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

这个职业项目的目标是开发一种对脑细胞和组织中的脂肪(脂肪酸)进行成像的方法。这种能力是必需的，因为脂质有助于神经生成和脉冲传导，而且它们是必不可少的信号分子和能量来源。该方法将在与阿尔茨海默病(AD)相关的神经细胞和组织样本上进行演示。目前AD的诊断方法要么是侵入性的、昂贵的、低分辨率的、低敏感性的，要么是主观的，不能探测复杂的分子作用，直到研究神经退行性疾病所需的水平。这些限制将通过结合两种现有的显微镜技术来解决，这两种技术能够在当前技术无法实现的深度进行精确测量。拟议技术的发展将促进我们对AD病理的理解，并可能导致AD的早期发现和更好的治疗方法。该职业项目的研究目标与教育目标相结合，通过与当地博物馆合作来吸引初中生和高中生。此外，该项目将涉及来自当地社区大学的学生、少数族裔和未被充分代表的群体进行脑研究。调查者的长期目标是开发第二次谐波产生(SHG)和基于拉曼的显微内窥镜探头系统以及统计分析软件，以亚毫秒的时间分辨率对体内脂质和代谢物进行微创成像。为了实现这一目标，这个职业项目的重点是开创一种多模式的无标记方法，以阐明脑细胞和组织中脂类的空间分布，从而能够探测复杂的分子作用，一直延伸到突触水平(纳米级)。一旦开发完成，3D成像平台将用于评估阿尔茨海默病(AD)小鼠模型神经细胞膜上蛋白质和脂质的动态变化，这可能有助于更好地理解AD的发病机制。研究计划分为三个目标：(1)开发最先进的非线性显微镜系统，建立偏振激活二次谐波开关(PAR)快速开关二次谐波(SHG)/PAR-SHG方法，定位分子的精度为20 nm，深度为1 mm；(2)利用时间分辨的高分辨率SHG成像分子在单细胞膜上的吸附和传输，并实施一种新颖的光学生物标记物识别方法来对神经细胞的代谢进行定量描述；(3)将高分辨率SHG与拉曼显微镜相结合，对AD小鼠模型的体外和体内样品进行脂体成像。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成像，以分子分辨率研究亚细胞过程。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.