Optical probe and instrumentation development for in vivo near-infrared fluorescence imaging of Alzheimer's disease

项目摘要/摘要 阿尔茨海默病(AD)的主要神经病理特征是细胞外淀粉样蛋白的堆积 斑块沉积和神经原纤维缠结(NFTs)的细胞内形成。使疾病研究成为可能 阿尔茨海默病临床前小鼠模型的研究进展及治疗效果 侵入性的方法对活着的大脑的这两个标志进行成像。最成熟的非侵入性成像技术 在整个动物水平上的方法是磁共振成像(MRI)、正电子发射断层扫描 (PET)和单光子发射计算机断层扫描(SPECT)。然而，只有光学技术，如近- 红外(NIR)荧光成像具有成像单个淀粉样斑块的理论空间分辨率 和NFTs。与PET和SPECT相比，近红外荧光具有使用安全、非 电离辐射。以前用于AD的近红外荧光探针大多在600-650 nm波长发射 射程。虽然这些探针可以提供低分辨率，但在活体内对AD病理负担的批量估计 为了区分AD小鼠和野生型对照组，它们缺乏提供绝对定量的能力 并且不能用于显微分辨率的非侵入性成像。实现非侵入性光学成像 通过小鼠完整的头皮和完整的头骨，荧光探针的波长需要转移到 近红外II光谱(1,000-1,700 nm)。在这些较长的波长下，组织的穿透深度是 显著增加，而由本征自发荧光引起的背景最小。在这里我们 建议通过采用两种互补的策略来开发和确定用于NIR-II成像的候选探针。 在第一种方法中，我们将修改具有现有亲和力的有希望的候选探针的化学结构 用于淀粉样斑块和NFT，以增强其结合亲和力和NIR-II荧光发射。在第二个 方法，我们将使用化学结构-活性筛选现有的&gt；650近红外荧光团的化学库 建模以及体外和活体表征。为了在体内成像我们的候选NIR-II荧光团，我们将 构建一种结合了近红外共焦和多光子显微镜的定制多模显微镜，并完全 通过仿真和实验验证来表征其性能。我们将在体内进行纵向的 在AD小鼠身上进行的成像研究，以验证我们开发的探针和仪器，并首次展示 时间无创监测AD的病理与显微分辨率。优良的科学环境 和马萨诸塞州总医院和哈佛医学院提供的世界级资源，如 以及国际公认的申请者导师的领导，以及 拟议的合作者将是成功完成拟议研究的关键。这个K01奖将 有助于申请者通过光学探头开发和体内实验方面的培训来增强其技能 在AD小鼠中进行成像，并采取下一步行动，成为一名独立的研究人员。

PROJECT SUMMARY / ABSTRACT The main neuropathological hallmarks of Alzheimer's disease (AD) are the extracellular accumulation of amyloid plaque deposits and intracellular formation of neurofibrillary tangles (NFTs). To enable the study of disease progression and the effect of therapeutics in preclinical mouse models of AD, there is an urgent need for non- invasive methods of imaging both hallmarks in the living brain. The most established non-invasive imaging approaches at the whole animal level are magnetic resonance imaging (MRI), positron emission tomography (PET), and single photon emission computed tomography (SPECT). However, only optical techniques like near- infrared (NIR) fluorescence imaging have the theoretical spatial resolution to image individual amyloid plaques and NFTs. In contrast to PET and SPECT, NIR fluorescence has the additional advantage of using safe, non- ionizing radiation. Previous NIR fluorescence probes for AD have mostly emitted in the 600-650 nm wavelength range. While these probes can be used to provide low resolution, bulk estimates of AD pathology burden in vivo and to distinguish between AD mice and wildtype controls, they lack the ability to provide absolute quantitation and cannot be used to image non-invasively with microscopic resolution. To enable non-invasive optical imaging through the intact scalp and intact skull of mice, the wavelength of the fluorescent probe needs to be shifted into the NIR-II spectrum (1,000-1,700 nm). At these longer wavelengths, the penetration depth through tissue is increased dramatically while the background caused by intrinsic autofluorescence is minimized. Here we propose to develop and identify candidate probes for NIR-II imaging by employing two complimentary strategies. In the first approach, we will modify the chemical structure of a promising candidate probe, with existing affinity for amyloid plaques and NFTs to enhance its binding affinity and NIR-II fluorescence emission. In the second approach, we will screen an existing chemical library of > 650 NIR fluorophores using chemical structure-activity modeling and in vitro and in vivo characterization. To image our candidate NIR-II fluorophores in vivo, we will build a custom multi-modal microscope combining NIR confocal and multiphoton microscopy and fully characterize its performance with simulations and experimental validation. We will conduct longitudinal, in vivo imaging studies in AD mice to validate our developed probes and instrumentation and demonstrate for the first time non-invasive monitoring of AD pathology with microscopic resolution. The excellent scientific environment and world-class resources provided by the Massachusetts General Hospital and Harvard Medical School, as well as the internationally recognized leadership of the applicant's mentors, along with the expertise of the proposed collaborators, will be key to the successful completion of the proposed research. This K01 award will be instrumental for the applicant to strengthen his skillset with training in optical probe development and in vivo imaging in AD mice and to take the next steps towards becoming an independent researcher.