Matz Magnus, Schumacher Kirstin, Hatlapatka Kathrin, Lorenz Dirk, Baumann Knut, Rustenbeck Ingo
Institute of Medicinal and Pharmaceutical Chemistry, University of Braunschweig, Braunschweig D38106, Germany.
Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig D38106, Germany.
Microsc Microanal. 2014 Feb;20(1):206-18. doi: 10.1017/S1431927613013767. Epub 2013 Nov 13.
Total internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X-Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion.
对荧光标记的分泌颗粒进行全内反射荧光显微镜检查，可在一个实验中监测胞吐作用及之前的颗粒行为。虽然依赖观察者的评估可能足以量化胞吐作用，但视频文件中包含的大多数其他信息无法通过这种方式获取。本程序可对胞吐作用进行独立于观察者的检测，并对胰岛素颗粒的整个膜下群体进行追踪。一个前提条件是颗粒荧光峰值的精确定位。追踪基于峰值基部半径、峰值强度和交叉前的轨迹。通过原始颗粒模式的模拟轨迹展示了追踪的稳健性。X-Y维度的移动性由笼径描述，与广泛使用的均方位移不同，笼径具有固有的时间分辨率。在胰岛素-增强绿色荧光蛋白标记的MIN6细胞中，对胞吐作用进行独立于观察者的检测是基于荧光强度的最大下降以及释放物质消散云团质心的位置。将氯化钾诱导的胰岛素胞吐作用的量化与融合前移动性分析相结合表明，在胞吐前的最后3秒，胞吐前颗粒的笼径比对照颗粒小，并且在融合前立即显著增加。
