Presentation + Paper
4 March 2019 Hyperspectral imaging microscopy for measurement of localized second messenger signals in single cells
Author Affiliations +
Abstract
Ca2+ and cAMP are ubiquitous second messengers known to differentially regulate a variety of cellular functions over a wide range of timescales. Studies from a variety of groups support the hypothesis that these signals can be localized to discrete locations within cells, and that this subcellular localization is a critical component of signaling specificity. However, to date, it has been difficult to track second messenger signals at multiple locations within a single cell. This difficulty is largely due to the inability to measure multiplexed florescence signals in real time. To overcome this limitation, we have utilized both emission scan- and excitation scan-based hyperspectral imaging approaches to track second messenger signals as well as labeled cellular structures and/or proteins in the same cell. We have previously reported that hyperspectral imaging techniques improve the signal-to-noise ratios of both fluorescence and FRET measurements, and are thus well suited for the measurement of localized second messenger signals. Using these approaches, we have measured near plasma membrane and near nuclear membrane cAMP signals, as well as distributed signals within the cytosol, in several cell types including airway smooth muscle, pulmonary endothelial, and HEK-293 cells. We have also measured cAMP and Ca2+ signals near autofluorescent structures that appear to be golgi. Our data demonstrate that hyperspectral imaging approaches provide unique insight into the spatial and kinetic distributions of cAMP and Ca2+ signals in single cells.
Conference Presentation
© (2019) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Thomas C. Rich, J. R. Griswold, Joshua Deal, Naga Annamdevula, Kathleen McAlister, Samuel Mayes, Craig Browning, Marina Parker, and Silas J. Leavesley "Hyperspectral imaging microscopy for measurement of localized second messenger signals in single cells", Proc. SPIE 10881, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII, 108811F (4 March 2019); https://doi.org/10.1117/12.2508052
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KEYWORDS
Proteins

Signal detection

Signal to noise ratio

3D metrology

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