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: 8683516
• 负责人: PAUL R SELVIN
• 金额: $ 21.33万
• 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 2016-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8683516
• 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; 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; next generation; novel; particle; public health relevance; receptor; relating to nervous system; 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性质的荧光纳米颗粒，具有革新细胞显微镜和生物成像的潜力。这些荧光团与荧光显微镜中正在进行的革命同时出现，称为“超分辨率”成像，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，小至神经元突触间隙（~30 nm）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参与微小突触内的神经元-神经元通信的单个蛋白质，以揭示参与神经信号传导的异质的、动态变化的过程，以及在诸如阿尔茨海默氏症、帕金森氏症和中风的疾病中细胞间通信如何被破坏。然而，量子点用于活细胞的先进显微镜的实施受到其庞大尺寸（~20 nm）和非特异性标记的阻碍，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，小尺寸的量子点（~7 nm）具有更大的访问神经元突触，大大大于以前的笨重的点。这允许长时间（~1小时）跟踪单个神经递质受体。然而，在该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，该涂层用于使颗粒在溶液中胶体稳定。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个权衡。在这里，我们建议根据我们以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。这些聚合物和配体是超紧凑的，并通过强大的多齿结合来稳定;涂覆有这些新材料的量子点将使用一系列定量测定来测试光学稳定性、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异性结合到 AMPA神经递质受体在活的神经元和卡帕克能力，以保持天然受体的行为，通过直接比较紧凑（但不稳定）染料和荧光蛋白。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许多细胞和分子过程打开大门，这些细胞和分子过程是疾病的基础，而这些过程一直无法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性和转移、运动蛋白功能障碍和疾病中的神经元功能障碍。