High-resolution, large scanning atomic force microscope (AFM) for capturing cellular processes in action

近年来，原子力显微镜（AFM）已成为在纳米尺度上研究各种衬底形貌特性的关键分析工具。虽然最初的应用主要集中在表面科学和摩擦学应用上，但这项技术现在已经足够成熟，可以发展并接受新的挑战，例如理解管理单个细胞核心内发生的许多基本生物过程的分子机制的物理学基础。由于其规模庞大，跨越到约。由于高度为30微米，这些细胞测量受到当前原子力显微镜仪器可承受的（有限）成像尺寸的严重阻碍。在这里，我们的目标是获得一个高分辨率，快速，大型扫描原子力显微镜（AFM），将规避这些技术限制，从而使我们能够可视化和量化分子相互作用的整个活细胞和组织在高空间，时间和力的分辨率。它与高分辨率光学显微镜的独特组合将允许单分子纳米力学与单分子生物光子学耦合。由于扫描探针技术已经获得了分子/原子水平的实验访问，许多尚未探索的关键问题现在可以通过实验进行攻击。例如，虽然一般的热力学定律是针对大的分子集合推导的，但许多关键的生物过程只需要几个单独的分子就可以发生。因此，新的单分子实验，经常发生在非平衡条件下，将探索经典热力学定律的范围和有效性，以描述在活细胞的框架内真实的时间发生的非平衡生物过程。此外，通过推动仪器的极限，地形亚纳米分辨率将允许直接观察和测量不同的生物分子界面的物理特性与关键的体内影响。与光学显微镜的新组合将使联合收割机的两种显微镜技术的优势和捕获的单分子过程中发生的细胞基板（AFM）和那些发生在细胞内部，使用荧光显微镜。结合起来，这些实验将允许对细胞内发生的单个过程进行全面的观察，并进行前所未有的单分子检测。由这种新型仪器实现的研究是开放式的。特别是，它将有助于阐明细胞力学的分子机制，以及基质刚度决定单个干细胞命运的机械反馈机制。它还将允许直接探测几个基因被机械激活的假设，并且机械力可以以不依赖于简单阻尼扩散的有效方式从细胞外基质向下传递到细胞核。这些实验将着重强调机械传导和细胞粘附的机制，从而极大地补充和扩展了伦敦国王学院和伦敦地区其他领先机构（牛津、弗朗西斯克里克研究所）目前正在进行的世界领先的研究。此外，这种新仪器所允许的技术发展将使新的基于细胞的纳米技术应用成为可能，伦敦纳米技术中心（LCN）对此特别感兴趣。总而言之，这些设备将促进和鼓励与其他伦敦（和英国）的机构在机械生物学和生物物理学的密集和多产的研究领域进行富有成效的合作，允许跨学科的方法和居住从单细胞到单分子水平。

Atomic force microscopy (AFM) has become, in the last recent years, a key analytic tool to investigate the topographical properties of a wide variety of substrates, at the nanometer scale. While initial applications were basically focused on surface science and tribological applications, this technique has now matured enough to evolve and take on new challenges, such as the understanding of the physics underlying the molecular mechanisms governing a number of fundamental biological processes occurring within the core of an individual cell. Due to their large size, spanning up to ca. 30 micrometers in height, these cell measurements have been severely hampered by the (limited) imaging size affordable by current AFM instrumentation. Here we aim to acquire a high-resolution, fast, large scanning atomic force microscope (AFM) that will circumvent these technical limitations, thus enabling us to visualise and quantify molecular interactions on whole living cells and tissues at high spatial, temporal, and force resolution. Its unique combination with high-resolution optical microscopy will allow coupling single molecule nanomechanics with single molecule biophotonics. Since Scanning Probe techniques have gained experimental access to the molecular/atomic level, many crucial questions that remained unexplored can now be experimentally attacked. For example, while general thermodynamics laws were deducted for large ensembles of molecules, many key biological processes require only a few individual molecules to occur. Therefore, new single molecule experiments, often occurring under non-equilibrium conditions, will probe the extent and validity of classical thermodynamics laws to describe out-of-equilibrium biological processes occurring in real time within the framework of a living cell. Moreover, by pushing forward the instrumental limits, topographic sub-nanometer resolution will allow direct observation and measurement of the physical properties of distinct bio-molecular interfaces with key in-vivo implications. The novel combination with optical microscopy will enable to combine the strengths of both microscopy techniques and capture the single molecule processes occurring on the cell substrate (AFM) and those occurring in the cell interior, using fluorescence microscopy. Combined, these experiments will allow a comprehensive vista on individual processes occurring within a cell with unprecedented single molecule detection. The research enabled by this novel instrumentation is open ended. In particular, it will help elucidate the molecular mechanisms underlying cell mechanics, and the mechanical feedback mechanism by which substrate stiffness dictates the fate of individual stem cells. It will also allow to directly probe the hypothesis that several genes are mechano-activated, and that mechanical forces can transmit from the extracellular matrix down to the cell nucleus in an efficient way that does not rely on simple damped diffusion. These experiments will put a strong accent on the mechanisms governing mechanostranduction and cell adhestion, thus greatly complementing and expanding world-leading research being currently conducted in King's College London and other leading institutions in the London Area (Oxford, Francis Crick Institute). Moreover, the technical developments allowed by this new instrument will enable new cell-based nanotechnological applications, of particular interest for the London Centre for Nanotechnology (LCN). Altogether, this equipment will foster and encourage fruitful collaborations with other London- (and UK-) based institutions working on the intense and prolific research fields of mechanobiology and biophysics, allowing a cross-disciplinary approach and dwelling from the single cell to the single molecule level.