Core 1: Cellular diagnostics/imaging core

核心1：细胞诊断/成像核心

• 批准号: 10670739
• 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/10670739
• 关键词: 3-Dimensional; Animals; Atherosclerosis; Blood Vessels; Blood flow; Blood specimen; Bone Marrow; Cardiovascular Diseases; Cells; Circulation; Collaborations; Color; Communication; Data Analyses; Detection; Development; Diagnostic; Endothelium; Extracellular Matrix; Flow Cytometry; 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; Productivity; Proliferating; Proteins; Protocols documentation; Research Personnel; Resources; Scientific Advances and Accomplishments; Signal Transduction; Techniques; Technology; Time; Tissues; Vascular Endothelial Cell; Vascular Permeabilities; Work; bone cell; bone imaging; cardiovascular effects; cell type; design; fabrication; 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/成像核心的使命有两个。首先，我们将提供最先进的成像 技术和细胞分析工具，以满足本次 PPG 中四个项目的科学需求。我们将工作 与项目研究人员密切合作，进行定量测量和数据分析，定制 必要时使用仪器和优化实验方案。第二，我们将追求新技术 创新将带来未来科学进步，超越现有技术所能实现的 技术。该核心是一个独特的资源，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 与该 PPG 中的多名研究人员进行富有成效的合作的记录，包括 Scadden 博士（项目 1）， Nahrendorf 博士（项目 2）和 Swirski 博士（项目 3）。我们的实验室都位于同一地点 同一栋大楼，使核心成为来自不同实验室的团队成员的共同聚会场所 聚集在一起，不仅是为了使用设施，也是为了互动和交流想法。为履行使命 核心，我们提出以下具体目标。在目标 1 中，我们将与项目 1 和 4 合作进行克隆 基于多色荧光蛋白表达的造血细胞分析。我们将扩大 我们的体内流式细胞仪的多色功能是我们实验室开发的实时技术 无需抽取即可检测和定量活体动物循环中的荧光细胞 血液样本，能够无创监测外周循环中的克隆动态 白细胞群。在目标 2 中，我们将与项目 2 合作来评估骨髓 (BM) 脉管系统如何 因心血管疾病（CVD）而改变。我们将测量功能参数，例如血流量、 通过执行多光子测量血管通透性、血管反应性和跨内皮迁移 BM 脉管系统成像以及细胞外基质二次谐波成像 成分。我们还将与项目 3 合作来表征造血干细胞 (HSC) 定位和 使用由基因编辑的 HSC 提供的 MI 和动脉粥样硬化背景下 BM 的动态变化 基因组工程核心。在目标 3 中，我们建议开发一种新方法来实现图像引导激光 从 BM 中显微切割和提取活细胞进行单细胞分子分析。我们将首先 专注于捕获 BM 血管内皮细胞，因为它们构成 HSC 生态位的关键组成部分，并且 还调节白细胞运输和大分子运输。该技术将弥合现有的 一方面单细胞分析提供分子信息但不提供空间信息， 另一方面是实时成像，它提供了分子分析中缺乏的 3D 空间背景。这 该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。