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在环境的变化中(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。为了了解导致特定反应(如细胞死亡、分化、黏附、拓扑变化等)的分子机制，生物学家需要以谨慎控制的方式操纵环境并测量其影响，例如对细胞内蛋白质的水平和定位或对细胞表面结构特性的影响。每个单独的细胞可能会在不同的时间和不同的相邻细胞做出不同的反应，因此实时跟踪每个单独的细胞中的事件至关重要。这可以使用生物原子力显微镜成像，这是一项新兴技术，它结合了原子力显微镜(AFM)和先进的光学成像(如共焦，TIRF)。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的工具之一。信息是通过用机械探头“感觉”表面来收集的。然而，这项技术不提供细胞内事件的信息，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。尽管生物原子力显微镜技术在专业中心已经存在了十多年，但技术限制限制了将这些互补方法的测量结果轻松结合起来的能力。直到现在，通过开发用户友好的例程来捕获和覆盖信息，这项技术才得到了进步和完善，才达到了让生命科学界更广泛地使用这一技术的地步。我们计划购买英国第一台生物AFM显微镜之一，并将其安装在利物浦细胞成像中心(CCI)。CCI是一个开放获取设施，因此来自几所大学和公司的团体将可以访问该显微镜。为了说明这种设备将服务于科学的广度，我们简要介绍了我们的三个示范项目：1.抗菌素耐药性的新表面世卫组织和英国政府的首席医疗官反复发出警告，强调抗菌素耐药性增加的严重全球威胁。由于产品污染、能源损失和设备损坏，表面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。通过先进的抗微生物表面控制感染是对抗耐药性的关键策略。独一无二的是，生物原子力显微镜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，并开发出纳米级的材料来对抗感染。发展节水生物燃料作物迫切需要提高生物燃料植物在不适合主要粮食作物种植的边际土地上高效和可持续生长的能力。使用这种新的显微镜，我们将更好地了解适应干旱的沙漠植物通过在夜间打开气孔并在炎热、干燥的光照期间关闭气孔来保存水分的机制。然后，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.老化过程中关节的修复软骨的再生，作为关节间的软垫，取决于软骨细胞产生和维持软骨材料的能力。软骨细胞功能衰竭是在老年观察到的，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，我们将首次在分子水平上跟踪老化对软骨细胞应对压力的能力的影响，并开始剖析触发软骨产生的关键途径。

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.