Super-Resolution Microscopy of live cells in 3D

观察分子如何以及何时在亚细胞结构中运动，使我们能够精确地查明细胞是如何做出决定的；这一基础知识对人类健康、粮食安全和生物技术研究至关重要。虽然电子显微镜可以清楚地分辨两个距离小于1纳米（nm）的结构，但由于光的固有特性，光学显微镜只能分辨距离至少220纳米的结构。尽管存在这种巨大的局限性，生物学家通常更喜欢光学显微镜，因为它允许跟踪和共同染色多个生物分子（使用不同颜色的荧光探针）来比较和揭示它们在活细胞中的作用和命运。最近，一组革命性的技术突破了光学显微镜的220纳米分辨率障碍，这些技术统称为超分辨率显微镜方法；这些方法获得了2014年的诺贝尔化学奖。然而，超分辨率显微镜（SRM）主要用于生成静态快照或几分钟的微小电影爆发，因为两个关键障碍：(i) SRM将活标本暴露在光线下超过几秒钟导致损伤（光毒性）；（ii） SRM需要收集大量快照，这会减慢3D数据采集速度，无法可靠地跟踪3D中快速移动的结构。因此，SRM不能有效地用于对光高度敏感的快速生物过程的活细胞研究。例如，DNA损伤修复途径、细胞分裂机制和光合作用步骤的研究需要在不诱导光毒性的情况下快速获取图像的方法。最近，新型的超分辨率显微镜能够对光敏过程进行长期的实时成像。（提高灵敏度的工具，降低光毒性的数据采集速度）已经商业化。我们的目标是利用这一最新发展，建立一个多用户SRM设施，用于在几个模型系统中进行光敏活细胞研究。多用户SRM设备将推动正在进行的传统光学显微镜研究进入超分辨率制度，以便涉及微小亚细胞结构的生物过程，尺寸为100-130纳米，可以更详细地研究。例如，对细胞内部小于150纳米的研究将从根本上受益于这种新设备。我们期望这个多用户设施不仅能够在BBSRC资助的研究人员领导的17个不同研究领域进行高影响力的研究，而且还将有助于分享方法，以推动从细菌到人类细胞等一系列模式生物的广泛亚细胞结构的成像。该联盟的研究人员将共同帮助确定或修改软件工具，以推进超分辨率图像和电影的分析。确定细胞内生物分子的水平和定位何时发生变化，对于揭示生物分子如何自我组织、相互沟通和控制活细胞的功能至关重要。为此，研究人员将把超分辨率成像与延时显微镜结合起来——延时显微镜是一种方法，在一段时间内记录亚细胞结构的图像（使用荧光标记的生物分子），以揭示细胞内动态变化的顺序。总之，所要求的超分辨率显微镜将允许几个BBSRC资助的小组长时间观察光敏过程，以测量100-130 nm分辨率精度的生物分子的定量变化。因此，多用户SRM设备将为研究人员提供所需的工具，以前所未有的空间和时间细节扩展我们对亚细胞结构和多蛋白组织的知识。

Observing how and when molecules move within subcellular structures allows us to precisely pinpoint how cells make decisions; this fundamental knowledge is critical for human health, food security and biotechnology research. While Electron Microscopy can clearly resolve two structures that are separated by less than one nanometer (nm), Light Microscopy had been limited to resolving structures that are at least 220 nm apart, due to the intrinsic properties of light. Despite this dramatic limitation, biologists frequently prefer Light Microscopy as it allows the tracking and co-staining of multiple biomolecules (using differently coloured fluorescent probes) to compare and unravel their roles and fates in living cells. The 220 nm resolution barrier in Light Microscopy was recently broken by a set of revolutionary techniques which are collectively known as Super-Resolution Microscopy methods; the methods won the Nobel prize for Chemistry in 2014. Super-Resolution microscopy (SRM) however has been primarily used to generate static snapshots or tiny bursts of movies for a few minutes because of two key hurdles: (i) SRM exposes live specimens to light for longer than a few seconds inducing damage (photo-toxicity) and (ii) SRM requires the collection of a huge number of snapshots which slows 3D data acquisition, disallowing reliable tracking of fast moving structures in 3D. Hence, SRM could not be effectively used for live-cell studies of rapid biological processes that are highly sensitive to light. For example, studies of DNA damage repair pathways, cell division mechanisms and steps of photosynthesis require methods that allow fast image acquisition without inducing phototoxicity. Recently, newer Super-Resolution microscopes capable of long-term live-imaging of light sensitive processes (ie., tools rendering increased sensitivity, data acquisition speed that reduce phototoxicity) have become commercially available. We aim to take advantage of this recent development and establish a multi-user SRM facility for light sensitive live-cell studies in several model systems. The multi-user SRM facility will push forward ongoing conventional light microscopy studies into the super-resolution regime so that biological processes involving tiny sub-cellular structures, 100-130 nm in size, can be studied in greater detail. For example, studies of compartments inside cells that are all less than 150 nm in size will radically benefit from using this new facility. We expect this multi-user facility to not only enable high-impact research in 17 different research areas led by BBSRC funded investigators, it will also help share methodologies to push forward the imaging of a wide range of subcellular structures in a range of model organisms, from bacteria to human cells. The researchers of the consortium will together help identify or modify software tools to advance the analysis of Super-Resolution images and movies. Determining when changes in the levels and localisation of biomolecules occur within cells is crucial to reveal how biomolecules organize themselves, communicate with each other and control the function of living cells. For this purpose, researchers will combine super-resolution imaging with time-lapse microscopy - a method where images of sub-cellular structures (using fluorescently tagged biomolecules) are recorded through a period of time to reveal the sequence of dynamic changes within cells. In summary, the requested super-resolution microscope will allow several BBSRC funded groups to observe photosensitive processes for long period of hours to measure quantitative changes in biomolecules within 100-130 nm resolution accuracy. Thus, the multi-user SRM facility will provide researchers with the tools needed to expand our knowledge of subcellular structures and multiprotein organisation in unprecedented spatial and temporal detail.