Multispectral rapid 3D super-resolution imaging of nuclear biology

核生物学多光谱快速 3D 超分辨率成像

• 批准号: BB/X019705/1
• 负责人: Giolanta Markaki
• 金额: $ 119.57万
• 依托单位: University of Leicester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX019705%2F1
• 关键词: Multispectral; rapid; 3D; super; resolution

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.

在莱斯特大学，我们对调节核功能和基因组组织的机制进行前沿研究，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学或疾病的大分子复合物的高阶结构，这将使我们能够测试细胞内的结构-功能关系。研究大分子复合物和细胞内亚细胞区室的分子组织的挑战在于它们的衍射极限尺寸为几十纳米。因此，对细胞内必要规模的生命基本过程的研究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. 点阵结构照明显微镜 (SIM2) 达到前所未有的横向分辨率约 60 nm 和轴向约 200 nm（与传统成像相比，体积分辨率提高了 8 倍，是传统 SIM 的两倍）。 2. 3D-单分子定位显微镜（SMLM），通过单次采集实现 20-30nm 横向分辨率和 50-80nm 体积分辨率（通过 1.4 微米）。该仪器还可以进行光操纵实验，包括单分子光活化、DNA 损伤、FRAP 和单分子跟踪实验。它提供超快 2 色同步 SRM 和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、跨学科高级成像设施 (AIF) 配备 SRM 功能，而伦敦大学或我们的中部学术和工业合作伙伴目前尚不具备这些功能。这个平台将具有变革性，使我们能够回答支撑生命规则的基本开放问题，这些问题无法用其他方法解决，并将使大学走在尖端 SRM 研究的最前沿。我们的目标是：1。安装具有晶格 SIM2、3D-SMLM 和光学操纵模块2 的 SRM 平台。支持和促进伦敦大学基因组生物学及其他领域的前沿研究，这将使我们具有国际竞争力3。提供对多用户跨学科成像设施的访问和培训，了解尖端的体积次衍射、亚细胞和单分子成像4。加强我们与工业合作伙伴的联系显微镜将无缝集成到现有的 AIF 基础设施中，在现有的房间内进行气候控制和仪器的所有必要要求。该设施向国内外所有用户以及已建立成本核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，发表了许多关于细胞核定量 SRM 成像的同行评审文章，并将有助于在莱斯特实施 SRM，促进员工、教师和学生的发展。设施经理 co-I KS 的技术专业知识将是系统日常运行和培训成功的关键。我们已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 文件存储驱动器连接来支持外部用户，UoL 文件存储驱动器将在安装时连接到平台。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 平台将为伦敦大学和广大中部地区财团提供独特的机会，以提高国际竞争力。它将进一步加强我们与工业合作伙伴的牢固联系，在计划研究中采用新颖的读出方法。