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,?) 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 ?-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 ?-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 ?-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 ?-cell, where first phase secretory events appear to come from vesicles newly arriving at the plasma membrane. We hypothesize that vesicles in ?-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.
The human body is comprised of cells, so the ultimate cure for many diseases is dependent on a complete knowledge about the cell. Fluorescence microscopy permits interrogation of subcellular components within living cells on the functionally important time and length scales. We propose to create an innovative imaging and measurement method that will allow us to watch cellular components move and interact on a stop-action time scale, and apply this method to address current questions about insulin secretion.
