MRI: Development of a Pulsed Laser Source for Deep in Vivo Imaging, a Synergy of Physics and Brain Science

这是一个开发项目，旨在构建扫描二光子和三光子显微镜，用于大脑深层成像，以支持与神经元回路分析和神经血管耦合相关的活动。利用光学方法对大脑更深处进行成像的能力是我们破译神经元解剖结构、回路功能以及神经血管功能的关键技术。光学工具与特定大脑结构的标签一起，是在体内以非侵入性或部分侵入性方式探测单细胞的几何形状和状态变量（例如电压和第二信使）以及脑脉管系统动力学的唯一手段。目前选择的体内成像方法利用双光子显微镜和 100 飞秒脉冲激光源来观察小鼠上层约 500 微米皮质的结构和动态。然而，显然需要对整个皮层深度（小鼠的 1.0 至 1.2 微米）进行成像，以确定皮层处理中完整的信息流。还需要在不挖掘覆盖组织的情况下对海马和其他皮层下结构进行更深入的成像，以及确定整个灰色和白色物质中血管控制的位点。最初提出的实验全部取决于拟议的仪器，涉及基础脑科学和生物医学的主题。基本问题围绕神经元可塑性和记忆形成，包括：运动记忆的形成，其中行为任务的学习被认为是运动皮层中相关神经元输出模式的形成的结果；将皮质中的感觉信号转化为记忆痕迹，例如通过杏仁核习得的恐惧和通过缰核诱发的抑郁；特定基因产物（称为诱导转录因子）在突触可塑性中的作用；并了解嗅球中巨大的成人神经发生如何整合到持续的嗅觉功能中。更多的应用问题涉及单独接触尼古丁在改变记忆形成基础中的作用，以及血管动力学问题，包括神经元通过皮质脉管系统控制其自身营养供应的位点，以及微梗塞对白质内细胞死亡的影响，其中有髓纤维将信息从跨越皮质地幔的感觉区域传递到运动区域。该系统的实现将允许对研究生和博士后进行最先进的体内光学成像方面的培训。加州大学圣地亚哥分校与更大的拉霍亚科学界一起，支持一个大型且高度协作的神经科学社区，其中包括研究生和研究员，他们将在全县乃至全世界的研究所追求职业生涯。他们将受到启发，思考基于大脑新视野成像能力的新实验，以及新的相关技术，特别是在尚未测量的变量的光学探针的设计方面。最后，该社区内潜在用户的高密度将促进深度成像的意想不到的改进，并可能将拟议的开发项目转变为交钥匙设计，以造福全球神经科学界。 PI 提议建造一种仪器，其设计由三个工作线程驱动，能够在整个皮层深度和更深的结构中进行两光子和三光子成像。首先是使用波长为 1.3 或 1.7 微米的 100-fs 脉冲激光，其中散射最小化，但水的吸收仍然很弱；第二是使用光学放大器来增加每个脉冲的能量并在更大的深度驱动荧光，第三是使用像差校正光学器件来抵消入射光束随着进入脑组织的深度增加而产生的畸变。

This is a development project to construct a scanning two- and three-photon microscope for deep imaging in the brain in support of activities related to neuronal circuit analysis and neurovascular coupling. The ability to image ever deeper in the brain with optical methods is a key enabling technology in our ability to decipher neuronal anatomy and circuit function as well as neurovascular function. Optical tools, together with labels of specific brain structures, are the only means to probe the geometry and state variables of single cells, e.g., voltage and second messengers, and the dynamics of brain vasculature in a noninvasive or partially invasive manner in vivo. The current method of choice for in vivo imaging makes use of two-photon microscopy with a 100-femtosecond pulsed laser sources to observe structure and dynamics throughout the upper ~ 500 micrometers of cortex of mice. Yet there is a clear need to image throughout the full depth of cortex, 1.0 to 1.2 micrometers in mice, to determine the complete flow of information in cortical processing. There is also a need to image deeper still into hippocampus and other subcortical structures without excavated overlying tissue, as well as to determine the loci of vascular control throughout gray and while matter. The initial proposed experiments, all of which depend on the proposed instrument, address topics in fundamental brain science as well biomedicine. Fundamental issues revolve around neuronal plasticity and memory formation and include: the formation of motor memories, where the learning of a behavioral task is believed to follow from the formation of patterns of correlated neuronal output in motor cortex; the transformation of sensory signals in cortex into memory traces, such as learned fear via the amygdala and induction of depression via the habenula; the role of specific gene products, known as inducible transcription factors, in synaptic plasticity; and understanding how the prodigious adult neurogenesis in the olfactory bulb is integrated into ongoing olfactory function. More applied issues concern the role of exposure to nicotine alone in changing the basis for memory formation, as well as issues in vasodynamics, including the locus for neuronal control of its own nutriment supply through the cortical vasculature and the impact of microinfarctions on cell death within the white matter, where myelinated fibers traffic information from sensory to motor areas that span the cortical mantle. Realization of this system will permit training of graduate students and postdoctoral fellows in state of the art in vivo optical imaging. UC San Diego, along with the greater La Jolla scientific community, supports a large and highly collaborative neuroscience community with graduate students and fellows who will pursue careers at institutes throughout the county, even the world. They will be inspired to think of new experiments based on the capabilities of imaging new vistas in the brain, as well as new associated technologies, particularly in the design of optical probes of yet unmeasured variables. Lastly, the high density of potential users within this community will facilitate unanticipated refinements of deep imaging and perhaps transform the proposed development project into a turn-key design for the benefit of the global neurosciences communities. The PI proposes to build an instrument, whose design is motivated by three threads of work, that enables two- and three-photon imaging throughout the full depth of cortex and into deeper structures. First is the use of 100-fs pulsed laser light at wavelengths of 1.3 or 1.7 micrometers, where scattering is minimized but absorption by water is still weak; second is the use of an optical amplifier to increase the energy per pulse and drive fluorescence at greater depths, and third is the use of aberration corrective optics to counteract distortion of the incident beam with increasing depth into brain tissue.