Core 1: Cellular diagnostics/imaging core

核心1：细胞诊断/成像核心

• 批准号: 10238046
• 负责人: Charles P. Lin
• 金额: $ 35.86万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238046
• 关键词: 3-Dimensional; Animals; Atherosclerosis; Blood Circulation; Blood Vessels; Blood flow; Blood specimen; Bone Marrow; Cardiovascular Diseases; Cells; Collaborations; Color; Communication; Custom; Data Analyses; Detection; Development; Diagnostic Imaging; Endothelium; Extracellular Matrix; Fluorescence; Functional Imaging; Future; Genes; Genome engineering; Heart; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Image; Imaging technology; Laboratories; Lasers; Lesion; Leukocyte Trafficking; Leukocytes; Measurement; Measures; Methods; Microdissection; Mission; Molecular; Molecular Analysis; Molecular Profiling; Mus; Optics; Peripheral; Population; Production; Proteins; Protocols documentation; Research Personnel; Resources; Scientific Advances and Accomplishments; Signal Transduction; Techniques; Technology; Time; Tissues; Vascular Endothelial Cell; Vascular Permeabilities; Work; base; bone cell; bone imaging; cardiovascular effects; cell type; design; hematopoietic stem cell niche; image guided; image processing; imaging capabilities; imaging modality; in vivo; innovation; instrumentation; intravital microscopy; meetings; member; migration; multiphoton imaging; non-invasive monitor; receptor; second harmonic; single cell analysis; tool

The mission of the cellular diagnostics/imaging core is two fold. First, we will provide state-of-the art imaging technology and cell analysis tools to meet the scientific needs of the four projects in this PPG. We will work closely with project investigators to perform quantitative measurements and data analysis, customizing instrumentation and optimizing experimental protocols as necessary. Second, we will pursue new technical innovations that will lead to future scientific advances beyond what can be accomplished with existing technologies. The Core is a unique resource with deep expertise in optical technology, intravital microscopy, instrumentation design and fabrication, as well as image processing and data analysis. We also have a record of productive collaborations with multiple investigators in this PPG, including Dr. Scadden (Project 1), Dr. Nahrendorf (Project 2), and Dr. Swirski (Project 3). Our laboratories are all physically co-located in the same building, making the Core a common meeting place where team members from various laboratories converge, not just to use the facility but also to interact and exchange ideas. To carry out the mission of the Core, we propose the following Specific Aims. In Aim 1, we will work with Projects 1 and 4 to perform clonal analysis of hematopoietic cells based on their expression of multi-color fluorescent proteins. We will expand the multi-color capability of our in vivo flow cytometer, a technology developed in our laboratory for real-time detection and quantification of fluorescent cells in the circulation of live animals without the need to draw blood samples, to enable noninvasive monitoring of the clonal dynamics in the peripheral circulating leukocyte population. In Aim 2, we will work with Project 2 to assess how the bone marrow (BM) vasculature is altered by cardiovascular diseases (CVD). We will measure functional parameters such as blood flow, vascular permeability, vascular reactivity, and trans-endothelial migration, by performing multiphoton imaging of the BM vasculature together with second-harmonic imaging of the extracellular matrix component. We will also work with Project 3 to characterize hematopoietic stem cell (HSC) localization and dynamics in the BM in the settings of MI and atherosclerosis using gene-edited HSCs provided by the Genome Engineering Core. In Aim 3 we propose to develop a new method to enable image-guided laser microdissection and extraction of live cells from the BM for single cell molecular profiling. We will initially focus on capturing BM vascular endothelial cells, as they form a critical component of the HSC niche and also regulate leukocyte trafficking and macromolecular transport. The technique will bridge the existing divide between single-cell analysis on the one hand, which provides molecular but no spatial information, and live imaging on the other, which supplies the 3D spatial context lacking in the molecular analysis. The technique can be extended in future studies to the analysis of other cell types and in other tissues.

细胞诊断/成像核心的任务是两个倍。首先，我们将提供最先进的成像 技术和细胞分析工具可以满足本ppg中四个项目的科学需求。我们将工作 与项目调查人员紧密进行定量测量和数据分析，自定义 必要的仪器和优化实验协议。第二，我们将追求新的技术 创新将导致未来的科学进步，而不是现有的创新 技术。核心是一种独特的资源，具有光学技术，插入显微镜， 仪器设计和制造以及图像处理和数据分析。我们也有一个 与该PPG的多个研究人员的生产合作记录，包括Scadden博士（项目1）， Nahrendorf博士（项目2）和Swirski博士（项目3）。我们的实验室在物理上都在 同一建筑物，使核心成为各个实验室团队成员的共同聚会场所 融合，不仅是为了使用设施，还可以互动和交换想法。执行任务 核心，我们提出以下特定目标。在AIM 1中，我们将与项目1和4合作执行克隆 造血细胞基于其多色荧光蛋白的表达分析。我们将扩展 我们的体内流式细胞仪的多色能力，这是我们实验室实时开发的一项技术 在无需绘制的无需画 血液样本，以实现外围循环中克隆动力学的无创监测 白细胞种群。在AIM 2中，我们将与项目2合作评估骨髓（BM）脉管系统如何 通过心血管疾病（CVD）改变。我们将测量功能参数，例如血流， 血管通透性，血管反应性和跨内皮迁移，通过执行多光子 BM脉管系统的成像以及细胞外基质的第二次谐波成像 成分。我们还将与项目3合作，以表征造血干细胞（HSC）定位和 BM在MI和动脉粥样硬化的设置中使用基因编辑的HSC的动力学 基因组工程核心。在AIM 3中，我们建议开发一种新方法来启用图像引导激光器 从BM中进行微分解和提取单细胞分子分析。我们最初会 专注于捕获BM血管内皮细胞，因为它们构成了HSC生态位的关键组成部分 还调节白细胞运输和大分子运输。该技术将桥接现有的 一方面对单细胞分析进行划分，该分析提供分子但没有空间信息， 并在另一个上进行实时成像，该成像提供了分子分析中缺乏的3D空间环境。这 在将来的研究中可以扩展技术，以分析其他细胞类型和其他组织。