Visualizing cellular ultrastructure using light microscopy in hematology

在血液学中使用光学显微镜观察细胞超微结构

• 批准号: 10473885
• 负责人: Diane S Krause
• 金额: $ 20.5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-25 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10473885
• 关键词: 3-Dimensional; Address; Algorithmic Analysis; Antibodies; Architecture; Archives; Biological; Biomedical Research; Blood; Blood Cells; Blood Platelets; Bone Marrow; Bone Marrow Cells; Cells; Clinical Pathology; Collaborations; Color; Communities; Complex; Complex Analysis; Confocal Microscopy; Consumption; Data Set; Development; Diagnostics Research; Dimensions; Dyes; Electron Microscopy; Environment; Fluorescence; Fluorescence Microscopy; Goals; Head; Hematological Disease; Hematology; Hematopoietic; Histologic; Hour; Human; Image; Imaging technology; Immobilization; Immunofluorescence Immunologic; Individual; Investigation; Isotropy; Kidney Diseases; Knowledge; Letters; Light; Light Microscope; Location; Mammalian Cell; Marrow; Megakaryocytes; Methodology; Microscope; Microscopy; Molecular; Organelles; Pathology; Proteins; Protocols documentation; Research; Research Personnel; Resolution; Sampling; Slide; Stains; Subcellular structure; Swelling; Techniques; Technology; Time; Tissues; Training; Urologic Diseases; Vision; Visualization; cell type; cellular imaging; design; high resolution imaging; imaging approach; imaging capabilities; improved; insight; interest; light microscopy; macrophage; nanoscale; neglect; new technology; novel; peripheral blood; preservation; protein distribution; response; technology development; tool; tool development

Project Summary/Abstract Imaging is an important diagnostic and research tool in hematology. This R21 proposal, written in response to PAR-20-140 (Catalytic Tool and Technology Development in Kidney, Urologic, and Hematologic Diseases), is designed to optimize, validate, and deploy a groundbreaking light microscopy platform for visualization of proteins in their ultrastructural context in hematology. Visualizing specific proteins of interest in their ultrastructural (sub-organelle, ~sub-50 nm) context currently requires correlative light and electron microscopy (CLEM) which is extremely complicated, time-consuming and expensive and thereby inaccessible to most researchers in hematology. Pan Expansion Microscopy (PanExM) is a novel imaging approach that allows for imaging of proteins (and other biomolecules and organelles) in their ultrastructural context on a whole-cell basis with nanoscale (~10-30 nm) resolution using conventional (diffraction-limited) light microscopes. With PanExM, fixed cells are expanded up to 20-fold in the linear dimension (~8000 fold in volume) while maintaining subcellular organization, cellular protein content and antigenicity. Staining the preserved ultrastructure as well as the protein of interest in these samples in different colors and imaging them in three dimensions using standard light microscopy allows for visualization and analysis of what would otherwise be below the diffraction limit of light. Aim 1 is focused on developing and optimizing the methodology to expand individual blood and bone marrow cells adhered or immobilized on slides. Aim 2 is focused on optimizing expansion of bone marrow sections to assess hematopoietic cells in their microenvironment. Our highly synergistic research team is dedicated to training and dissemination of the latest technologies in hematology research. Application of this powerful new technology to blood and bone marrow cells will be transformative by enabling hematology investigators to quantify the location, distribution and interactions of molecules of interest in their sub-cellular context. By extending these high-resolution imaging capabilities from a handful to hundreds of research groups and increasing the throughput from weeks per data set to hours, PanExM will democratize the hematology research community’s access to 3D structural information and will drive discoveries across biomedical research ranging from fundamental cell biological findings to histological applications in clinical pathology.

项目概要/摘要 成像是血液学中重要的诊断和研究工具。此 R21 提案是为了回应 PAR-20-140（肾脏、泌尿和血液疾病的催化工具和技术开发） 旨在优化、验证和部署突破性的光学显微镜平台，用于可视化 血液学中蛋白质的超微结构。可视化其感兴趣的特定蛋白质 超微结构（亚细胞器，~sub-50 nm）背景目前需要相关的光学和电子显微镜 （CLEM）极其复杂、耗时且昂贵，因此大多数人无法获得 血液学研究人员。泛膨胀显微镜 (PanExM) 是一种新颖的成像方法，可以 在全细胞基础上对蛋白质（以及其他生物分子和细胞器）的超微结构进行成像 使用传统（衍射限制）光学显微镜获得纳米级（~10-30 nm）分辨率。与 PanExM 一起， 固定细胞在线性尺寸上扩展至 20 倍（体积约 8000 倍），同时保持亚细胞状态 组织、细胞蛋白质含量和抗原性。对保存的超微结构和蛋白质进行染色 对这些不同颜色的样本感兴趣，并使用标准光对它们进行三维成像 显微镜可以对低于光衍射极限的物质进行可视化和分析。 目标 1 专注于开发和优化扩大个体血液和骨髓的方法 细胞粘附或固定在载玻片上。目标 2 的重点是优化骨髓切片的扩张 评估造血细胞的微环境。我们高度协同的研究团队致力于 培训和传播血液学研究的最新技术。应用这一强大的新功能 血液和骨髓细胞技术将带来变革，使血液学研究人员能够 量化感兴趣分子在其亚细胞环境中的位置、分布和相互作用。经过 将这些高分辨率成像能力从少数几个研究小组扩展到数百个研究小组， 将每个数据集的吞吐量从数周增加到数小时，PanExM 将使血液学研究民主化 社区对 3D 结构信息的访问，并将推动生物医学研究的发现 从基本的细胞生物学发现到临床病理学中的组织学应用。