Multispectral rapid 3D super-resolution imaging of nuclear biology

在莱斯特大学，我们提供关于调控核功能和基因组组织的机制的尖端研究，并在结构、化学和单分子研究方面拥有独特的专业知识，以了解和操纵它们的功能。我们已经解决了许多涉及生理或疾病的大分子复合体的高阶结构，这将使我们能够测试细胞内的结构-功能关系。研究大分子复合体和细胞内亚细胞隔间的分子组织的挑战在于它们的衍射限制尺寸只有几十纳米。因此，在细胞内必要的尺度上研究生命的基本过程依赖于超分辨率显微镜(SRM)方法。我们申请了一个提供多种SRM模式的SRM平台。其中包括：1.晶格结构照明显微镜(SIM2)达到前所未有的分辨率，横向分辨率为~60 nm，轴向分辨率为~200 nm(体积分辨率比常规成像提高8倍，是常规SIM的2倍)。2.三维单分子定位显微镜(SMLM)，一次采集即可获得20-30 nm的横向分辨率和50-80 nm的体积分辨率(1.4微米)。该仪器还允许进行光操纵实验，包括单分子光激活、DNA损伤、FRAP和单分子跟踪实验。它提供超快的双色同步SRM，具有前所未有的温和成像，以保持活细胞观察的细胞完整性。我们的目标是为我们的多用户、跨学科的先进成像设备(AIF)配备目前UOL或我们的米德兰兹郡学术和工业合作伙伴无法提供的SRM可能性。我们的目标是：1.安装一个具有格子SIM2、3D-SMLM和光操控模块的SRM平台。支持和促进UOL在基因组生物学及其他领域的前沿研究，使我们具有国际竞争力3。提供对多用户跨学科成像设备的访问和培训，内容涉及尖端体积亚衍射、亚细胞和单分子成像4。加强我们与工业合作伙伴的联系，该显微镜将无缝集成到现有的AIF基础设施中，在一个具有气候控制和仪器所有必要要求的现有房间中。该设施向国内和国际上的所有用户以及已经建立了成本计算和在线预订系统的工业合作伙伴开放。Markaki博士(Pi)拥有12年以上使用SRM方法的经验，发表了大量关于核定量SRM成像的同行评议文章，并将对莱斯特SRM的实施以及教职员工和学生的发展起到重要作用。设施经理co-I KS的技术专长将是该系统成功日常运行和培训的关键。我们已经准备好解决大数据处理和传输问题，特别是通过专用GPU和UOL文件存储驱动器的连接来支持外部用户，UOL文件存储驱动器将在安装后连接到平台。为AIF配备SRM平台将为正在进行的BBSRC和UKRI资助的项目带来巨大好处。KS和YM将举办研讨会和讲座，包括支持MIBTP研究生项目。在AIF建立SRM平台将为UOL和广大的米德兰兹财团提供一个独特的机会，使其具有国际竞争力。它将进一步加强我们与工业合作伙伴的强大联系，在计划中的研究中采用新的读出方法。

At the University of Leicester we deliver cutting-edge research on mechanisms that regulate nuclear function and genome organization and hold unique expertise in structural, chemical and single-molecule investigations to understand and manipulate their function. We have solved numerous higher-order structures of macromolecular complexes involved in physiology or disease that will allow us to test structure-function relationships within cells. The challenge of studying macromolecular complexes and the molecular organization of subcellular compartments within cells resides in their diffraction-limited sizes of few dozens of nanometers. Therefore, the study of fundamental processes of life at the necessary scale within cells relies on super-resolution microscopy (SRM) methods.We apply for a SRM platform that offers multiple SRM modalities. These include: 1. Lattice Structured Illumination Microscopy (SIM2) reaching unprecedented resolution down to ~60 nm laterally and ~200 nm axially (8-times increase in volumetric resolution compared to conventional imaging and two times that of conventional SIM). 2. 3D-Single-Molecule Localization Microscopy (SMLM), achieving 20-30nm lateral resolution and 50-80nm volumetric resolution through 1.4 microns with a single acquisition. The instrument additionally allows for photomanipulation experiments, including single-molecule photoactivation, DNA damage, FRAP and single-molecule tracking experiments. It offers ultrafast 2-color simultaneous SRM with unprecedented mild imaging to preserve cellular integrity for live-cell observations. We aim at equipping our multi-user, interdisciplinary advanced imaging facility (AIF) with SRM possibilities that are not currently available at UoL or our Midlands academic and industrial partners. This platform will be transformative in allowing us to answer fundamental open questions underpinning the rules of life that cannot be addressed with other methods and will bring the University to the forefront of cutting-edge SRM research.Our objectives are to:1. Install a SRM platform featuring lattice SIM2, 3D-SMLM with a photomanipulation module2. Support and promote cutting-edge research on genome biology and beyond at UoL that will make us internationally competitive3. Provide access and training to a multi-user interdisciplinary imaging facility on cutting-edge volumetric subdiffraction, subcellular and single-molecule imaging4. Strengthen our links with industrial partnersThe microscope will integrate seamlessly into the existing AIF infrastructure, in an existing room with climate control and all necessary requirements of the instrument. The facility is open to all users nationally and internationally as well as industrial partners with already established costing and online reservations booking systems in place. Dr Markaki (PI) has 12+ years of experience using SRM methods, has published numerous peer-reviewed articles on quantitative SRM imaging of the nucleus and will be instrumental in the implementation of SRM in Leicester the development of staff, faculty and students. The technical expertise of the facility manager, co-I KS, will be key to the successful daily running and training on the system. We have prepared to address big data processing and transfer particularly for supporting external users by the dedicated GPUs and connections to the UoL File Storage Drive, which will be connected to the platform upon its installation. Equipping the AIF with a SRM platform will be of huge benefit to ongoing BBSRC and UKRI-funded projects. KS and YM will run workshops and lectures, including supporting the MIBTP graduate program. The establishment of a SRM platform at the AIF will provide UoL and the broad Midlands consortia with a unique opportunity to become internationally competitive. It will further strengthen our strong links to industrial partners with novel readout methods in planned research.