Small Quantum Dots for Super-Resolution of Neuronal Sub-Synaptic Structures

描述(申请人提供)：量子点是具有独特光学性质的荧光纳米颗粒，有可能给细胞显微镜和生物成像带来革命性的变化。这些荧光团出现的同时，荧光显微镜正在进行的一场名为超分辨率成像的革命，使分子、细胞和组织现在可以以接近单个蛋白质的分辨率进行光学成像，具有分子特异性。例如，小到神经元突触裂隙(~30 nm)的细胞结构现在可以在活细胞中动态分辨，这在以前需要通过电子显微镜进行静态、固定的细胞成像。量子点在这里有一个独特的利基市场：与荧光蛋白质和有机染料不同，它们的发射强度非常明亮，可以很容易地观察到单个分子，而且它们的发射不会光漂白。理论上，这些颗粒可以用来成像和跟踪微小突触内参与神经元-神经元通信的单个蛋白质，以揭示神经信号传递中涉及的不同的、动态变化的过程，以及阿尔茨海默氏症、帕金森氏症和中风等疾病的细胞间通信是如何中断的。然而，由于量子点体积庞大(~20 nm)和非特异性标记，极大地限制了对拥挤的神经元突触的特异性访问，阻碍了量子点用于先进的活细胞显微镜的实施。我们的初步数据显示，小尺寸量子点(~7 nm)与神经元突触的接触要大得多，比以前的大量子点要大得多。这使得对单个神经递质受体的跟踪可以持续很长时间(~1小时)。然而，这种尺寸范围的颗粒的生产仍然是一个主要问题，主要是由于它们的涂层，这有助于在溶液中胶体稳定颗粒。在尺寸、稳定性和非特异性之间有一个基本的权衡，目前的涂层还没有克服这一点。在这里，我们建议在我们以前的工作和迄今为止最好的文献结果的基础上生成一系列新的涂层。这些聚合物和配体是超致密的，通过强大的多齿结合来稳定；涂有这些新材料的量子点将使用一系列定量分析来测试光学稳定性、胶体稳定性和非特异性相互作用。我们将评估这些新粒子特定结合到 活神经元上的AMPA神经递质受体，以及通过与紧凑(但不稳定的)染料和荧光蛋白进行直接比较来保持天然受体行为的能力。这项提议是显微镜、操作学和生物物理学专家Paul Selvin教授和量子点开发和胶体合成专家Andrew Smith教授共同努力的结果。成功将为超分辨率观察疾病背后的多种细胞和分子过程打开大门，这些过程一直拒绝使用经典的分子和细胞生物学方法来理解。这些包括肿瘤细胞的趋化和转移，运动蛋白功能障碍，以及疾病中的神经元功能障碍。

DESCRIPTION (provided by applicant): Quantum dots are fluorescent nanoparticles with unique optical properties that have the potential to revolution- ize cellular microscopy and bioimaging. These fluorophores have emerged simultaneously with an ongoing revolution in fluorescence microscopy called 'super-resolution' imaging whereby molecules, cells, and tissues can now be optically imaged at resolutions approaching that of individual proteins, with molecular specificity. For example, cellular structures as small as the neuronal synaptic cleft (~30 nm) can now be resolved dynami- cally in live cells, which previously required static, fixed cell imaging through electron microscopy. Quantum dots have a unique niche here: unlike fluorescent proteins and organic dyes, their emission intensity is so bright that individual molecules can be readily observed, and their emission does not photobleach. Theoretical- ly, these particles can be used to image and track single proteins involved in neuron-neuron communication within the tiny synapse to reveal the heterogeneous, dynamically changing processes involved in neural signal- ing and how intercellular communication is disrupted in diseases such as Alzheimer's, Parkinson's and in strokes. However the implementation of quantum dots for advanced microscopy of live cells has been hindered by their bulky size (~20 nm) and non-specific labeling, which greatly restricts specific access to the crowded neuronal synapse. Our preliminary data show that small-sized quantum dots (~7 nm) have much greater ac- cess to the neuronal synapse, substantially greater than previous bulky dots. This allows tracking of individual neurotransmitter receptors for long durations (~1 hour). However the production of particles in this size range remains a major problem primarily due to their coating, which serves to stabilize the particles colloidally in solu- tion. There is simply a fundamental tradeoff between size, stability, and nonspecificity that has yet to be over- come with current coatings. Here we propose to generate a new series of coatings based on our previous work and the best literature results to date. These polymers and ligands are ultra-compact and stabilized by strong multidentate binding; quantum dots coated with these new materials will be tested for optical stability, colloidal stability, and nonspecific interactions using a battery of quantitative assays. We will assess the capacity of these new particles to bind specifically to the AMPA neurotransmitter receptor on living neurons and the capac- ity to preserve native receptor behavior through direct comparisons with compact (but unstable) dyes and fluo- rescent proteins. This proposal is a collaborative effort between Prof. Paul Selvin, an expert in microscopy, op- tics, and biophysics, and Prof. Andrew Smith, an expert in quantum dot development and colloidal synthesis. Success will open the door to super-resolution observation of a multitude of cellular and molecular processes underlying disease that have resisted understanding using classical molecular and cellular biology approaches. These include tumor cell chemotaxis and metastasis, motor protein dysfunction, and neuronal dysfunction in diseases.