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。该提案代表了一种整合的方法，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态。该开发基于以下观察结果：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（Zn 2+）。在刺激时，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，Zn 2+被共同释放。通过设计锌传感器来专门报告质膜附近的局部Zn 2+升高，我们能够以细胞和亚细胞分辨率连续监测Zn 2+颗粒释放。为了研究缝隙连接偶联对同步分泌的调节作用，我们将应用光遗传学技术控制细胞膜的兴奋性，并结合光激活和锌成像的方法。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如何偶联细胞分泌。最后，为了表征细胞如何协调其分泌活动在生理制剂或在组织中，正常的细胞-细胞接触保持，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，检查Zn 2+颗粒释放在三维细胞和亚细胞分辨率。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。 公共卫生相关性：异常的细胞偶联与许多疾病有关，包括心律失常、耳聋、神经元脱髓鞘和白内障。了解调节细胞连接通讯及其功能的机制仍然是一个重大的生物学挑战，对健康和疾病都有重要意义。该提案的重点是开发新的成像探针和技术，以研究间隙连接耦合如何协调细胞分泌。

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. PUBLIC HEALTH RELEVANCE: Abnormal cell coupling has been implicated in a number of diseases including cardiac arrhythmia, deafness, neuronal demyelination, and cataracts. Understanding mechanisms regulating cell junctional communication and its functions remains a significant biological challenge which has important implications in both health and disease. The focus of this proposal is to develop new imaging probes and techniques to study how gap junction coupling coordinates cell secretion.