Perinetti Giuseppe, Müller Tobias, Spaar Alexander, Polishchuk Roman, Luini Alberto, Egner Alexander
Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (CH), Italy.
Traffic. 2009 Apr;10(4):379-91. doi: 10.1111/j.1600-0854.2008.00875.x.
Two problems have hampered the use of light microscopy for structural studies of cellular organelles for a long time: the limited resolution and the difficulty of obtaining true structural boundaries from complex intensity curves. The advent of modern high-resolution light microscopy techniques and their combination with objective image segmentation now provide us with the means to bridge the gap between light and electronmicroscopy in cell biology applications. In this study, we provide the first comparative correlative analysis of three-dimensional structures obtained by 4Pi microscopy and segmented by a zero-crossing procedure with those of transmission electron microscopy (TEM). The distribution within the cisternae of isolated Golgi stacks of the cargo protein procollagen 3 was mapped by both 4Pi microscopy and TEM for a detailed comparative analysis of their imaging capabilities. A high correlation was seen for the structures, indicating the particular accuracy of the 4Pi microscopy. Furthermore, for the first time, transport of a cargo molecule (vesicular stomatitis virus G protein-pEGFP) through individual Golgi stacks (labeled by galactosyl transferase-venus-YFP) was visualized by 4Pi microscopy. Following the procedures validated by the correlative analysis, our transport experiments show that (i) VSVG-pEGFP rapidly enter/exit individual Golgi stacks, (ii) VSVG-pEGFP never fills the GalT-venusYFP compartments completely and (iii) the GalT-venusYFP compartment volume increases upon VSVG-pEGFP arrival. This morphological evidence supports some previous TEM-based observations of intra-Golgi transport of VSVG-pEGFP and provides new insights toward a better understanding of protein progression across Golgi stacks. Our study thus demonstrates the general applicability of super resolution fluorescence microscopy, coupled with the zero-crossing segmentation procedure, for structural studies of suborganelle protein distributions under living cell conditions.
长期以来，有两个问题一直阻碍着利用光学显微镜对细胞器进行结构研究：分辨率有限以及难以从复杂的强度曲线中获取真实的结构边界。现代高分辨率光学显微镜技术的出现以及它们与客观图像分割技术的结合，现在为我们提供了在细胞生物学应用中弥合光学显微镜和电子显微镜之间差距的手段。在本研究中，我们首次对通过4Pi显微镜获得并通过过零过程分割的三维结构与透射电子显微镜（TEM）的三维结构进行了比较相关分析。通过4Pi显微镜和TEM对货物蛋白原胶原3在分离的高尔基体堆叠的扁平囊泡中的分布进行了映射，以对它们的成像能力进行详细的比较分析。观察到结构之间具有高度相关性，表明4Pi显微镜具有特别的准确性。此外，首次通过4Pi显微镜观察到货物分子（水泡性口炎病毒G蛋白 - pEGFP）通过单个高尔基体堆叠（由半乳糖基转移酶 - 金星 - YFP标记）的运输。按照相关分析验证的程序，我们的运输实验表明：（i）VSVG - pEGFP迅速进入/离开单个高尔基体堆叠；（ii）VSVG - pEGFP从未完全充满GalT - 金星YFP区室；（iii）在VSVG - pEGFP到达后，GalT - 金星YFP区室体积增加。这一形态学证据支持了先前一些基于TEM对VSVG - pEGFP在高尔基体内部运输的观察结果，并为更好地理解蛋白质在高尔基体堆叠中的进展提供了新的见解。因此，我们的研究证明了超分辨率荧光显微镜结合过零分割程序在活细胞条件下对亚细胞器蛋白质分布进行结构研究的普遍适用性。
