Multi-dimensional Dynamics of Pancreatic Islet Cells Measured by Image Mapping diSPIM

摘要 我们对细胞动力学的理解通过活细胞荧光显微镜得到了显著的进步 实验这些实验已经在细胞膜上的囊泡运输和胞吐作用中产生了发现。 功能上重要的时间和长度尺度，与胰岛功能的具体影响。活细胞 超光谱成像允许同时测量具有信噪比的多个动态过程 比率等于或优于基于滤波器的方法。目前，最方便的高光谱成像 系统使用受光漂白和成像速度慢限制的共焦显微镜。我们提出 为了开发一种新颖的五维（x，y，z，t，λ）荧光成像系统， 时间和光谱分辨率与最小可能的光漂白。我们将优化其性能 用于研究长期存在的胰岛素分泌调节问题。这个仪器将联合收割机 两种技术：双视图选择平面照明显微镜（diSPIM），产生各向同性衍射- 三维扩展视图上的有限成像，以及允许 整个领域的高光谱检测在一个单一的快照。我们将构建、测试和优化这部小说 通过两个具体的目标。具体目标1将侧重于建立和优化一个新的 与diSPIM一起使用的高光谱IMS系统，以及适用于五维数据的软件模块 采集和分析。为了证实IMS/diSPIM方法的优势，我们将采集图像 同时具有高时间和空间分辨率的至少五种生物传感器颜色。测试和引导 目标1，具体目标2的发展将把这种新工具应用于β细胞生物学中的问题， 目前可用的方法，重点是两个问题，胰岛素囊泡运输和 分泌：a）β细胞中胰岛素囊泡的正常生命周期是什么？由于<10%的胰岛素囊泡 被分泌，已经假设新形成的囊泡优先分泌，我们提出 寿命较长的囊泡是一个信号平台。我们将使用IMS/diSPIM进行定量测量， 到6个荧光探针，这将使我们能够跟踪β细胞中的每一个囊泡，因为它从高尔基体发芽，成熟， 并且被分泌或不可逆地移动到长寿命池中。B）做到"易于释放"， “储备”囊泡池导致葡萄糖刺激胰岛素分泌的两个阶段？二的概念 池来自突触囊泡研究，这可能不同于β细胞的拥挤环境， 第一阶段分泌事件似乎来自新到达质膜的囊泡。我们 假设β细胞中的小泡沿着微管移动到胞吐部位，这些移动是 受细胞内游离钙活性（[Ca 2 +] i和cAMP水平）调节。为了验证这个假设，我们将使用 IMS/diSPIM可同时测量多达6个荧光探针，并允许定量相关 胰岛素囊泡运动和分泌与[Ca2 +] i，cAMP和细胞骨架结构之间的关系。

ABSTRACT Our understanding of cellular dynamics has been advanced significantly by live-cell fluorescence microscopy experiments. These experiments have yielded discoveries in vesicle trafficking and exocytosis on the functionally important time and length scales, with specific implications for pancreatic islet function. Live-cell hyperspectral imaging permits simultaneous measurements of multiple dynamic processes with signal-to-noise ratios equivalent or superior to filter-based approaches. Currently, the most expedient hyperspectral imaging systems use confocal microscopy, which is limited by photobleaching and slow imaging speeds. We propose to develop a novel five-dimensional (x,y,z,t,λ) fluorescence imaging system that provides high spatial, temporal, and spectral resolution with the minimal possible photobleaching. We will optimize its performance for investigations of long-standing questions about regulation of insulin secretion. This instrument will combine two technologies: dual-view Selective-Plane Illumination Microscopy (diSPIM) that yields isotropic diffraction- limited imaging over extended views in three dimensions, and image mapping spectroscopy (IMS) that permits whole field hyperspectral detection in a single snapshot. We will build, test, and optimize this novel instrumentation through two specific aims. Specific aim 1 will focus on building and optimizing a new hyperspectral IMS system for use with diSPIM, and also adapting software modules for five-dimensional data acquisition and analysis. To substantiate the advantages of the IMS/diSPIM approach, we will acquire images simultaneously for at least five biosensor colors with high temporal and spatial resolution. To test and guide the developments in Aim 1, Specific aim 2 will apply this new instrument to issues in β-cell biology that cannot be addressed with currently available methods, focusing on two questions of insulin vesicle trafficking and secretion: a) What is the normal life cycle of an insulin vesicle in the β-cell? Since <10% of the insulin vesicles are secreted, it has been hypothesized that newly formed vesicles are preferentially secreted, and we propose that longer-lived vesicles act as a signaling platform. We will use the IMS/diSPIM to measure quantitatively up to 6 fluorescent probes, which will allow us to track every vesicle in a β-cell as it buds from the Golgi, matures, and is either secreted or moves, putatively irreversibly, into a long-lived pool. b) Do “readily releasable” and “reserve” vesicle pools lead to the two phases of glucose-stimulated insulin secretion? The concept of two pools comes from synaptic vesicle studies, which may differ from the crowded environment of the β-cell, where first phase secretory events appear to come from vesicles newly arriving at the plasma membrane. We hypothesize that vesicles in β-cells move along microtubules to sites of exocytosis, and these movements are regulated by intracellular free calcium activity ([Ca2+]i and cAMP levels. To test this hypothesis, we will use the IMS/diSPIM to measure up to 6 fluorescent probes simultaneously and permit quantitative correlations between insulin vesicle motions and secretion with [Ca2+]i, cAMP, and cytoskeletal architecture.