Live cell imaging for infectious disease research

在过去，对病原体与宿主的细胞事件和分子相互作用的分析受到检测特定蛋白质的合适抗体的可用性或来自生化研究的推断的限制。然而，这些方法代表了实验性的“尸检”，其中必须从固定样本中推断出动态的细胞过程。然而，活细胞分子事件成像技术的发展，现在意味着有可能实时跟踪细胞内分子的位置、运动和相互作用，以及细胞之间的相互作用——例如，当病毒或细菌感染人体细胞时。这些发展是通过工程和在细胞内表达与自然荧光蛋白融合的蛋白质以及显微镜技术的改进而实现的。特别是，目前的共聚焦显微镜系统既可以在固定样品中传统的高分辨率定位蛋白质，也可以在限制荧光照射导致的细胞损伤的条件下检测活样品中的荧光蛋白。活细胞成像技术的应用对于病原体与其宿主或宿主细胞的相互作用的分析尤其有效。最近，在技术上已经可以设计感染因子，如病毒、细菌或原生动物寄生虫来表达一种或多种与荧光蛋白结合的蛋白质。当与哺乳动物宿主细胞内不同荧光蛋白表达的现有技术相结合时，就有可能跟踪病原体与宿主之间的分子相互作用，或者跟踪病原体通过不同宿主细胞区室移动时的感染途径。这些技术在传染病研究中有非常广泛的应用，研究人员对这些应用程序感兴趣的是不同的过程，如：病毒蛋白与其他病毒蛋白和宿主细胞蛋白的完全相互作用。-利用荧光标记病毒跟踪宿主细胞和组织的感染过程。-抗体产生细胞向免疫系统呈递抗原，免疫效应细胞和调节细胞的信号事件和蛋白质相互作用。-利什曼原虫、锥虫和疟疾等原生动物病原体的感染过程、发育生物学和进化策略。这项技术的优势——能够对活的病原体及其与宿主细胞的相互作用成像——也有一个限制——需要对病原体进行安全遏制。因此，大学院系常规提供的设施不能用于处理传染性病原体。本申请建议建立一个活细胞成像设施，将感染因子安置在适当的条件下，以实现有效和安全的控制。所要求的设备是徕卡TMC-SP5，具有固定和活体样品的高分辨率成像能力，以及以高分辨率高速捕获动态细胞事件的能力。该设备将提供给爱丁堡大学传染病中心的一大群研究人员，并提供一个高端设施，有可能鼓励更广泛的群体共享资源、技术和知识。

In the past, the analysis of the cellular events and molecular interactions of pathogens with their hosts was limited by the availability of suitable antibodies to detect particular proteins, or by inference from biochemical studies. These approaches, however, represent experimental 'autopsies' in which dynamic cellular processes must be deduced from a fixed sample. Developments in the imaging of molecular events in live cells, however, now mean that it is possible to follow, in real time, the location, movement and interactions of molecules within cells, and the interactions between cells- for example when a virus or bacterium infects cells of the body. These developments have been made possible by the engineering and expression within cells of proteins fused to naturally fluorescent proteins and by improvements in microscope technology. In particular, current confocal microscope systems enable both conventional high resolution positioning of proteins within fixed samples and the detection of fluorescent proteins in live samples under conditions that limit the cell damage that results from fluorescence illumination. The application of live cell imaging technology is particularly powerful for analysis of the interactions of a pathogen with its host, or host cell. Recently, it has become technically possible to engineer infectious agents such as viruses, bacteria or protozoan parasites to express one or more proteins conjugated to a fluorescent protein. When combined with existing technology for the expression of distinct fluorescent proteins within a mammalian host cell, it is possible to track molecular interactions between the pathogen and host, or track the infection pathway as a pathogen moves through distinct host cellular compartments, for example. These technologies have very wide ranging applications in infectious disease research, with researchers comprising this application being interested in such diverse processes as: - The complete interactions of viral proteins with other viral proteins and the proteins of the host cell. - The use of fluorescently labelled viruses to track the infection process in host cells and tissues. - The presentation of antigens by antibody producing cells to the immune system and the signalling events and protein interactions of immune effector and regulatory cells. - The infection process, developmental biology and evolutionary strategies of protozoan pathogens such as Leishmania, trypanosomes and malaria. The advantage of this technology- the ability to image live pathogens and their interactions with host cells- also presents a limitation- the need for safe containment of the pathogen. In consequence facilities available routinely in University departments are not available for use with infectious agents. This application proposes to establish a live cell imaging facility for infectious agents housed under suitable conditions for effective and safe containment. The equipment requested, a Leica TMC-SP5, has the capability for high resolution imaging of fixed and live samples and of high-speed capture, at excellent resolution, of dynamic cellular events. The equipment will be available to a large group of researchers contained within the Centre for Infectious Diseases at the University of Edinburgh, and provide a high-end facility with the potential to encourage resource, technology and knowledge sharing among a far wider group.