excitation, 3P excitation supports imaging deep into macaque cortex. In a future R01 application, we will use this 3P microscope to image large volumes of macaque primary visual cortex, studying how motion information is pooled across neurons in layer 4B and pairing 3P microscopy with serial-section electron microscopy to study the processing of color information, particularly in layer 4Cβ. We will pursue four specific aims: - Specific aim 1: Construct and test a multi-plane 3P microscope - Specific aim 2: Measure visual tuning of ~50,000 neurons in layers 1-4 in a macaque. - Specific aim 3: Extend multi-plane 3P imaging through layers 5 and 6 of macaque cortex. - Specific aim 4: Make hardware designs, software and protocols freely available.