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

描述（由申请人提供）：本研究计划通过设计窄发射光谱的模块化可调谐纳米传感器，提出了离子动力学荧光成像的重大技术进步。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、半自主的神经元隔室，它们的结构和处理突触信号的功能之间表现出一种迷人的关系。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，从而产生神经回路的可塑性。了解脊柱生理学可以极大地提高我们对从阿尔茨海默氏症到神经性疼痛以及学习和记忆的基本过程的广泛疾病的因果知识。为了获得对钠通量成像的能力，我们需要设计能够进入脊柱并快速测量局部钠浓度的荧光纳米传感器。出于这个原因，我们将使用一种新颖的设计，我们称之为punq，即光稳定、超快、纳米光电、量子点。这些新的punq是模块化的，可调的，并且具有窄发射光谱。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目标，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，并且它们可以复用在一起。这项研究将对树枝状棘中难以捉摸的钠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。本研究的具体目标是：1)设计具有最佳尺寸和化学性质的punq，以实现与生理相关的分析物敏感性和选择性；2)以有效浓度将punq输送到小细胞亚室中。3)测量树突和体细胞对谷氨酸能刺激的Na+通量。这个跨学科的研究

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. PUBLIC HEALTH RELEVANCE: This research plan proposes a major technical advancement in fluorescent imaging of ion dynamics by designing modular, tunable nanosensors with narrow emission spectra. These nanosensors will be used to image sodium dynamics in dendritic spines, and they will be expanded to imaging multiple ions simultaneously in the future.