CAREER: Multimodal Approach for Label-free Imaging of Lipidomic Changes in Brain

这个CAREER项目的目标是开发一种方法来成像脑细胞和组织中的脂质（脂肪酸）。 这种能力是必需的，因为脂质有助于神经生成和冲动传导，并且它们是必需的信号分子和能量来源。该方法将在与阿尔茨海默病（AD）相关的神经元细胞和组织样本上进行验证。目前AD的诊断方法要么是侵入性的、昂贵的、低分辨率的、低灵敏度的，要么是主观的，并且不能探测复杂的分子作用到研究神经退行性疾病所需的水平。这些限制将通过结合现有的两种显微镜技术，使精度测量深度不可能与目前的技术来解决。该技术的发展将促进我们对AD病理学的理解，并可能导致AD的早期检测和更好的治疗方法。职业项目的研究目标与教育目标相结合，通过当地博物馆的合作，吸引初中和高中学生。此外，该项目将涉及来自当地社区学院，少数民族和代表性不足的群体在大脑研究的学生。研究者的长期目标是开发一个二次谐波发生（SHG）和拉曼为基础的显微内窥镜探头系统沿着统计分析软件，以执行脂质和代谢物在亚毫秒的时间分辨率在体内微创成像。为了实现这一目标，该CAREER项目的重点是开创一种多模式无标记方法，以阐明脑细胞和组织中脂质的空间分布，从而能够探测到突触水平（纳米级）的复杂分子作用。 一旦开发出来，3D成像平台将用于评估阿尔茨海默病（AD）小鼠模型中蛋白质和脂质沿着神经元膜的动态，这可能会提高对AD发病机制的理解。 本课题的主要研究目标是：（1）建立一套先进的非线性光学显微镜系统，建立一种偏振激活快速开关的二次谐波产生（PAR-SHG）方法，分子定位精度为20 nm，深度为1 mm;（2）利用时间─解析的高分辨率SHG成像分子穿过单细胞膜的吸附和运输，并实现新的光学生物标志物识别方法来进行神经元细胞的定量代谢谱分析;和（3）将高分辨率SHG与拉曼显微术相结合来进行来自AD小鼠模型的体外和体内样品的脂质组学成像。从长远来看，这种变革性的方法将能够对活体大脑进行超分辨率体内成像，以分子分辨率研究亚细胞过程。该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估的支持。

The objective of this CAREER project is to develop a method to image lipids (fatty acids) in brain cells and tissues. This capability is needed because lipids contribute to nerve generation and impulse conduction, and they are essential signaling molecules and sources of energy. The method will be demonstrated on neuronal cell and tissue samples relevant to Alzheimer’s Disease (AD). Current diagnosis methods of AD are either invasive, expensive, low-resolution, low-sensitivity, or subjective and are not capable of probing complex molecular actions down to the level needed to study neurodegenerative diseases. These limitations will be addressed by combining two existing microscopy techniques that enable precision measurements at depths not possible with current techniques. The development of the proposed technology will advance our understanding of the pathology of AD and could lead to early detection as well as better therapeutics for AD. The research objectives of the CAREER project are integrated with educational objectives to engage middle school and high school students by partnering through a local museum. Further, the project will involve students from local community colleges, minority, and underrepresented groups on brain research.The long-term goal of the Investigator is to develop a Second Harmonic Generation (SHG) and Raman-based micro-endoscopy probe system along with statistical analysis software to perform minimally invasive imaging of lipids and metabolites in vivo at sub-millisecond time resolution. Towards this goal, the focus of this CAREER project is on pioneering a multimodal label-free approach to elucidate the spatial distribution of lipids in brain cells and tissues that will enable probing of complex molecular actions down to the synaptic level (nanoscale). Once developed, the 3D imaging platform will be used to assess the dynamics of proteins and lipids along the neuronal membrane in a mouse model of Alzheimer's Disease (AD), which could lead to improved understanding of AD pathogenesis. The Research Plan is organized under three objectives: (1) Develop a state-of-the-art nonlinear microscope system and establish a polarization activated rapid (PAR) switching of second harmonic generation (SHG) / PAR-SHG method with localize molecules with 20 nm accuracy, depth 1mm; (2) Utilize time-resolved high resolution SHG to image the adsorption and transport of molecules across the membrane of a single cell and to implement a novel optical biomarker identification method to perform quantitative metabolic profiling of neuronal cells; and (3) Combine high resolution SHG with Raman microscopy to perform lipidomic imaging of in vitro and in vivo samples from an AD mouse model. In the long term, the transformative approach will be able to perform super resolution in vivo imaging of living brain to study sub-cellular processes at molecular resolution..This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.