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

描述（由申请人提供）：PI引入新的仪器来成像，然后分析和量化神经元的全皮层电动力学和类似的脑血管血管运动动力学。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（当时为毫米尺度）。大范围的脑活动包含了血液再分配到代谢需要区域的神经血管问题，以及继发于血管舒缩活动的血液流动模式。它进一步关注注意力和决策过程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，以及在更抽象的层面上，诸如大脑中电波模式的问题。此外，该仪器作为一种诊断跨越不同皮层区域的神经元和神经血管处理的手段，具有广泛的智力价值。我们的方法包括设计和实现一种新颖的，大视场的体内双光子显微镜，具有前所未有的10毫米视场和一微米的分辨率。这足以以亚细胞分辨率成像小鼠的整个皮质地幔。我们的设计和实现必须满足复杂但并非不可克服的挑战，即在大扫描角度上保持无像差的光束，这通常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。我们的新显微镜将与转基因动物的使用相结合，其中个体特定细胞类型表达功能报告。这将使我们能够从特定的细胞类型记录-深层到枕部表面-从而为神经元和神经血管动力学提供解剖学基础。我们的方法对过去的全场成像技术进行了定性改进，在全场成像技术中，均匀染色的神经元组织既没有深度也没有细胞特异性信息。超出了我们对神经处理的科学兴趣

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.