A broadly accessible facility microscope to probe nanoscale cellular dynamics by combined live cell super-resolution microscopy and photomanipulation

显微镜是现代生物学研究的基石，因为它能够在亚细胞水平上可视化生命的动态。显微镜在生物学的每个领域都取得了突破，但它传统上受到衍射极限的限制，直到最近还被认为是无法逾越的250 nm分辨率障碍。在过去的几十年里，超分辨率显微镜技术已经发展起来，使我们能够可视化细胞及其内部工作方式，远远超出了衍射极限。超分辨率显微镜可以在纳米尺度上观察细胞组织和动力学，并揭示了细菌、植物和动物中具有生物学意义的、以前看不见的细胞组织。在华威大学，我们拥有世界级的研究人员，整个机构都在研究对我们经济和健康的未来至关重要的领域，如抗生素耐药性、食品安全和老龄化。由于细胞是高度动态的，直接在活细胞中进行高速超分辨率测量的能力是尖端细胞生物学研究的重要工具。华威大学目前缺乏这一关键能力。我们已经定义了几种互补的活细胞超分辨率显微镜能力，这些能力将增强华威大学大量团队的研究能力：-以100纳米或更高的分辨率成像的能力，因为许多细胞组件，如细胞骨架元素或细胞细胞器，都是以这种规模组织起来的。-高速成像以捕捉动态过程的能力，因为细胞中分子和细胞器的运动可以非常快。-能够在低激光照度下为细胞成像-大多数细胞具有高度的光敏性。显微镜实验中过量的光剂量会损害细胞--就像人类的晒伤一样--并由于激活了不想要的细胞反应而混淆了实验。-在受控区域选择性地操纵荧光蛋白的能力。细胞是一个密集、拥挤的环境，这阻碍了追踪特定蛋白质或细胞结构的能力。光操纵技术使我们能够在密集的细胞环境中通过突出显示或光漂白它们周围的特定蛋白质来分离它们。然后，可以发现并跟踪高密度区域中突出显示的蛋白质的运动。我们请求资金购买具有所有这些功能的蔡司晶格结构照明显微镜(SIM^2)系统。我们将在华威大学生命科学学院的成像设施中建立这个仪器。我们将为广大当地用户提供方便的使用途径，并向全国提供该系统的使用途径。拟议的显微镜将解决我们目前在活细胞超分辨率显微镜方面的能力差距，并为我们提供全国独一无二的联合晶格-SIM成像和光操纵能力。该系统还将增强英国的生物科学研究能力，因为我们将提供并促进全国使用这一仪器。

Microscopy is a cornerstone of modern biological research because it enables visualisation of the dynamics of life at the subcellular level. Microscopy has led to breakthroughs in every area of biology, but it has traditionally been constrained by the diffraction limit, a 250 nm resolution barrier that was until recently thought insurmountable. Over the past few decades, super-resolution microscopy techniques have been developed that allow us to visualise cells and their inner workings far beyond the diffraction limit. Super-resolution microscopes allow observation of cellular organization and dynamics at the nanometre scale, and have revealed biologically critical, previously invisible, cellular organization in bacteria, plants and animals. At the University of Warwick, we have world class researchers across the institution investigating areas pivotal for the future of our economy and health, such as antibiotic resistance, food security and aging. As cells are highly dynamic, the ability to perform high speed super-resolution measurements directly in live cells is an essential tool in cutting edge cell biology research. This critical capability is currently lacking at the University of Warwick. We have defined several complementary live cell super-resolution microscopy capabilities which would enhance the research capabilities of a large number of groups at University of Warwick: - The ability to image at a resolution of 100 nm or better, because many cellular components, such as cytoskeletal elements or cellular organelles, are organized on this scale. - The ability to image at high speed to capture dynamic processes, as the movements of molecules and organelles in the cell can be extremely fast. - The ability to image cells with low laser illumination - most cells are highly photosensitive. Excessive light dose in microscopy experiments damages cells - just like sunburn in humans - and confounds experiments due to activation of unwanted cellular responses. - The ability to selectively photomanipulate fluorescent proteins in a controlled region. The cell is a dense, crowded environment which hampers the ability to track specific proteins or cellular structures. Photomanipulation techniques enable us to isolate specific proteins within the dense cellular environment by either highlighting them or photobleaching their surroundings. The movement of highlighted proteins within dense regions can then be revealed and followed over time. We request funds to purchase a Zeiss Lattice Structured Illumination Microscope (SIM^2) system with all these capabilities. We will establish this instrument at the University of Warwick School of Life Sciences Imaging Facility. We will provide user-friendly access to a wide local user base, as well as providing national access to the system. The proposed microscope will address our current capabilities gap in live cell super-resolution microscopy and give us nationally unique capabilities for joint lattice-SIM imaging and photomanipulation. The system will also enhance UK bioscience research capabilities as we will provide and promote national access to this instrument.