Imaging cellular structure and function beyond the diffraction limit

超分辨显微镜(SRM)代表了光学成像领域的一场革命，对生命科学有着深远的影响。它使研究人员能够在几乎分子尺度上可视化活细胞和有机体的生物结构和功能。通常，生物过程是通过将荧光分子附着到感兴趣的蛋白质上来可视化的，通过这些荧光“标记”发出的光形成图像。光学分辨率定义了可以可视化的最小结构，长期以来，人们一直认为，由于光波的衍射，光学分辨率从根本上限制在&gt；200 nm以内。然而，最近在荧光显微镜方面的突破利用了系统地打开和关闭荧光分子发射的能力，以打破“衍射极限”，并达到10‘S纳米的分辨率-几乎是典型生物蛋白质的规模，这些蛋白质参与了决定细胞和有机体命运的信号过程，因此对疾病过程至关重要。该项目建立在MRC临床科学中心(CSC)和伦敦帝国理工学院之间的合作伙伴关系之上，以开发一系列SRM仪器，通过解决细胞核内、活细胞内和相互作用细胞的突触中的基本生物机制来促进我们对表观遗传学、基因和细胞的调节、免疫学和新陈代谢的理解。它还旨在将超分辨率显微镜作为CSC和帝国理工学院研究界广泛使用的工具。第一种SRM技术将是结构照明显微镜(SIM)，它需要用光的图案照明样品，并观察这些图案与样品结构之间的干涉图案。这有效地使普通显微镜的分辨率翻倍。第二种方法是激光扫描共焦显微镜的进展，即通过扫描聚焦的激光束穿过样品，并通过阻止焦点外或散射光的针孔逐个像素地收集荧光来对样品进行成像。聚焦激光光斑的大小决定了这台显微镜的分辨率。通过使用第二束共线的特殊形状的激光束，该激光束被同步扫描，并且可以关闭来自第一束焦点外部的分子的荧光，荧光激发被限制在比衍射极限更小的光点，从而可以实现低于50 nm的分辨率。由于这项技术可以阻挡散射或散焦的光，因此特别适合在活体中成像。第三种SRM模式依赖于在任何时候只有一小部分荧光标记被打开，以便可以像单分子阵列一样对待它们。它们各自的位置可以通过定位代表图像中每个分子的“斑点”的中心来确定-这是可能的，精确度为几纳米。通过依次打开荧光标记的不同子集并确定它们的单独位置，人们可以建立一个超分辨率的“图像”，它只是所有荧光分子的位置图。在这项提议中，我们将开发和应用这些新的成像技术来可视化传统显微镜无法获得的太小或无法获得的生物结构和过程。从细胞表面开始，研究人员将能够观察免疫细胞如何相互沟通和神经细胞之间的相互作用，以及肿瘤细胞如何与正常细胞相关。观察细胞内部，我们将可视化传达细胞能量状态和基因组组织方式的信号。这将有可能解决染色体在生殖细胞发育中的行为，这与最常见的不孕和出生缺陷原因有关。这项研究将对正常的生物过程和疾病状态提供新的见解。

Super-resolved microscopy (SRM) represents a revolution in optical imaging with profound implications for the life sciences. It allows researchers to visualise biological structure and function on almost the molecular scale in live cells and organisms. Typically biological processes are visualised by attaching fluorescent molecules to proteins of interest forming images from the light emitted by these fluorescent "labels". The optical resolution, which defines the smallest structures that can be visualised, has long been regarded as fundamentally limited to >200 nm by the diffraction of light waves. Recent breakthroughs in fluorescence microscopy, however, have exploited the ability to systematically switch the emission of fluorescent molecules on and off in order to break the "diffraction limit" and achieve resolutions down to 10's of nm - almost the scale of the typical biological proteins that are involved in signalling processes that determine the fate of cells and organisms and so are essential to disease processes. This project builds on the partnership between the MRC Clinical Sciences Centre (CSC) and Imperial College London to develop a range of SRM instruments to advance our understanding of epigenetics, regulation of genes and cells, immunology and metabolism by resolving fundamental biological mechanisms inside the nucleus, inside live cells and at the synapses of interacting cells. It also aims to establish super-resolved microscopy as a widely accessible tool for the research community at the CSC and Imperial. The first SRM technique would be structured illumination microscopy (SIM) that entails illuminating samples with patterns of light and observing the interference patterns between these patterns and the structure of the sample. This effectively doubles the resolution of a normal microscope. The second approach is an advance on laser scanning confocal microscopy, in which as sample is imaged by scanning a focused laser beam across a sample and collecting the fluorescence pixel by pixel through a pinhole that blocks out of focus or scattered light. The size of this focussed laser beam spot defines the resolution of this microscope. By using a second, collinear, specially shaped laser beam that is scanned synchronously and can switch off the fluorescence from molecules around the outside of the first beam focus, the fluorescence excitation is restricted to a smaller spot than the diffraction limit and thus resolutions below 50 nm can be realised. Because this technique can block scattered or out of focus light, it is particularly suitable for imaging in live organisms. The third SRM modality relies on having only a small fraction of the fluorescent labels being switched on at any time such that they can be treated like arrays of single molecules. Their individual positions can be determined by locating the centres of the "blobs" that represent each molecule in an image - which is possible to a precision of a few nm. By sequentially switching on different subsets of the fluorescent labels and determining their individual positions, one can build up a super-resolved "image" that is simply the map of the locations of all the fluorescent molecules.In this proposal, we will develop and apply these new imaging techniques to visualise biological structures and processes that are simply too small or inaccessible for conventional microscopes. Starting at the surface of cells, researchers will be able to observe how immune cells communicate and nerve cells interact with each other, and how tumour cells relate to normal cells. Looking inside cells, we will visualise signals that communicate the energy status of cells and how the genome is organised. It will be possible to resolve how chromosomes behave in developing germ cells, which is relevant to the most frequent causes of infertility and birth defects. The research will deliver new insights into both normal biological processes and disease states.