Masedunskas Andrius, Porat-Shliom Natalie, Tora Muhibullah, Milberg Oleg, Weigert Roberto
Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research, National Institutes of Health.
J Vis Exp. 2013 Sep 1(79):50558. doi: 10.3791/50558.
Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level. In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.
在此，我们描述了一种基于共聚焦活体显微镜技术对活体啮齿动物亚细胞结构进行成像的方法。作为模型器官，我们使用活体小鼠的唾液腺，因为它们具有多个优点。首先，它们易于暴露以便接触光学元件，并且可以稳定下来以促进减少由于心跳和呼吸引起的运动伪影。这极大地便于对小的亚细胞结构进行成像和追踪。其次，唾液腺的大多数细胞群体可从器官表面接触到。这允许使用共聚焦显微镜，其空间分辨率高于用于体内成像的其他技术，如双光子显微镜。最后，唾液腺可以很容易地进行药理学和遗传学操作，从而提供一个强大的系统来在分子水平上研究生物学过程。在本研究中，我们专注于一种方案，旨在追踪腺泡细胞中分泌颗粒胞吐作用的动力学以及在β-肾上腺素能受体刺激下分泌颗粒融合的顶端质膜的动态变化。具体而言，我们使用了一种共表达胞质绿色荧光蛋白（GFP）和与荧光蛋白串联番茄红素融合的膜靶向肽的转基因小鼠。然而，我们用于稳定和成像唾液腺的方法可以扩展到其他小鼠模型，并与其他标记体内细胞成分的方法相结合，从而能够可视化各种亚细胞结构，如内体、溶酶体、线粒体和肌动蛋白细胞骨架。
