Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy
Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy
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DOI：
10.3791/58355
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发表时间：
2018-08-01
影响因子：
1.2
通讯作者：
Gonzalez, Santiago F.
中科院分区：
文献类型：
--
作者：
Palomino-Segura, Miguel;Virgilio, Tommaso;Gonzalez, Santiago F.
体内细胞-细胞或细胞-病原体相互作用的分析是理解对感染的免疫应答动力学的重要工具。双光子活体显微镜（2 P-IVM）允许观察活体动物深部组织中的细胞相互作用，同时最大限度地减少图像采集过程中产生的光漂白。迄今为止，已经描述了淋巴和非淋巴器官的2 P-IVM的不同模型。然而，呼吸器官的成像仍然是一个挑战，由于运动与呼吸cycle of the animal. There，我们描述了一个协议，可视化体内免疫细胞相互作用的小鼠感染流感病毒的气管使用2 P-IVM。为此，我们开发了一个定制的成像平台，其中包括手术暴露和气管插管，然后在粘膜上皮中的中性粒细胞和树突状细胞（DC）的动态图像的采集。此外，我们详细介绍了流感鼻内感染和气管免疫细胞流式细胞术分析所需的步骤。最后，我们分析了中性粒细胞和DC运动以及他们的相互作用过程中的电影。该方案允许生成评估气管中细胞-细胞相互作用所需的稳定和明亮的4D图像。
The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.