Photonic probe and techniques for biological imaging applications

项目描述（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。本研究将分子设计、有机合成、光遗传学和先进的荧光显微镜相结合，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，用于成像具有非常高的灵敏度和时空分辨率的调节胞吐动力学。这一进展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（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，在此过程中Zn2+被共同释放。通过工程锌传感器专门报告局部Zn2+在质膜附近的上升，我们能够在细胞和亚细胞分辨率下连续监测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方法相结合。结合药理学和遗传学方法来操纵细胞偶联强度，我们将研究间隙连接偶联如何同步细胞分泌。最后，为了描述细胞在生理制剂或维持正常细胞-细胞接触的组织中如何协调其分泌活性，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盘共聚焦显微镜，以细胞和亚细胞分辨率在三维空间上检查Zn2+颗粒释放。这里开发的新探针和新方法应该在细胞和神经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.