Broadband multispectral microscopy of insulin granule dynamics in live pancreatic islets

糖尿病是一种以长期高血糖为特征的慢性疾病。截至2019年，全球估计有4.63亿人患有糖尿病，占世界成年人口的近9%，而且发病率还在继续上升。糖尿病的标志是胰腺中分泌胰岛素的细胞的衰竭。β细胞是体内唯一分泌胰岛素的细胞，而胰岛素又是唯一能够降低血糖水平的激素。鉴于β细胞的重要性，许多方法被应用于研究它们的生理学。β细胞将胰岛素储存在称为胰岛素颗粒的小隔间中，当血糖水平升高时，这些颗粒与细胞膜融合，使β细胞一次释放大量胰岛素。不幸的是，胰岛素颗粒的直接可视化并不简单，因为目前的方法需要外部标签，可以影响颗粒的功能和动力学。研究小组一直在开发一种直接的光学显微镜技术，称为共聚焦宽带后向散射显微镜（CBBM），它可以在没有任何标签的情况下可视化胰岛素颗粒，并在数小时甚至数天内监测它们的动态，这可能会导致一种新的糖尿病治疗方法。项目成果和努力将纳入生物学光学显微镜课程；为本科生、研究生和研究生提供培训机会；提高K-12学生的研究经验。该项目的总体目标是开发一种无标签显微镜系统，用于在定制的胰岛芯片中对活胰岛中的胰岛素颗粒进行功能成像和连续监测。该方法将新型共聚焦宽带后向散射显微镜（CBBM）与现有的共聚焦FRET和超分辨率STORM模式结合起来，三者共享光学序列的主要部分并具有共同的视野。CBBM技术将提供β细胞的功能图像，分解胰岛素颗粒，监测胰岛素原在颗粒内转化为胰岛素，并可视化基于颗粒的胰岛素细胞间运输。该项目还将开发一个物理和数学模型来描述无标签显微镜系统共聚焦体积内胰岛素颗粒的光散射，并开发一个逆算法来区分无标签胰岛素颗粒和非胰岛素颗粒。这些算法将用于监测颗粒的时间变化，并在胰岛素细胞间运输和释放过程中连续地以3D方式可视化颗粒。为了更好地了解胰岛的功能和可移植胰岛的最佳环境，葡萄糖交换将与人体胰岛的体内功能成像相结合，从而研究葡萄糖/胰岛素循环中的重要反馈回路。实验还将研究糖尿病患者和健康受试者胰岛之间的关键差异。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Diabetes is a chronic disease characterized by a high blood sugar level over an extended period of time. As of 2019, it was estimated that 463 million people worldwide or almost 9% of the world adult population, had diabetes, with rates continuing to rise. The hallmark of diabetes is the failure of insulin secreting beta cells located in the pancreas. The beta cell is the only cell in the body that secretes insulin, which in turn is the only hormone able to lower plasma glucose levels. Given the importance of beta cells, numerous methods have been applied to study their physiology. Beta cells store insulin in small compartments called insulin granules and when blood sugar levels rise, these granules fuse with the cell membrane, allowing beta cells to release large quantities of insulin at once. Unfortunately, direct visualization of insulin granules is not straightforward, since current methods require external labels that can affect granule function and dynamics. The team of investigators has been developing a straightforward optical microscopic technique called Confocal Broadband Backscattering Microscopy (CBBM) that can visualize insulin granules without any labels and monitor their dynamics over the course of hours and even days, which could potentially lead to a new treatment for diabetes. Project outcomes and efforts will be integrated into a course on optical microscopy in biology; provide training opportunities for undergraduate, graduate, and post graduate students; and enhance research experiences for K-12 students. The overarching goal of this project is to develop a label-free microscopy system for functional imaging and continuous monitoring of insulin granules in live pancreatic islets in a custom islet-on-a-chip. The approach will combine novel confocal broadband backscattering microscopy (CBBM) with the existing confocal FRET and super-resolution STORM modalities, with all three sharing a major part of the optical train and having a common field of view. The CBBM technique will provide a functional image of a beta cell, resolve insulin granules, monitor the conversion of proinsulin into insulin inside the granules, and visualize granule-based insulin intercellular transport. The project will also develop a physical and mathematical model to describe light scattering from insulin granules within the confocal volume of the label-free microscopy system and an inverse algorithm to differentiate insulin and non-insulin granules label-free. These algorithms will be used to monitor temporal changes in granules and continuously visualize them in 3D during insulin intercellular transport and release. To better understand islet function and the optimal environments for transplantable islets, glucose exchanges will be combined with in vivo functional imaging in human pancreatic islets, allowing investigation of important feedback loops in glucose/insulin cycles. Experiments will also be performed to investigate critical differences between islets derived from diabetic and healthy subjects.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.