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

摘要 在纳米空间尺度上测量神经元通讯的分子机制的能力 将对基础生物科学和未来的临床神经科学产生巨大影响。尤其是AMPA- 而NMDA型谷氨酸受体(AMPAR/NMDARs，又称iGluR)参与神经元到神经元的传递 突触之间的交流，这些受体在哪里对学习和记忆做出贡献，以及何时 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(或突触的一部分)的运输 在一个叫做突触可塑性的动态过程中。一场革命正在进行，因为最近有能力 使用荧光超分辨显微镜(FSRM)在纳米尺度上解决这些事件。 然而，这项技术的重大内在问题导致了令人困惑的结果和错误的信息。 最大的问题是用来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；商用(和大)量子点(BQD)，尽管它们 超乎寻常的亮度和光稳定性，直径超过20纳米，太大了，无法放入突触内 在iGluR活跃的地方裂开。我们最近通过R21解决了这个问题，这使我们能够 开发直径为10纳米的小量子点。他们在突触中特别标记了iGluR 裂隙，它只有20-30 nm宽。SQD以极高的亮度和稳定性做到了这一点，导致 连续激发超过2分钟的时间分辨率为100毫秒的FSRM三维图像。 相反，BQD标记的AMPAR主要卡在突触外空间，因为立体 障碍物使他们无法进入室内。我们最近用更新的SQD扩展了这些发现 是完全稳定的，我们现在展示的小有机荧光团在活的神经元上是足够稳定的 (之前，它太不适合进行这样的测量了。)我们的发现，其中一些已经被 发表在由我们的R21基金产生的3篇论文中，可能会对基础科学和 健康：iGluR的表面流动性和贩运取决于内外扩散的容易程度 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，使用我们的新的sQD和其他新的可光激活 荧光蛋白和一些有机荧光团。为此，在光学、探测器等方面取得了一些新进展 设计，以及对受体单价的护理是必要的。在这些技术问题解决之后( 将有助于回答许多不同的生物学问题)，我们将验证我们已有的生物学 观察到的，并将这些应用于涉及两个关键生物学问题的原则证明实验：1) 在动态平衡和突触可塑性过程中，受体以什么方式进入突触并围绕突触移动？ 内吞受体在同一神经元上的突触之间相互通信？

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?