Super-Resolution Microscopy of Neuronal Synapses with Small Quantum Dots and Advanced Imaging Tools

使用小量子点和先进成像工具对神经元突触进行超分辨率显微镜检查

• 批准号: 9384063
• 负责人: Hee Jung Chung
• 金额: $ 33.06万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9384063
• 关键词: 3-Dimensional; AMPA Receptors; Alzheimer' s Disease; Basic Science; Behavior; Biological; Biological Sciences; Biology; Caliber; Caring; Cells; Chemicals; Clinical; Color; Communication; DLG1 gene; DLG4 gene; Detection; Diffusion; Dyes; Ensure; Event; Fluorescence; Fluorescent Probes; Future; Generations; Glutamate Receptor; Goals; Grant; Grant Review; Health; Hour; Image; Imaging Device; Integral Membrane Protein; Label; Learning; Ligands; Long-Term Depression; Long-Term Potentiation; Measurement; Measures; Memory; Methods; Microscope; Microscopy; Minority; Misinformation; Molecular; Monitor; N-Methylaspartate; Neurodegenerative Disorders; Neurons; Neurosciences; Neurotransmitter Receptor; Optics; Output; Paper; Parkinson Disease; Photons; Problem Solving; Process; Proteins; Publishing; Quantum Dots; Regulation; Resolution; S-nitro-N-acetylpenicillamine; Sampling; Site; Stroke; Surface; Synapses; Synaptic Cleft; Synaptic plasticity; Technology; Temperature; Testing; Time; base; density; design; experimental study; fluorophore; microscopic imaging; nanometer; nanoscale; prevent; protein E; receptor; single molecule; tool; trafficking

Abstract The ability to measure the molecular mechanisms of neuronal communication at the nanometer spatial scale will have enormous impact in both basic bioscience and in future clinical neuroscience. In particular, AMPA- and NMDA-type glutamate receptors (AMPARs/NMDARs, known as iGluRs) are involved in neuron-to-neuron communication across synapses, where these receptors contribute to learning and memory, and when dysregulated, to neurodegenerative diseases including Alzheimer's, Parkinson's and complications from strokes. A critical mechanistic event is the transport of iGluRs into and out of synapses (or parts of synapses) in a dynamic process called synaptic plasticity. A revolution is underway because of the recent ability to resolve these events at the nanometer-scale using fluorescence super-resolution microscopy (FSRM). However significant inherent problems with this technology have led to confounding results and misinformation. The biggest problem has been with the fluorescent probes used to image receptors: conventional organic fluorescent probes last only a few seconds; commercial (and big) quantum dots (bQDs), despite their exceptional brightness and photostability, are over 20 nm in diameter and are too large to fit inside the synaptic cleft where iGluRs are active. We recently overcame this problem through an R21, which enabled us to develop small quantum dots (sQDs) that are <10 nm in diameter. They specifically label iGluRs in the synaptic cleft, which is just ~20-30 nm wide. The sQDs do this with tremendous brightness and stability, resulting in FSRM images in 3-dimensions with 100 ms time-resolution for greater than 2 minutes of continuous excitation. In contrast, bQD-labeled AMPARs are predominantly stuck in the extra-synaptic space because steric hindrance prevents them from going inside. We have recently extended these findings with a newer sQD that is completely stable, and with small organic fluorophores that we now show are stable enough, on live neurons (which previously had been too photolabile for such measurements.) Our findings, some of which have been published in 3 papers resulting from our R21 grant, may have tremendous implications for basic science and health: the surface mobility and trafficking of iGluRs, which depend on the ease of diffusion inside and outside of synapses, regulates synaptic efficacy. Here we wish to understand the distribution and dynamics of iGluRs, both within the synapses and between synapses, using our new sQDs and other new photoactivatable fluorescent proteins and some organic fluorophores. For this, a number of new advances in optics, probe design, and care with receptor monovalency are necessary. After these technical problems are solved (which will be useful to answer many different biological questions), we will validate the biology that we have observed, and to apply these to proof-of-principle experiments involved in two key biological questions: 1) In what way do receptors move into and around the synapse during homeostatic and synaptic plasticity? 2) Do endocytosed receptors communicate with each other between synapses on the same neuron?

摘要 在纳米空间尺度上测量神经元通信的分子机制的能力 将对基础生物科学和未来的临床神经科学产生巨大的影响。特别是，AMPA- 和NMDA型谷氨酸受体（AMPAR/NMDAR，称为iGluRs）参与神经元到神经元的相互作用。 这些受体有助于学习和记忆， 失调，神经退行性疾病，包括阿尔茨海默氏症，帕金森氏症和 中风一个关键的机制事件是iGluR进出突触（或部分突触）的转运 一个叫做突触可塑性的动态过程。一场革命正在进行中，因为最近的能力， 使用荧光超分辨率显微镜（FSRM）在纳米尺度上解决这些事件。 然而，这项技术的重大固有问题导致了混淆的结果和错误的信息。 最大的问题是用于成像受体的荧光探针：传统的有机 荧光探针只能持续几秒钟;商业（和大）量子点（bQD），尽管它们 异常的亮度和光稳定性，直径超过20纳米，太大而无法放入突触内 iGluRs活跃的地方。我们最近通过R21解决了这个问题， 开发直径<10 nm的小量子点（sQD）。它们特异性标记突触中的iGluRs 裂缝，其仅约20-30 nm宽。sQD以极大的亮度和稳定性做到这一点， FSRM图像在3维与100毫秒的时间分辨率大于2分钟的连续激发。 相比之下，bQD标记的AMPAR主要停留在突触外空间，因为空间位阻 障碍阻止他们进入。我们最近用一个新的sQD扩展了这些发现， 是完全稳定的，我们现在已经证明，小的有机荧光团在活的神经元上足够稳定， （以前对于这种测量来说，它太不对光稳定了。）我们的发现，其中一些已经被 发表在我们的R21赠款产生的3篇论文中，可能对基础科学产生巨大的影响， 健康：iGluRs的表面流动性和运输，这取决于内部和外部扩散的难易程度 调节突触的功效。在这里，我们希望了解iGluRs的分布和动态， 在突触内和突触之间，使用我们的新sQD和其他新的光活化 荧光蛋白和一些有机荧光团。为此，在光学、探测器、 设计和照顾受体单价是必要的。这些技术问题解决后（ 将有助于回答许多不同的生物学问题），我们将验证我们所拥有的生物学 观察，并将其应用于涉及两个关键生物学问题的原理验证实验：1）在 在稳态和突触可塑性过程中，受体是如何进入和环绕突触的？2)做 内吞受体在同一个神经元的突触之间相互交流？