Rich Thomas C, Griswold J R, Deal Joshua, Annamdevula Naga, McAlister Kathleen, Mayes Samuel, Browning Craig, Parker Marina, Leavelsey Silas J
Pharmacology, University of South Alabama, AL 36688.
Center for Lung Biology, University of South Alabama, AL 36688.
Proc SPIE Int Soc Opt Eng. 2019 Feb;10881. doi: 10.1117/12.2508052. Epub 2019 Mar 4.
Ca 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 Ca 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 Ca signals in single cells.
钙（Ca）和环磷酸腺苷（cAMP）是普遍存在的第二信使，已知它们能在广泛的时间尺度上差异性地调节多种细胞功能。来自多个研究小组的研究支持这样一种假说，即这些信号可定位于细胞内的离散位置，且这种亚细胞定位是信号特异性的关键组成部分。然而，迄今为止，在单个细胞内的多个位置追踪第二信使信号一直很困难。这一困难主要是由于无法实时测量多重荧光信号。为克服这一限制，我们利用了基于发射扫描和激发扫描的高光谱成像方法，在同一细胞中追踪第二信使信号以及标记的细胞结构和/或蛋白质。我们之前报道过高光谱成像技术可提高荧光和荧光共振能量转移（FRET）测量的信噪比，因此非常适合测量局部第二信使信号。使用这些方法，我们在包括气道平滑肌、肺内皮细胞和人胚肾293（HEK-293）细胞在内的几种细胞类型中，测量了靠近质膜和靠近核膜的cAMP信号，以及胞质溶胶内的分布信号。我们还测量了靠近似乎是高尔基体的自发荧光结构附近的cAMP和Ca信号。我们的数据表明，高光谱成像方法为深入了解单个细胞中cAMP和Ca信号的空间和动力学分布提供了独特的视角。
