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，以构建一个扫描双光子和三光子显微镜，用于大脑中的深度成像，以支持与神经元电路分析和神经血管耦合相关的活动。用光学方法在大脑中更深处成像的能力是我们能够破译神经元解剖结构和电路功能以及神经血管功能的关键技术。光学工具以及特定大脑结构的标签是探测单个细胞的几何形状和状态变量的唯一手段，例如，电压和第二信使，以及在体内以非侵入性或部分侵入性方式的脑脉管系统的动力学。目前选择的体内成像方法是使用双光子显微镜和100飞秒脉冲激光源来观察小鼠皮层上部~ 500微米的结构和动力学。然而，显然需要在小鼠大脑皮层的整个深度（1.0至1.2微米）进行成像，以确定大脑皮层处理过程中的完整信息流。还需要在没有挖掘覆盖组织的情况下更深地成像到海马和其他皮质下结构，以及确定整个灰质和白质中血管控制的位点。最初提出的实验，所有这些都取决于拟议的仪器，解决基础脑科学以及生物医学的主题。基本问题围绕着神经元的可塑性和记忆的形成，包括：运动记忆的形成，其中行为任务的学习被认为是从运动皮层中相关神经元输出模式的形成而来的;皮层中的感觉信号转化为记忆痕迹，例如通过杏仁核学习恐惧和通过缰核诱导抑郁;特定的基因产物，被称为诱导转录因子，在突触可塑性中的作用;以及了解嗅球中巨大的成人神经发生如何整合到正在进行的嗅觉功能中。更多的应用问题涉及单独暴露于尼古丁在改变记忆形成基础方面的作用，以及血管动力学问题，包括神经元通过皮层血管系统控制自身营养供应的位点，以及微梗塞对白色物质内细胞死亡的影响，其中有髓鞘纤维将信息从感觉区传递到跨越皮层外套的运动区。该系统的实现将允许在最先进的体内光学成像的研究生和博士后研究员的培训。加州大学圣地亚哥分校，沿着与更大的拉霍亚科学界，支持一个大型和高度协作的神经科学社区与研究生和研究员谁将追求事业在全国各地的研究所，甚至世界。他们将受到启发，考虑基于大脑中成像新景象的能力的新实验，以及新的相关技术，特别是在设计尚未测量变量的光学探针方面。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社区。PI建议建立一个仪器，其设计是由三个线程的工作，使整个皮层的整个深度和更深的结构的双光子和三光子成像的动机。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;其次是使用光学放大器来增加每个脉冲的能量并在更深的深度驱动荧光，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失真。

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.