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+...