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，我们提供关于调节核功能和基因组组织的机制的前沿研究，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高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几十纳米的衍射极限尺寸。因此，在必要的尺度上研究细胞内的基本生命过程依赖于超分辨率显微镜（SRM）方法。这些措施包括：1.晶格结构照明显微镜（SIM 2）达到前所未有的分辨率，横向分辨率低至约60 nm，轴向分辨率低至约200 nm（体积分辨率比传统成像提高8倍，是传统SIM的2倍）。2. 3D单分子定位显微镜（SMLM），实现20- 30 nm的横向分辨率和50- 80 nm的体积分辨率通过1.4微米的一次采集。该仪器还允许光操纵实验，包括单分子光活化，DNA损伤，FRAP和单分子跟踪实验。它提供了超快的2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。我们的目标是装备我们的多用户，跨学科的先进成像设施（AIF）与SRM的可能性，目前还没有在UoL或我们的米德兰学术和工业合作伙伴。这个平台将是变革性的，让我们回答基本的开放性问题，这些问题支撑着无法用其他方法解决的生活规则，并将把大学带到尖端SRM研究的最前沿。我们的目标是：1.安装一个SRM平台，该平台具有lattice SIM 2、3D-SMLM和光操纵模块2。支持和促进基因组生物学和超越UoL的前沿研究，这将使我们具有国际竞争力3。提供多用户跨学科成像设施的使用和培训，包括尖端的体积亚衍射、亚细胞和单分子成像4。加强我们与工业合作伙伴的联系显微镜将无缝集成到现有的AIF基础设施中，在现有的房间中进行气候控制和仪器的所有必要要求。该设施向国内和国际所有用户以及已经建立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，促进教职员工和学生的发展。设施经理co-I KS的技术专长将是成功进行系统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和连接到UoL文件存储驱动器来支持外部用户，该驱动器将在安装后连接到平台。为AIF配备SRM平台将对正在进行的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研究生课程。在AIF建立SRM平台将为UoL和广泛的米德兰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紧密联系，在计划的研究中采用新的读出方法。