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

用于神经元亚突触结构超分辨率的小量子点

• 批准号: 8804970
• 负责人: PAUL R SELVIN
• 金额: $ 18.89万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8804970
• 关键词: AMPA Receptors; Alzheimer' s Disease; Behavior; Binding; Biological; Biological Assay; Biophysics; Biotinylation; Caliber; Cancer Diagnostics; Cells; Cellular Structures; Charge; Chemotaxis; Clinical Medicine; Communication; Communities; Complex; Confined Spaces; Crowding; Data; Development; Dimensions; Disease; Drops; Dyes; Electron Microscopy; Ensure; Event; Figs - dietary; Fluorescence Microscopy; Fluorescent Dyes; Functional disorder; Genetic; Health; Hour; Hydroxyl Radical; Image; Immunoglobulin Fragments; Individual; Label; Learning; Letters; Life; Ligands; Literature; Measures; Memory; Microscopy; Modification; Molecular; Molecular Weight; Molecular and Cellular Biology; Morphologic artifacts; Motor; Neoplasm Metastasis; Nerve; Neurologic; Neuronal Dysfunction; Neurons; Neurosciences; Neurotransmitter Receptor; Optics; Parkinson Disease; Photobleaching; Polymers; Process; Production; Property; Proteins; Quantum Dots; Reporting; Research Personnel; Resolution; Semiconductors; Series; Signal Transduction; Site; Solutions; Source; Specificity; Streptavidin; Stroke; Structure; Sulfhydryl Compounds; Surface; Synapses; Synaptic Cleft; Techniques; Testing; Therapeutic; Tissues; Work; base; bioimaging; cellular imaging; density; fluorophore; globular protein; intercellular communication; meetings; millisecond; nanobodies; nanocrystal; nanoparticle; neoplastic cell; neurotransmission; next generation; novel; particle; receptor; research study; single molecule; small molecule; stability testing; success; tool

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.

描述（由申请人提供）：量子点是具有独特光学特性的荧光纳米粒子，有可能彻底改变细胞显微镜和生物成像。这些荧光团的出现与荧光显微镜领域正在进行的一场名为“超分辨率”成像的革命同时出现，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，并具有分子特异性。例如，像神经元突触间隙（~30 nm）这样小的细胞结构现在可以在活细胞中动态解析，而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：与荧光蛋白和有机染料不同，它们的发射强度非常明亮，可以很容易地观察到单个分子，并且它们的发射不会光漂白。理论上，这些粒子可用于成像和跟踪微小突触内参与神经元间通讯的单个蛋白质，以揭示神经信号传导中涉及的异质、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中细胞间通讯如何被破坏。然而，量子点在活细胞先进显微镜中的应用因其体积大（~20 nm）和非特异性标记而受到阻碍，这极大地限制了对拥挤的神经元突触的特异性访问。我们的初步数据表明，小尺寸量子点（~7 nm）比以前的大量子点更容易进入神经元突触。这允许长时间（约 1 小时）跟踪单个神经递质受体。然而，这种尺寸范围内的颗粒的生产仍然是一个主要问题，这主要是由于它们的涂层，其作用是稳定溶液中的胶体颗粒。目前的涂层尚未克服尺寸、稳定性和非特异性之间的根本权衡。在这里，我们建议根据我们之前的工作和迄今为止最好的文献结果生成一系列新的涂层。这些聚合物和配体非常紧凑，并通过强多齿结合来稳定；将使用一系列定量分析来测试涂有这些新材料的量子点的光学稳定性、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异性结合的能力 活神经元上的 AMPA 神经递质受体以及通过与致密（但不稳定）染料和荧光蛋白直接比较来保留天然受体行为的能力。该提案是显微镜、光学和生物物理学专家 Paul Selvin 教授与量子点开发和胶体合成专家 Andrew Smith 教授之间的合作成果。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，而这些过程一直难以使用经典的分子和细胞生物学方法来理解。这些包括肿瘤细胞趋化和转移、运动蛋白功能障碍和疾病中的神经元功能障碍。