PUNQs: Photostable, Ultrafast, Nano-optode, Quantum-dots to image Na in dendrites

描述(由申请人提供)：这项研究计划通过设计具有窄发射光谱的模块化、可调纳米传感器，提出了离子动力学荧光成像的主要技术进步。在双光子显微镜下，这些纳米传感器将定量成像树突棘中钠通量的时空动态，这是以前从未做过的。树突棘是微小的、半自主的神经元间隔，在处理突触信号的过程中，它们的结构和功能之间显示出令人着迷的关系。这种动态的结构-功能关系提供了非常高的输入特异性，同时也允许快速修改，从而引起神经电路的可塑性。了解脊椎生理学可以极大地增强我们对从阿尔茨海默氏症到神经病理性疼痛等一系列疾病的因果知识，以及学习和记忆的基本过程。为了能够对钠离子通量进行成像，我们需要设计能够进入脊柱并快速测量局部钠离子浓度的荧光纳米传感器。为此，我们将使用一种新的设计，我们称之为PUNQ，用于光稳定，超快，纳米光，量子点。这些新的PUNQ是模块化的，可调的，并且具有窄的发射光谱。因此，在现有的化学条件下，它们可以很容易地进行修改，以研究新的离子和小分子靶标，它们的动态范围可以根据目标分析物的生理浓度进行调整，并且它们可以被多路复用在一起。这项研究将对树突棘中难以捉摸的钠的动力学产生新的见解，PUNQ平台将适用于任何涉及离子动力学的研究。这项研究的具体目的是：1)设计具有最佳大小和化学性质的PUNQ，以实现与生理相关的分析物的灵敏度和选择性；2)以有效浓度将PUNQ输送到小的细胞亚室。3)测定树突棘对谷氨酸能兴奋反应的树突和体细胞的Na+通量。这项跨学科的研究 结合了Clark实验室在纳米传感器开发方面的经验，Bhatia实验室在多功能纳米颗粒开发方面的专业知识，以及Sabatini实验室在神经生物学方面的专业知识。拟议的研究和培训计划将阐明钠在树突棘中的作用，并为研究单个细胞内的离子动力学提供一个高价值、灵活的工具。最后，研究将为我提供宝贵的经验，为我的未来做好准备 作为生物医学研究的独立研究员。

DESCRIPTION (provided by applicant): This research plan proposes a major technical advancement in fluorescent imaging of ion dynamics by designing modular, tunable nanosensors with narrow emission spectra. Under two-photon microscopy, these nanosensors will quantitatively image the spatio-temporal dynamics of sodium fluxes in dendritic spines, which has never been done before. Dendritic spines are tiny, semi-autonomously neuronal compartments that exhibit a fascinating relationship between their structure and their function in processing synaptic signals. This dynamic structure-function relationship provides extraordinarily high input specificity while also allowing for rapid modifications that give rise t plasticity in neural circuits. Understanding spinal physiology may vastly enhance our causal knowledge for a broad range of diseases from Alzheimer's to neuropathic pain as well as basic processes in learning and memory. In order to gain be able to image sodium fluxes, we need to design fluorescent nanosensors that can enter into spines and rapidly measure local sodium concentrations. For this reason, we will use a novel design we call PUNQs for Photostable, Ultra-fast, Nano-optode, Quantum dots. These new PUNQs are modular, tunable, and have narrow emission spectra. Thus, with existing chemistry, they can be easily modifiable to study new ion and small-molecule targets, their dynamic ranges can be adjusted for the target analyte's physiologic concentration, and they can be multiplexed together. This research will produce new insight into the elusive dynamics of sodium in dendritic spines, and the PUNQ platform will be applicable to any research involving ion dynamics. The specific aims of this research are: 1) Designing PUNQs with optimal size and chemical properties to achieve physiologically-relevant analyte sensitivity and selectivity 2) Delivering PUNQs into small cellular subcompartments at effective concentrations. 3) Measuring dendritic and somatic Na+ fluxes in response to glutamatergic excitation at dendritic spines. This interdisciplinary research merges the Clark laboratory's experience with nanosensor development, the Bhatia laboratory's expertise in multifunctional nanoparticle development, and the Sabatini laboratory's expertise in neurobiology. The proposed research and training plan will elucidate the role of sodium in dendritic spines, and provide a high-value, flexible tool to study ion dynamics within individual cells. Finally, research will provide me with valuable experience that will prepare me for a future as an independent investigator in biomedical research.