Photo-oxidation and cryofluorescence for Correlative Light Electron Microscopy.

相当大的技术发展使我们能够对活细胞进行成像，以获得对时间敏感的细胞事件（毫秒时间尺度）的令人难以置信的细节。电子显微镜技术的进步意味着我们可以以非常高的分辨率对事件进行成像，但仅限于固定的、经过处理的样品。这可以在纳米分辨率下实现细胞结构的三维重建。结合这些方法，一种称为相关光电子显微镜（CLEM）的技术使我们能够获得细胞动力学的高分辨率电影和相同事件的高分辨率超微结构。布里斯托大学在开发CLEM方法方面处于领先地位，这使我们处于开发该领域新技术的理想位置。新的发展允许结合活细胞荧光成像和高分辨率超微结构成像的电子显微镜使用单个探针。令人兴奋的是，这些探针也可以用于蛋白质组学实验。蛋白质组学提供了分类和定量样品中所有蛋白质的方法。这些新探针使我们朝着将成像与蛋白质组学相结合的长期目标迈进。我们现在可以表达一个探针来标记特定的亚细胞区室。然后，我们将能够通过活细胞成像来定义其动力学，使用电子显微镜在3D中定义其超微结构，并在蛋白质组学鉴定之前标记探针空间受限区域内的所有蛋白质。这对于定义蛋白质机器的组成如何随着结构的移动、形状的改变或成熟成不同的形式而变化具有巨大的潜力。这些新方法需要两种模式的专业设备：高灵敏度和专门配置的光学显微镜设备，结合专业样品制备和成像设备，为电子显微镜制备样品。幸运的是，我们在布里斯托尔拥有出色的成像和蛋白质组学设备，这些设备被布里斯托尔和英国各地的研究人员大量使用。因此，提供额外的设备来连接LM， EM和蛋白质组学的成本并不像从一个设备不太好的中心开始那么高。我们在这个应用程序中提出的项目范围从基础研究到复杂的细胞生物学，再到研究癌细胞与免疫系统相互作用的实验，骨骼的发育，以及新设计的纳米结构与细胞相互作用的方式。我们的工作涉及BBSRC职权范围内的多个领域，包括支持正常人类和动物健康、感染和衰老的基础生物科学。项目组合包括拥有BBSRC资助记录的研究人员，涵盖与BBSRC职权范围和战略直接相关的领域，以及在类似方向发展的早期职业研究人员。申请人队列为这项工作的未来可能性提供了展示，我们希望从布里斯托尔内外获得重要的额外使用。事实上，这项工作的早期阶段已经通过主要的国际生物成像计划吸引了大量的外部兴趣。这项工作也将与徕卡微系统谁拥有强大的跟踪记录和持续的兴趣，这些方法的商业化合作伙伴关系；这确保了未来的技术和商业发展。因此，该提案虽然嵌入了现有技术，但对工业部门以及生物科学研究本身具有重大影响潜力。

Considerable technical developments have enabled us to image live cells to gain incredible detail of time-sensitive cellular events (on millisecond time scales). Advances in electron microscopy mean that we can image events at very high resolution, but only in fixed, processed samples. This can achieve 3D reconstructions of cellular structures at nanometre resolution. Combining these approaches, a technique called correlative light electron microscopy (CLEM), allows us to achieve high resolution movies of cell dynamics and high resolution ultrastructure of the same events. The University of Bristol is at the forefront of developing methods for CLEM, making us ideally placed to exploit new technologies in this area. New developments allow combined live cell fluorescence imaging and high resolution ultrastructure imaging by electron microscopy using a single probe. Excitingly, these same probes can also be used for proteomics experiments. Proteomics provides methodologies to catalogue and quantify all proteins within a sample. These new probes allow us to move towards our long term goal of combining imaging with proteomics. We would now be able to express a probe to label a specific sub-cellular compartment. We would then be able to define its dynamics by live cell imaging, its ultrastructure in 3D using electron microscopy, and to label all proteins within the spatially restricted area of the probe prior to proteomic identification. This has enormous potential to define how the composition of protein machines changes as structures move, change shape, or mature into different forms. These new approaches require specialist equipment for both modes: high sensitivity and specifically configured light microscopy equipment, combined with specialist sample preparation and imaging equipment to prepare samples for electron microscopy. We are in the fortunate position in Bristol of having outstanding facilities for imaging and proteomics that are used heavily by researchers in Bristol as well as across the UK. Thus, the costs of providing additional equipment to link LM, EM and proteomics are not as high as they would be if starting from a less well equipped centre. We propose projects within this application that range from fundamental studies into complex cell biology through to experiments to examine interactions of cancer cells with the immune system, the development of the skeleton, and the way in which newly designed nanostructures interact with cells. Our work has implications for multiple areas within BBSRC remit including basic bioscience underpinning normal human and animal health, infection, and aging. The portfolio of projects includes researchers with a strong track record of BBSRC funding and covers areas of direct relevance to BBSRC remit and strategy, as well as early career researchers whose work is developing in similar directions. The cohort of applicants provides a showcase for the future possibilities of this work from which we expect to derive significant additional use from within Bristol and beyond. Indeed, the early phases of this work have already attracted significant external interest through major international Bioimaging schemes. This work will also be a partnership with Leica Microsystems who have a strong track record and ongoing interest in commercialization of these methodologies; this ensures future technical and commercial development. While embedded in existing technology, this proposal therefore has significant impact potential to the industrial sector as well as from the bioscience research itself.