Leeds integrated atomic force and confocal microscopy for life science applications

眼见为实：自现代科学开始以来，成像技术一直是生物学进步的工具。该项目为利兹大学的研究人员提供了一种独特的仪器，该仪器集成了两种先进的显微镜，用于记录图像，并在非常小的尺度上进行机械和光学测量。有了这种能力，传统技术难以解决的生物学问题就可以得到解决。光学显微镜（OMs），自伽利略时代以来不断发展，现在非常强大和多功能。它们的放大能力使人们能够更清楚地看到生物结构，这种能力今天仍然很受欢迎，可以看到活细胞及其内部结构。然而，可以吸收和发射光的荧光标签，以及以精确的分辨率测量吸收和发射之间时间的方法，为OM提供了一个新的维度。随着时间分辨荧光，生物分子运动的动力学，生物分子的化学环境，生物分子之间的相互作用和距离现在可以非常详细地研究。然而，一些根本性的限制依然存在。从单个生物分子到细胞和组织，机械力和弹性等物理特性对生物系统的功能也至关重要，但这些都无法用光学显微镜观察到。自三十年前发明以来，原子力显微镜（AFM）已经成为一种独特的技术，可以直接测量长度缩小到单个分子的机械性能。基于扫描样品的微小尖端，它还能够产生高度（“地形”）图像，其分辨率优于光学显微镜，小于1纳米（典型细菌大小的1/1000）。原子力显微镜作为一种光学显微镜，可以应用于液体环境，因此可以对生物样品进行活体探测。OM和AFM是互补的技术，因为它们测量不同的物理参数。将它们结合到一个设备中，并同时使用两种技术对同一样品进行表征，从而能够将数据关联起来，并获得单独使用任何一种技术都无法获得的新见解，甚至无法单独使用两种技术。在更大的范围内，细胞和组织可以用独特的方法进行研究。例如，我们将描述神经周围网络的结构和机械特性，神经周围网络是覆盖神经元表面并调节神经元连接形成方式的绝缘护套。这一发现可能有助于开发延缓痴呆症患者记忆丧失或修复脊髓损伤的方法。在中等尺度上，分子组合可以被探测，例如，血凝块是如何在纳米尺度上构成的，以及构成血凝块的单个纤维的机械特性是什么。这将有助于更好地了解血栓形成及其解决方案，这是预防和治疗心脏病发作的关键。在更小的尺度上，我们将研究生物膜，这是一种由许多松散相互作用的分子（脂质和蛋白质）组成的迷人结构，形成一种超薄薄膜，这种薄膜是高度动态的，是细胞通信的关键，但又足够稳定，可以分隔细胞和组织。使用该组合仪器，我们将能够研究生物膜的结构和动力学，例如了解脂质组织（对细胞信号传导很重要），膜蛋白的结晶（促进其结构分析，这是药物开发的重要步骤），以及光收集植物膜如何工作，用于“下一代”生物启发能源生成方法。

Seeing is believing: imaging techniques have been instrumental for progress in biology since the inception of modern science. This project provides researchers at the University of Leeds with a unique instrument that integrates two types of advanced microscope for recording images and making mechanical and optical measurements at very small scales. With this capability, biological problems can be solved that have proven intractable with conventional techniques.Optical microscopes (OMs), continuously developed since the time of Galileo, are now extremely powerful and versatile. Their ability to magnify enabled a clearer view of biological structures and this capacity remains much appreciated today to visualize live cells and the structures inside them. However, fluorescent tags that can absorb and emit light, and methods to measure the time between absorption and emission with exquisite resolution, have given OM a new dimension. With time-resolved fluorescence, the dynamics of biomolecular motion, the chemical environment of biomolecules, and the interactions and distances between biomolecules can now be studied in great detail.Some fundamental limits remain, however. Physical properties such as mechanical forces and elasticity are also vital for the function of biological systems, from single biomolecules to cells and tissues, yet these are not accessible with optical microscopes. Since its invention three decades ago, atomic force microscopy (AFM) has emerged as a unique technique to directly measure mechanical properties at length scales down to individual molecules. Based on a tiny tip that scans across the sample, it is also able to produce height ('topographical') images with a resolution that is superior to that of optical microscopes, of less than one nanometer (1/1000 the size of a typical bacterium). AFM, as OM, can be applied in liquid environment and so biological samples can be probed alive. OM and AFM are complementary techniques, because they measure distinct physical parameters. Combining them into one device and characterizing the same sample at the same time with both techniques affords the ability to correlate data and gain new insight that cannot be obtained with either technique alone, or even with the two techniques applied separately one after the other.On a larger scale, cells and tissues can be studied in unique ways. For example, we will characterize the structure and mechanical properties of the perineuronal net, an insulation sheath that covers the surface of neurons and modulates how neuronal connections form. Such insight may help to develop ways to delay memory loss in dementia or to repair spinal cord injury. On an intermediate scale, molecular assemblies can be probed, for example how blood clots are structured on the nanometer scale and what the mechanical properties of the individual fibres are that make up the clot. This will help to better understand thrombosis and its resolution, key to the prevention and treatment of heart attack. On yet smaller scales, we will study biological membranes, fascinating structures made from many loosely interacting molecules (lipids and proteins) forming an ultrathin film that is highly dynamic and key to cellular communication yet stable enough to compartmentalize cells and tissues. With the combined instrument, we will be able to study the structure and dynamics of biological membranes, for example to understand lipid organization (important for cell signaling), crystallization of membrane proteins (to facilitate their structural analysis, an important step for drug development), and how light-harvesting plant membranes work, for 'next-generation' bio-inspired energy generation methods.