Broadband multispectral microscopy of insulin granule dynamics in live pancreatic islets

糖尿病是一种慢性疾病，其特征是在较长时间内血糖水平较高。截至2019年，据估计，全球有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。糖尿病的标志是位于胰腺的分泌胰岛素的β细胞失效。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，许多方法被用来研究它们的生理学。β细胞将胰岛素储存在称为胰岛素颗粒的小隔间中，当血糖水平上升时，这些颗粒与细胞膜融合，使β细胞能够立即释放大量胰岛素。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影响颗粒功能和动力学的外部标记。研究团队一直在开发一种名为共焦宽带背向散射显微镜(CBBM)的简单光学显微镜技术，它可以显示没有任何标签的胰岛素颗粒，并监测它们在几个小时甚至几天的过程中的动态，这可能会导致一种治疗糖尿病的新方法。项目成果和努力将被整合到生物学中的光学显微镜课程中；为本科生、研究生和研究生提供培训机会；并增强K-12学生的研究经验。该项目的主要目标是开发一种无标记显微镜系统，用于对定制芯片上胰岛的活胰岛中的胰岛素颗粒进行功能成像和连续监测。该方法将把新型的共焦宽带后向散射显微镜(CBBM)与现有的共焦FRET和超分辨率风暴模式相结合，这三种模式都共享光学序列的主要部分，并具有共同的视场。CBBM技术将提供β细胞的功能图像，解析胰岛素颗粒，监测颗粒内胰岛素原向胰岛素的转化，并可视化基于颗粒的胰岛素细胞间转运。该项目还将开发一种物理和数学模型来描述无标记显微镜系统共焦体积内胰岛素颗粒的光散射，并开发一种反向算法来区分无标记胰岛素颗粒和非胰岛素颗粒。这些算法将被用来监测胰岛素颗粒的时间变化，并在胰岛素细胞间运输和释放过程中以3D形式连续显示它们。为了更好地了解胰岛功能和可移植胰岛的最佳环境，葡萄糖交换将与人体胰岛体内功能成像相结合，从而能够研究葡萄糖/胰岛素周期中的重要反馈回路。还将进行实验，以调查糖尿病患者和健康患者的胰岛之间的关键差异。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.