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，旨在构建一个扫描双光子和三光子显微镜，用于大脑深部成像，以支持与神经元回路分析和神经血管耦合相关的活动。利用光学方法在大脑深处成像的能力是我们破译神经元解剖和电路功能以及神经血管功能的关键技术。光学工具，加上特定大脑结构的标签，是在体内以非侵入性或部分侵入性的方式探测单个细胞的几何和状态变量的唯一手段，例如电压和第二信使，以及脑血管的动力学。目前的活体成像方法是利用100飞秒脉冲激光源的双光子显微镜观察小鼠皮层上~ 500微米的结构和动力学。然而，为了确定皮质处理过程中的完整信息流，我们显然需要对皮层的整个深度进行成像，在小鼠中为1.0至1.2微米。此外，还需要在不挖掘覆盖组织的情况下，对海马体和其他皮层下结构进行更深入的成像，并确定整个灰质和白质的血管控制位点。最初提议的实验，所有这些都依赖于提议的仪器，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在运动皮层相关神经元输出模式的形成之后进行的；大脑皮层的感觉信号转化为记忆痕迹，如通过杏仁核习得的恐惧和通过缰核诱导的抑郁；特定基因产物的作用，被称为诱导转录因子，在突触可塑性；以及了解嗅球中巨大的成人神经发生是如何整合到持续的嗅觉功能中的。更多的应用问题涉及尼古丁单独暴露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置，以及微梗死对白质内细胞死亡的影响，在白质中，有髓鞘纤维将信息从感觉区传递到跨越皮质套的运动区。该系统的实现将允许培养研究生和博士后在最先进的体内光学成像。加州大学圣地亚哥分校，连同更大的拉霍亚科学社区，支持一个大型和高度协作的神经科学社区，研究生和研究员将在全县甚至世界各地的研究所追求职业生涯。他们将受到启发，想到新的实验，这些实验基于对大脑新远景的成像能力，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，该社区内高密度的潜在用户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对全球神经科学界有利的交钥匙设计。PI计划建造一种仪器，其设计是由三个工作线程驱动的，可以在整个皮层深度和更深的结构中进行双光子和三光子成像。第一种是使用波长为1.3或1.7微米的100秒脉冲激光，这种激光散射最小，但水的吸收仍然很弱；第二种是使用光学放大器来增加每个脉冲的能量，并在更深的深度上驱动荧光，第三种是使用像差校正光学来抵消入射光束随着进入脑组织深度的增加而产生的畸变。

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.