Photonic probe and techniques for biological imaging applications

描述（由申请人提供）：该项目的长期目标是设计光子探针以实现生物发现。该提案代表了一种综合方法，通过结合分子设计、有机合成、光遗传学和先进的荧光显微镜来开发荧光探针和成像技术，并将其应用于研究间隙连接耦合的重要功能：间隙连接细胞间通讯如何介导同步细胞分泌。为此，我们将首先开发一类新型荧光探针，用于以非常高的灵敏度和时空分辨率对受调节的胞吐作用的动力学进行成像。该开发基于以下观察：包括胰岛β细胞在内的许多分泌细胞的分泌颗粒中含有高水平的锌离子(Zn2)。受到刺激后，这些细胞将其分泌颗粒的内容物释放到细胞外介质中，在此期间共同释放 Zn2。通过设计锌传感器来专门报告质膜附近局部 Zn2 的升高，我们能够以细胞和亚细胞分辨率连续监测 Zn2 颗粒的释放。为了研究间隙连接耦合如何调节同步分泌，我们将应用光遗传学技术来控制膜的兴奋性，并将光激活方法与锌成像相结合。结合药理学和遗传学方法来操纵细胞耦合强度，我们将研究间隙连接耦合如何同步细胞分泌。最后，为了表征细胞如何在生理制剂或维持正常细胞-细胞接触的组织中协调其分泌活动，我们将使用高空间选择性的成像方法，包括双光子激光扫描显微镜和转盘共聚焦显微镜，以细胞和亚细胞分辨率在三个维度上检查Zn2颗粒的释放。这里开发的新探针和方法应该在细胞和神经元生物学以及不同的生物系统中具有广泛的应用。

DESCRIPTION (provided by applicant): The long term goal of this project is to engineer photonic probes to enable biological discovery. This proposal represents an integral approach by combining molecular design, organic synthesis, optogenetics, and advanced fluorescence microscopy to develop fluorescent probes and imaging techniques, and to apply them to study an important function of gap junction coupling: how gap junction intercellular communication mediates synchronized cell secretion. To this end, we will first develop a new class of fluorescent probes for imaging the dynamics of regulated exocytosis with very high sensitivity and spatiotemporal resolution. The development is based on the observation that a number of secretory cells, including pancreatic islet beta cells, contain a high level of zinc ion (Zn2+) in their secretory granules. Upon stimulation, these cells release the contents of their secretory granules into extracellular medium, during which Zn2+ is co-released. By engineering zinc sensors to specifically report local Zn2+ rise near plasma membranes, we are able to monitor Zn2+ granule release continuously at cellular and subcellular resolution. To examine how gap junction coupling regulates synchronized secretion, we will apply the technique of optogenetics to control the membrane excitability, and to integrate the method of photo-activation with zinc imaging. Combined with pharmacological and genetic approaches to manipulate cell coupling strength, we will investigate how gap junction coupling synchronizes cell secretion. Finally, to characterize how cells coordinate their secretory activity in physiological preparations or in tissues where normal cell-cell contact is maintained, we will use imaging methods of high spatial selectivity, including two photon laser scanning microscopy and spinning disk confocal microscopy, to examine Zn2+ granule release in three dimensions at cellular and subcellular resolution. New probes and methods developed here should have broad applications in cellular and neuronal biology and in different biological systems.