Core 1: Cellular diagnostics/imaging core

核心1：细胞诊断/成像核心

• 批准号: 10469358
• 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/10469358
• 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/成像核心的任务是双重的。首先，我们将提供最先进的成像技术 技术和细胞分析工具，以满足这一PPG中四个项目的科学需求。我们会工作的 与项目调查人员密切合作，执行定量测量和数据分析，定制 必要时进行仪器和优化实验方案。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 技术。核心是一种独特的资源，在光学技术、活体显微镜、 仪器设计和制造，以及图像处理和数据分析。我们还有一个 在此PPG中与多名研究人员进行富有成效的合作记录，包括斯卡登博士(项目1)， 纳伦多夫博士(项目2)和斯维尔斯基博士(项目3)。我们的实验室都实际位于 同一建筑，使核心成为来自不同实验室的团队成员的公共会议场所 融合，不仅是为了使用设施，也是为了互动和交流思想。执行国家安全局的使命 核心是，我们提出了以下具体目标。在目标1中，我们将与项目1和4一起进行克隆 基于多色荧光蛋白表达的造血细胞分析。我们将扩大规模 我们的活体流式细胞仪的多色能力，这是我们实验室开发的一项实时技术 活体动物循环中荧光细胞的检测和量化 血液样本，以实现外周循环中克隆动态的非侵入性监测 白细胞数量。在目标2中，我们将与项目2合作，评估骨髓(BM)血管系统如何 是由心血管疾病(CVD)改变的。我们将测量功能参数，如血流量， 通过执行多光子，血管渗透性、血管反应性和跨内皮细胞迁移 骨髓血管成像与细胞外基质的二次谐波成像 组件。我们还将与项目3合作，以确定造血干细胞(HSC)的定位和 使用由提供的基因编辑的HSCs在心肌梗死和动脉粥样硬化环境中的骨髓动力学 基因组工程核心。在目标3中，我们建议开发一种新的方法来实现图像制导激光 从骨髓中显微解剖和提取活细胞，用于单细胞分子图谱。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利基和 也调节白细胞的运输和大分子的运输。这项技术将在现有的 一方面，单细胞分析提供了分子信息，但没有空间信息， 另一方面是实时成像，这提供了分子分析所缺乏的3D空间背景。这个 这项技术可以在未来的研究中扩展到其他细胞类型和其他组织的分析。