Photonic probe and techniques for biological imaging applications

描述(申请人提供)：该项目的长期目标是设计光子探测器，使之能够进行生物发现。这一建议代表了一种将分子设计、有机合成、光遗传学和先进的荧光显微镜相结合的综合方法，以开发荧光探针和成像技术，并将其应用于研究缝隙连接耦合的一个重要功能：缝隙连接细胞间通讯如何调节细胞同步分泌。为此，我们将首先开发一类新的荧光探针，用于以非常高的灵敏度和时空分辨率成像调节胞吐的动力学。这一发育是基于观察到许多分泌细胞，包括胰岛β细胞，在其分泌颗粒中含有高水平的锌离子(锌离子)。在刺激下，这些细胞将其分泌颗粒的内容释放到细胞外介质中，在此过程中，锌离子被共同释放。通过设计锌传感器来专门报告质膜附近局部锌离子的上升，我们能够在细胞和亚细胞分辨率下连续监测锌颗粒的释放。为了研究缝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偶联强度，我们将研究缝隙连接偶联如何同步细胞分泌。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旋转圆盘共聚焦显微镜，在细胞和亚细胞分辨率下研究三维锌颗粒的释放。这里开发的新的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.