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

基本信息

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
项目类别：
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&apos 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

项目摘要

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）：量子点是具有独特的光学特性的荧光纳米颗粒，具有革命的细胞显微镜和生物成像。这些荧光团已经与持续的荧光显微镜革命同时出现，称为“超分辨率”成像，从而以分子特异性在分辨率下以分辨率接近单个蛋白质的分辨率，分子，细胞和组织现在可以光学成像。例如，现在可以在活细胞中动态解析像神经元突触裂口（〜30 nm）一样小的细胞结构，该细胞以前需要通过电子显微镜进行静态的固定细胞成像。量子点在这里具有独特的利基市场：与荧光蛋白和有机染料不同，它们的发射强度是如此明亮，以至于可以很容易地观察到个体分子，并且它们的发射不会光泽。理论上，这些颗粒可用于图像和跟踪微小突触中涉及神经元神经元通信的单一蛋白质，以揭示神经信号的异质性，动态变化的过程，以及在诸如阿尔茨海默氏症，帕克森（Parkinson）和帕克森（Parkinson）等疾病中如何破坏细胞间通信。然而，实施量子点用于实时细胞的高级显微镜，其庞大的大小（约20 nm）和非特异性标记受到了阻碍，这极大地限制了对拥挤的神经元突触的特定访问。我们的初步数据表明，小型量子点（〜7 nm）对神经元突触具有更大的影响，大大比以前的笨重点要大得多。这允许在长时间（〜1小时）中跟踪单个神经递质受体。但是，这种尺寸范围内的颗粒的产生主要是由于其涂层，这是一个主要问题，这是在溶液中胶体稳定的。尺寸，稳定性和非特异性之间的基本权衡只是目前的涂料。在这里，我们建议根据我们以前的工作和迄今为止最佳文献结果生成新的涂料系列。这些聚合物和配体是超压缩的，并通过强多介质结合而稳定。涂有这些新材料的量子点将通过一系列定量测定量进行测试，以进行光学稳定性，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通过直接比较紧凑（但不稳定的）染料和氟化蛋白，可以保留天然受体行为的AMPA神经递质受体以及能够保留天然受体行为的能力。这项建议是显微镜，专家和生物物理学专家保罗·塞尔文（Paul Selvin）与量子点开发和胶体合成专家安德鲁·史密斯（Andrew Smith）教授之间的合作努力。成功将为超分辨率观察的大门观察到许多细胞和分子过程，这些细胞和分子过程使用经典的分子和细胞生物学方法抵抗了理解。这些包括肿瘤细胞趋化性和转移，运动蛋白功能障碍以及疾病中的神经元功能障碍。