Super-Resolution Microscopy of live cells in 3D

观察分子如何以及何时在亚细胞结构内移动，使我们能够精确地确定细胞如何做出决定;这一基础知识对人类健康，食品安全和生物技术研究至关重要。虽然电子显微镜可以清晰地分辨出两个相距小于1纳米（nm）的结构，但由于光的固有特性，光学显微镜仅限于分辨相距至少220 nm的结构。尽管有这种巨大的局限性，生物学家通常更喜欢光学显微镜，因为它允许跟踪和共染色多个生物分子（使用不同颜色的荧光探针），以比较和解开它们在活细胞中的作用和命运。光学显微镜中的220 nm分辨率障碍最近被一组革命性的技术所打破，这些技术被统称为超分辨率显微镜方法;这些方法在2014年获得了诺贝尔化学奖。然而，超分辨率显微镜（SRM）主要用于生成几分钟的静态快照或微小的电影突发，因为有两个关键障碍：（i）固体火箭发动机将活样品暴露在光下超过几秒钟，导致损伤（ii）SRM需要收集大量的快照，这减慢了3D数据采集，不允许在3D中可靠地跟踪快速移动的结构。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，对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允许快速图像采集而不诱导光毒性的方法。最近，能够对光敏过程进行长期实时成像的新型超分辨率显微镜（即，提供增加的灵敏度、降低光毒性的数据采集速度的工具）已经可商购获得。我们的目标是利用这一最新的发展，并建立一个多用户SRM设施，在几个模型系统的光敏活细胞研究。多用户SRM设施将推动正在进行的传统光学显微镜研究进入超分辨率领域，以便更详细地研究涉及100-130 nm大小的微小亚细胞结构的生物过程。例如，研究细胞内的隔间，都小于150纳米的大小将从使用这种新的设施从根本上受益。我们希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，还将有助于分享方法，推动从细菌到人类细胞的一系列模型生物中广泛的亚细胞结构的成像。该联盟的研究人员将共同帮助识别或修改软件工具，以推进对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组织自己，相互交流和控制活细胞的功能至关重要。为此，研究人员将联合收割机结合超分辨率成像与延时显微镜-一种通过一段时间记录亚细胞结构图像（使用荧光标记的生物分子）以揭示细胞内动态变化序列的方法。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分辨率精度内生物分子的定量变化。因此，多用户SRM设施将为研究人员提供所需的工具，以扩大我们对亚细胞结构和多蛋白组织的知识，在前所未有的空间和时间细节。

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.