Ultra-large field two-photon microscope to image transcortical brain dynamics

描述（由申请人提供）：PI引入了新型仪器来成像，然后分析和量化神经元的全皮层电动力学和脑血管系统的类似血管动力学。该仪器所涵盖的空间尺度从细胞分辨率（1微米尺度）到整个小鼠皮层（毫米尺度）。大规模的大脑活动包括将血流重新分配到代谢需要增加的区域的神经血管问题，以及继发于血管活动的血流模式。它还涉及注意力和决策中相关神经元活动的神经学问题，多感觉处理中的远距离信号，以及更抽象的问题，如大脑中电波的模式。此外，大规模因此，所提出的仪器作为诊断跨越不同皮层区域的神经元和神经血管处理的手段获得了广泛的智力价值。我们的方法涉及到一种新型的，大视场的体内双光子显微镜的设计和实现，具有前所未有的10毫米视场，1微米的分辨率跨越字段。这足以以亚细胞分辨率对小鼠中的整个皮质套成像。我们的设计和实现必须满足复杂但并非不可逾越的挑战，即在大扫描角度上保持无像差光束，这在传统上与色差和空间像差有关。虽然在已发表的文献中关于纠正这些问题的指导有限，但我们能够以系统的方式成功地解决这些问题。我们的新型显微镜将与转基因动物的使用相结合，在转基因动物中，个体特定的细胞类型表达功能性报告基因。这将使我们能够记录特定类型的细胞-深入到软膜表面-从而为神经元和神经血管动力学提供解剖学基础。我们的方法提供了一个定性的改进，过去，全场成像技术，其中均匀染色的神经元组织进行了研究，既没有深度，也没有细胞特异性的信息。除了我们对神经元加工的科学兴趣之外 和神经血管耦合，我们独特的仪器将服务于整个系统生理学的许多其他主题的研究，包括例如发育和再生中的细胞迁移以及肿瘤发生和转移中的细胞和组织流动。工作的技术方面将通过PI及其同事在研究生和研究生暑期学校的参与得到广泛传播。

DESCRIPTION (provided by applicant): The PI introduces novel instrumentation to image and then analyze and quantify the whole- cortex electrical dynamics of neurons and the analogous vasomotor dynamics of brain vasculature. The spatial-scales encompassed by this instrument span from cell resolution - at the one-micrometer scale - to all of mouse cortex - at the then-millimeter scale. Large-scale brain activity encompasses the neurovascular issue of the redistribution of blood flow to areas of heightened metabolic need as well as patterns of blood flow secondary to vasomotor activity. It further concerns neurological issues of correlated neuronal activity in attention and decision making, distant signaling in multisensory processing and, at a more abstract level, issues such as the patterning of electrical waves in the brain. Further, large- scale Thus the proposed instrument gains broad intellectual merit as a means to diagnose neuronal and neurovascular processing that extends across different cortical areas. Our approach involves the design and realization of a novel, large-field in vivo two-photon microscope with an unprecedented ten-millimeter field of view with one micrometer resolution across the field. This is sufficient to image the entire cortical mantle in mouse with subcellular resolution. Our design and realization must meet the complex yet not insurmountable challenges of maintaining an aberration-free beam across large scan-angles, which are traditionally associated with chromatic and spatial aberrations. While there is limited guidance in the published literature on correcting such problems, we are able to successfully address these issues in a systematic way. Our novel microscopy will be combined with the use of transgenic animals in which individual specific cell types express a functional reporter. This will allow us t record from specific cell types - deep to the pial surface - and thus provide an anatomical basis for neuronal and neurovascular dynamics. Our approach provides a qualitative improvement of past, whole-field imaging techniques in which uniformly stained neuronal tissue was studied with neither depth nor cell-specific information. Beyond our scientific interests in neuronal processing and neurovascular coupling, our unique instrument will be of service to investigations of many other topics in the physiology of whole systems, including for example cell migration in development and regeneration as well as the flow of cells and tissue in tumor genesis and metastasis. Technical aspects of work will be broadly disseminated through the involvement of the PI and colleagues in graduate and post-graduate summer schools.