Liverpool BioAFM: an integrated optical and atomic force microscope for research across the life sciences

植物或动物中的单个细胞暴露于其环境的变化（机械力，生化信号，温度，光变化......）。细胞必须解释这些信息，以适应和做出适当的反应。为了理解导致特定反应的分子机制（例如细胞死亡，分化，粘附，拓扑结构变化等），生物学家需要以仔细控制的方式操纵环境并测量影响的方法，例如对细胞内蛋白质的水平和定位或对细胞表面的结构特性的影响。每个细胞可能在不同的时间做出不同的反应，所以在真实的时间和每个细胞中跟踪事件是至关重要的。这可以使用BioAFM成像来实现，这是一种新兴技术，它将原子力显微镜（AFM）与先进的光学成像（例如共聚焦，TIRF）相结合。原子力显微镜是在纳米尺度上成像、测量和操纵物质的最重要工具之一。信息是通过用机械探针“感觉”表面来收集的。然而，这种技术并不能提供有关细胞内事件的信息，只能使用先进的光学成像技术，使用荧光标记的分子进行成像。虽然BioAFM技术已经在专业中心存在了十多年，但技术限制限制了从这些互补方法中容易地结合联合收割机测量的能力。只有现在的技术已经先进，并通过开发用户友好的例程来捕获和叠加信息进行了改进，它达到了它可以更广泛地提供给生命科学界的地步。我们建议购买第一台BioAFM显微镜在英国和安装在利物浦中心细胞成像（CCI）。CCI是一个开放获取的设施，因此来自几所大学和公司的团体可以使用显微镜。为了说明该设备将服务于科学的广度，我们简要介绍了以下三个示例项目：1。抗微生物耐药性的新型表面世界卫生组织和英国政府首席医疗官反复警告，强调日益增加的抗微生物耐药性对全球构成严重威胁。由于产品污染、能源损失和设备损坏，表面上的微生物活动和生物膜每年给英国工业造成数十亿英镑的损失。通过先进的抗菌表面控制感染是对抗耐药性的关键策略。独特的是，BioAFM将使我们能够了解有害细菌如何附着在表面并对其做出反应，并开发出可以对抗感染的纳米级材料。开发节水型生物燃料作物迫切需要提高生物燃料植物的能力，使其在不适合主要粮食作物的边际土地上高产和可持续地生长。使用新的显微镜，我们将更好地了解干旱适应的沙漠植物通过在夜间打开气孔并在炎热干燥的光照期关闭气孔来保存水分的机制。这些原理将被应用于生产具有这些特性的生物燃料作物。3.老化时关节的修复软骨的再生，作为关节之间的弹性垫，取决于软骨细胞产生和维持软骨物质的能力。软骨细胞功能的衰竭在老年人中观察到，并与骨关节炎有关，骨关节炎是英国最常见的关节炎类型，每年约有100万人受到影响。有了新的显微镜，我们将第一次跟踪衰老对软骨细胞在分子水平上对压力做出反应的能力的影响，并开始解剖触发软骨生产的关键途径。

Individual cells in a plant or an animal are exposed to changes in their environment (mechanical forces, biochemical signals, temperature, light variations...). Cells have to interpret this information to adapt and respond appropriately. To understand the molecular mechanisms leading to a particular response (e.g. cell death, differentiation, adhesion, changes in topology etc), biologists need means of manipulating the environment in a carefully controlled manner and measuring the effects, for instance on the levels and localisation of proteins inside cells or on the structural properties of the cell surface. Each individual cell might respond differently from its neighbour and at a different time so it is crucial to follow the events in real time and in each individual cell. This can be achieved using BioAFM imaging, an emerging technology, which combines Atomic Force Microscopy (AFM) with advanced optical imaging (e.g. confocal, TIRF). AFM is one of the foremost tools for imaging, measuring, and manipulating matter at the nanoscale. Information is gathered by "feeling" the surface with a mechanical probe. However, this technique does not provide information about the events inside cells, which can only be imaged using advanced optical imaging using fluorescently labelled molecules. Although BioAFM techniques have existed for over a decade in specialist centres, technological limitations restricted the ability to readily combine measurements from these complementary approaches. Only now that the technology has advanced and been refined through the development of user-friendly routines to capture and overlay information has it reached the point where it can be made available more widely to the life science community.We propose to purchase one of the first BioAFM microscopes in the UK and to install it in the Liverpool Centre for Cell Imaging (CCI). The CCI is an open access facility, so the microscope will be accessible to groups from several universities and companies. To illustrate the breadth of the science that will be served by this equipment, we briefly present below three of our exemplar projects:1. Novel surfaces for anti-microbial resistanceRepeated warnings from the WHO and the UK Government's Chief Medical Officer emphasise the serious global threat of increasing antimicrobial resistance. Microbial activity and biofilms on surfaces cost UK industry billions of pounds each year due to product contamination, energy losses and equipment damage. Infection control, via advanced anti-microbial surfaces, is a key strategy to combat resistance. Uniquely, the BioAFM will enable us to learn how harmful bacteria attach to and respond to surfaces, and develop materials engineered at the nanoscale that can combat infection.2. Developing water-efficient biofuel cropsThere is an urgent and pressing need to improve the ability of biofuel plants to grow productively and sustainably on marginal land that is unsuitable for major food crops. Using the new microscope, we will better understand the mechanisms used by drought-adapted desert plants to conserve water by opening their stomatal pores at night and closing them during the hot, dry light period. These principles will then be applied to generate biofuel crops with these properties. 3. Repair of joints during ageingRegeneration of cartilage, which acts as a flexible cushion between joints, depends on the ability of chondrocytes to produce and maintain the cartilaginous material. Failure in chondrocyte cell function is observed in old age and is associated with osteoarthritis, the most common type of arthritis in the UK, which afflicts around 1 million people every year. With the new microscope, we will follow for the first time, the effects of ageing on the ability of chondrocytes to respond to compression forces at the molecular level and begin to dissect the key pathways that trigger cartilage production.