Lavagnino Zeno, Dwight Jason, Ustione Alessandro, Nguyen Thuc-Uyen, Tkaczyk Tomasz S, Piston David W
Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee; Cell Biology and Physiology, Washington University in St. Louis, St. Louis, Missouri.
Department of Bioengineering, Rice University, Houston, Texas.
Biophys J. 2016 Jul 26;111(2):409-417. doi: 10.1016/j.bpj.2016.06.014.
The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and β-cell function. Insulin secretion from β-cells depends on the firing of action potentials and consequent rises of intracellular calcium activity ([Ca(2+)]i). Zinc (Zn(2+)) is cosecreted with insulin, and has been postulated to play a role in cell-to-cell cross talk within an islet, in particular inhibiting glucagon secretion from α-cells. Thus, measuring [Ca(2+)]i and Zn(2+) dynamics from both α- and β-cells will elucidate mechanisms underlying islet hormone secretion. [Ca(2+)]i and intracellular Zn(2+) can be measured using fluorescent biosensors, but the most efficient sensors have overlapping spectra that complicate their discrimination. Hyperspectral imaging can be used to distinguish signals from multiple fluorophores, but available hyperspectral implementations are either too slow to measure the dynamics of ionic signals or not suitable for thick samples. We have developed a five-dimensional (x,y,z,t,λ) imaging system that leverages a snapshot hyperspectral imaging method, image mapping spectrometry, and light-sheet microscopy. This system provides subsecond temporal resolution from deep within multicellular structures. Using a single excitation wavelength (488 nm) we acquired images from triply labeled samples with two biosensors and a genetically expressing fluorescent protein (spectrally overlapping with one of the biosensors) with high temporal resolution. Measurements of [Ca(2+)]i and Zn(2+) within both α- and β-cells as a function of glucose concentration show heterogeneous uptake of Zn(2+) into α-cells that correlates to the known heterogeneities in [Ca(2+)]i. These differences in intracellular Zn(2+) among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.
对完整胰岛中离子信号动态的观察极大地促进了我们对α细胞和β细胞功能的理解。β细胞分泌胰岛素依赖于动作电位的发放以及细胞内钙活性（[Ca(2+)]i）的随之升高。锌（Zn(2+)）与胰岛素共同分泌，并被推测在胰岛内的细胞间相互作用中发挥作用，特别是抑制α细胞分泌胰高血糖素。因此，测量α细胞和β细胞中的[Ca(2+)]i和Zn(2+)动态将阐明胰岛激素分泌的潜在机制。可以使用荧光生物传感器测量[Ca(2+)]i和细胞内Zn(2+)，但最有效的传感器具有重叠光谱，这使得它们的区分变得复杂。高光谱成像可用于区分来自多个荧光团的信号，但现有的高光谱实现方式要么太慢无法测量离子信号的动态，要么不适合厚样品。我们开发了一种五维（x、y、z、t、λ）成像系统，该系统利用了快照高光谱成像方法、图像映射光谱法和光片显微镜。该系统从多细胞结构深处提供亚秒级的时间分辨率。使用单一激发波长（488 nm），我们以高时间分辨率从用两种生物传感器和一种基因表达荧光蛋白（光谱与其中一种生物传感器重叠）三重标记的样品中获取图像。作为葡萄糖浓度函数的α细胞和β细胞内[Ca(2+)]i和Zn(2+)的测量结果显示，Zn(2+)进入α细胞的摄取存在异质性，这与[Ca(2+)]i中已知的异质性相关。α细胞之间细胞内Zn(2+)的这些差异可能导致在葡萄糖水平升高时观察到的胰高血糖素分泌受到抑制。
