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了尖端研究，并在结构，化学和单分子研究中拥有独特的专业知识，以了解和操纵其功能。我们已经解决了与生理或疾病有关的大分子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隔室的分子组织的挑战在于其衍射有限的尺寸，几十个纳米。因此，对细胞内必要尺度的生命基本过程的研究依赖于超分辨率显微镜（SRM）方法。我们申请提供多种SRM模态的SRM平台。其中包括：1。晶格结构化照明显微镜（SIM2），横向下降至〜60 nm，轴向降低到〜60 nm（轴向左右〜200 nm）（与常规成像相比，体积分辨率增加了8倍，而常规SIM的两倍）。 2。3D单分子定位显微镜（SMLM），通过1.4微米实现20-30nm的横向分辨率和50-80nm的体积分辨率，并具有单个采集。该仪器还允许进行光感实验，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2色同时SRM，并具有前所未有的轻度成像，以保留用于活细胞观察的细胞完整性。我们旨在为我们的多用户跨学科高级成像设施（AIF）提供SRM的可能性，这些可能性目前在UOL或我们的Midlands学术和工业合作伙伴。该平台将具有变革性，使我们能够回答基本的基本开放问题，该问题基于无法用其他方法解决的生活规则，并将大学成为尖端SRM Research的最前沿。我们的目标是：1。安装带有晶格SIM2，3D-SMLM的SRM平台，带有光感模块2。支持并促进对基因组生物学及其超越的尖端研究，这将使我们在国际上具有竞争力3。为多用户跨学科成像设施提供访问和培训，以实现尖端的体积亚缩写，亚细胞和单分子成像4。加强与工业伙伴的联系，显微镜将在具有气候控制和仪器的所有必要要求的现有房间中无缝集成到现有的AIF基础设施中。该设施在国内和国际上以及工业合作伙伴以及已经建立的成本核算和在线预订预订系统的工业合作伙伴开放。 Markaki博士（PI）使用SRM方法拥有12年以上的经验，并发表了许多有关该细胞核定量SRM成像的同行评审文章，并将在莱斯特在莱斯特的实施中发挥作用。设施经理Co-I KS的技术专业知识将是成功进行系统进行日常运行和培训的关键。我们已经准备好通过专用的GPU和连接到UOL文件存储驱动器，以解决大数据处理和转移，尤其是用于支持外部用户，该驱动器将在安装后将其连接到平台。为AIF配备SRM平台，将对正在进行的BBSRC和乌克里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Broad Midlands Consortia提供具有国际竞争力的独特机会。这将进一步加强我们通过计划研究中的新读数方法与工业伙伴的牢固联系。