A super-resolution multi-scale in vitro and in vivo imaging platform at Harwell: building models of development and disease from molecules to mammals

捕捉一种疾病的“原因”和一种治疗的“方法”需要建立疾病及其在治疗过程中的演变的模型。理想情况下，这个模型需要涵盖一切，从单个分子与分子网络的相互作用，到支撑表型发育的细胞间通信网络。为了实现这一目标，我们需要在生物运作的不同尺度上架起桥梁--分子、细胞和整个有机体。为此，一种方法是在不同的尺度上对细胞和生物成像：(I)纳米尺度，以显示细胞如何在分子水平上组织以及分子治疗如何影响分子间的相互作用；(Ii)微型尺度，以显示细胞器的功能及其在治疗挑战下的重组；(Iii)中尺度尺度，以研究细胞在发育和/或疾病进展过程中的行为；(Iv)整个生物体水平，以监测整个有机体对治疗(例如肿瘤缓解)的反应的变化和福祉。只有把这些拼图拼在一起，人们才能理解突变的基因和蛋白质如何影响发育，预测“下一代”疗法如何取得突破性进展，并阐明如何将治疗剂输送到疾病的局部区域，以最大限度地提高临床效益，同时限制副作用。细胞是罗伯特·胡克(Robert Hooke，1635-1703)在光学显微镜下发现的。瑞利勋爵(1842-1919)经验性地确定，衍射限制显微镜的分辨率不能优于定义微尺度的光波长(即200 nm)的~1/2。几个世纪以来，这是光学显微镜的一个基本限制，因为这种分辨率不足以解决支撑生物学的纳米级过程。尽管如此，由于许多有机标记的出现和绿色荧光蛋白的发现，荧光显微镜几十年来一直是许多基于体外的生物医学科学关键发现的基础。上个千年末，利用一种具有挑战性的技术--受激发射耗尽显微镜，其分辨率可达~20 nm，随后是破坏性较小的90 nm分辨率的结构照明显微镜，光学显微镜的分辨率极限被打破。在过去的十年里，通过使用分子比光的波长小得多的基本原理，更简单的超分辨率显微镜模式获得了类似的分辨率，因此可以被认为是“单点”发射体。这一点非常重要，因为它们在空间中的位置因此可以通过对显微镜光学产生的类似斑点的图像进行反卷积，以纳米级的精度确定，顺便说一句，这是与光学显微镜相关的低分辨率的根源。纳米尺度的高分辨率图像是通过将单独的分子图像组合在一起形成的，这是一个耗时的过程，但已经产生了壮观的结果。中尺度的高分辨率成像对于了解基本的哺乳动物生物学至关重要。OCT的开发是为了满足对快速成像工具的需求，以表征整个生物体中细胞之间的相互作用，以便对人类病理生理学进行建模，并在临床前研究中评估治疗方法产生的益处。我们已经形成了跨学科的合作伙伴关系，寻求利用新一代世界领先的超分辨率显微镜与最先进的活体成像方法(如OCT)相结合。我们的原则是打破领域之间的障碍，以方便更广泛的生物医学界利用这些新技术，并将英国置于成像领域的前沿。哈威尔校区跨学科的环境和科学家的集中精力将有助于我们在未来十年努力巩固基本发现。

Capturing the 'why' of a disease and the 'how' of a treatment requires building a model of the disease and its evolution during treatment. This model ideally needs to capture everything, from the interplay of a single molecule with a molecular network, to inter-cellular communication networks that underpin the development of a phenotype. To reach this goal we need to bridge the different scales on which biology operates - the molecular, the cellular and the whole organism. One way to do this is to image cells and organisms at different scales: (i) the nanoscale to show how cells are organised at the molecular level and how molecular therapies affect molecular interactions; (ii) the microscale to show the functions of cellular organelles and their reorganisation under therapeutic challenge; (iii) the mesoscale to investigate cellular behaviour during development and/or the progress of disease; (iv) and the whole organisms level, to monitor changes and the well-being of the entire organism in response to therapy (e.g. tumour remission). Only when these jigsaw pieces are put together should one be able to understand how mutated genes and proteins affect development, predict how the "next generation" therapies can deliver breakthrough advances and elucidate how therapeutic agents can be delivered to focal areas of disease to maximize clinical benefit while limiting side effects.Cells were discovered by Robert Hooke (1635-1703) under the optical microscope. Lord Rayleigh (1842-1919) empirically determined that the resolution of a diffraction-limited microscope can be no better than ~ 1/2 of the wavelength of light (i.e. >200 nm), which defines the microscale. For centuries this was a fundamental limitation of light microscopy, as this resolution is insufficient to resolve the nanoscale processes underpinning biology. Despite this, through the availability of many organic labels and the discovery of green fluorescent protein, fluorescence microscopy has been fundamental for decades to many of the in vitro-based key discoveries in the biomedical sciences.The 'resolution limit' of light microscopy was broken at the end of the last millennium using a challenging technique, stimulated emission depletion microscopy, which showed ~20 nm resolution, followed by the less damaging structured illumination microscopy with 90 nm resolution. During the last decade, 'simpler' modes of super-resolution microscopy achieved similar resolutions by using the fundamental principle that molecules are much smaller than the wavelength of light and therefore can be considered 'single point' emitters. This is very important because their position in space can therefore be determined with nanometre accuracy via deconvolution of the 'blob'-like spot image created by the microscope optics, which incidentally is the origin of the poor resolution associated with light microscopy. High resolution images at the nanoscale are formed by putting together individual molecular images, a time-consuming process which nevertheless has already delivered spectacular results.High resolution imaging at the mesoscale is critical to understand basic mammalian biology. OCT was developed to address the need for fast imaging tools to characterise the inter-play between cells in a whole organisms in order to model human pathophysiology, and assess benefits resulting from therapeutic treatments in pre-clinical research.We have formed an interdisciplinary partnership that seeks to exploit a new generation of world-leading super-resolution microscopy in combination with state-of-the-art in vivo imaging methods (like OCT). Our principle is to break the barriers between fields to ease the exploitation of these new technologies by the wider biomedical community and to place the UK at the imaging forefront. The interdisciplinary environment and concentration of scientists at the Harwell Campus will help in our efforts to underpin fundamental discoveries in the next decade.