Turnkey video-rate atomic force microscopy for nanometre resolution imaging of functional biomolecules and cellular surfaces

显微镜是我们进入细胞的窗口。它们的重要性从最近诺贝尔奖的超分辨率荧光显微镜奖(2014)和低温电子显微镜奖(2017)中可见一斑。这些诺贝尔奖还突出了目前在生物学中使用的主要类型的显微镜：荧光显微镜，它可以提供关于活细胞的信息，但分辨率相对较低(~100纳米)，以及电子显微镜，它可以提供生物分子和细胞的高分辨率图像，但需要冰冻样本。使用原子力显微镜，我们通过使用非常锋利的针有效地跟踪样本(生物或其他)的表面，从而绕过了其他类型显微镜的一些限制，使我们能够以~1 nm的分辨率成像功能生物分子和活细胞。由于我们逐行追踪这些样品来构建完整的图像，这是一种相对较慢的显微镜类型。在这里，我们申请资金购买所谓的高速原子力显微镜，它结合了多种开发技术，将图像采集速度从每帧几分钟提高到不到几秒。我们将在一个完善的用户设施中安装这些设备，并预计将在不同学科和机构中广泛使用。在中短期内，我们计划的第一批实验涉及决定细胞中DNA包装的蛋白质-DNA相互作用，为促进化学反应而开发的基于DNA的商业纳米材料，生物膜的重塑，定义活细胞如何与底物结合的分子相互作用，下一代抗生素对细菌表面的破坏，以及定义细胞形状和力学的分子结构。

Microscopes are our windows to the cell. Their importance is illustrated by the recent awards of Nobel prizes for super-resolution fluorescence microscopy (2014) and for cryo-electron microscopy (2017). These Nobel prizes also highlight the main types of microscopy that are currently used in biology: fluorescence microscopy, which can provide information on live cells, but at relatively poor resolution (~100s of nm), and electron microscopy, which can provide high-resolution images of biological molecules and cells, but requires samples to be frozen. Using atomic force microscopy, we circumvent some of the limitations of other types of microscopy by effectively tracing the surface of a sample (biological or other) with a very sharp needle, enabling us to image functional biological molecules and living cells as ~1 nm resolution. Since we trace these samples line by line to build an entire image, this is a relatively slow type of microscopy.Here we apply for funding to purchase a so-called high-speed atomic force microscope, which incorporates multiple developments to speed image acquisition up from minutes to less than seconds per frame. We will install the equipment in a well-established user facility and anticipate wide use across different disciplines and institutions. For the short-to-medium term, first experiments we plan are related to protein-DNA interactions that determine DNA packing in the cell, commercial DNA-based nanomaterials developed to facilitate chemical reactions, reshaping of biological membranes, molecular interactions that define how living cells bind to substrates, disruption of bacterial surfaces by next-generation antibiotics, and the molecular structures that define cell shape and mechanics.