Multiparametric advanced fluorescence imaging strategies for in situ analysis of live cell signalling

要了解和战胜人类疾病的原因，我们必须了解组成人体组织和器官的单个细胞的基本结构和功能。例如，为了设计针对癌症的有效治疗方法，我们首先需要回答有关如何控制细胞生长、分裂和移动的基本问题。1665年，罗伯特·胡克第一个使用显微镜描述细胞结构，从那时起，显微镜已经成为全世界细胞生物学家最强大的工具之一。当然，自从光学显微镜发明以来，它们的能力一直在不断增加，但值得注意的是，最戏剧性的改进发生在过去十年左右。在此期间，物理学家已经研究出了如何测量单个蛋白质在细胞中的位置，其精度大约是之前认为的可能的十倍。这一点很重要，因为我们现在可以更详细地了解细胞的内部结构和组织。与此同时，物理学家和生物学家合作开发了显微镜方法，这种方法不只是绘制一张特定蛋白质在细胞内的位置图，还可以绘制出蛋白质A与蛋白质B结合的准确位置图。这是一个根本性的进步，因为细胞功能由特定蛋白质之间的这种相互作用的路径和网络控制。潜在地，这些新的显微镜提供了一个了解细胞内部工作的窗口，使我们能够看到这些蛋白质网络。然而，目前最详细的图像只能从化学保存的细胞中获得，而不是从活细胞中获得，而且每一张图像都需要几分钟的时间来记录。这是一个严重的问题，因为控制细胞功能的蛋白质之间的相互作用在几秒钟的时间尺度上是瞬时发生的。要了解细胞的功能，我们需要的是电影而不是静止的图像。在目前的提议中，生物学家和物理学家将共同开发这项技术，使我们能够在毫秒内记录活细胞中蛋白质位置和相互作用的详细地图，而不是几分钟或几小时。我们认为，这些新的发展将释放这些显微镜的潜力，向我们展示细胞如何在分子水平上工作。

To understand and combat the causes of human disease, we must understand the basic structure and function of the individual cells that make up the tissues and organs of the human body. For example, to allow the design of effective therapies to target cancer we first need to answer fundamental questions about how the growth, division and movement of cells are controlled. Robert Hooke was the first to use microscopes to describe cell structure in 1665, and since then microscopy has become one of the most powerful tools for cell biologists across the world. The power of light microscopes has of course continued to increase since their invention but, remarkably, the most dramatic improvement has come in the last ten years or so. In that period physicists have worked out how to measure the location of a single protein in a cell with a precision about ten times better that was previously thought possible. This is important because we can now see the internal structure and organisation of cells in much more detail. In parallel, physicists working together with biologists developed microscopical methods that, instead of just producing a map of the locations of one particular protein inside a cell, can produce a map of precisely where protein A is bound to protein B. This is a fundamental advance, because cell function is controlled by pathways and networks of such interactions between specific proteins. Potentially then, these new microscopes provide a window into the internal workings of a cell that allow us to see these protein networks. However, at the moment, the most detailed images can only be obtained from chemically preserved rather than living cells, and each image takes many minutes to record. This is a serious problem, because the interactions between proteins that control cell function take place transiently on the time scale of seconds. To understand cell function, we need movies rather than still images. In the present proposal, biologists and physicists will work together to develop the technology to allow us to record the detailed maps of protein locations and interactions in live cells in milli-seconds rather than minutes or hours. We think that these new developments will unlock the potential of these microscopes to show us how cells work at the molecular level.