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.

项目摘要/摘要 成像是血液学的重要诊断和研究工具。此R21提案，以回应 PAR-20-140（肾脏，泌尿科和血液疾病的催化工具和技术开发）是 旨在优化，验证和部署开创性的光学显微镜平台，以可视化 在血液学的超微结构环境中的蛋白质。可视化他们感兴趣的特定蛋白质 当前需要相关的光和电子显微镜 （CLEM）非常复杂，耗时且昂贵，因此大多数人都无法访问 血液学研究人员。 PAN膨胀显微镜（Panexm）是一种新型的成像方法，可用于 在整个细胞的基础上，在其超微结构环境下蛋白质（以及其他生物分子和细胞器）成像 使用常规（衍射限制）光显微镜使用纳米级（〜10-30 nm）分辨率。使用panexm， 固定的单元在线性尺寸（〜8000倍的体积）中扩展到20倍，同时保持亚细胞 组织，细胞蛋白质含量和抗原性。染色保留的超微结构以及蛋白质 对这些样品的不同颜色感兴趣，并使用标准光在三个维度上成像它们 显微镜可以可视化和分析原本低于光的衍射极限。 AIM 1的重点是开发和优化扩展单个血液和骨髓的方法 粘附或固定在载玻片上的细胞。 AIM 2的重点是优化骨髓切片的扩展 评估其微环境中的造血细胞。我们高度协同的研究团队致力于 培训和传播血液学研究中最新技术。应用这个有力的新 血液和骨髓细胞的技术将通过使血液学研究者能够转化 量化感兴趣的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功能从少数数百个研究小组扩展到 将吞吐量从每个数据集增加到几个小时，Panexm将使血液学研究民主化 社区访问3D结构信息，并将推动生物医学研究的发现 从基本细胞生物学发现到临床病理学中的组织学应用。