Core 1: Cellular diagnostics/imaging core

细胞诊断/成像核心的使命有两个方面。首先我们会提供最先进的成像技术 技术和细胞分析工具，以满足该PPG中四个项目的科学需求。我们将 与项目调查人员密切合作，进行定量测量和数据分析， 仪器和必要时优化实验方案。第二，我们将追求新的技术 这些创新将导致未来的科学进步，超越现有技术所能实现的。 技术.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。我们也有一个 在本PPG中与多名研究者（包括Scadden博士（项目1））的富有成效的合作记录， 博士Nawerdorf（项目2）和Swirski博士（项目3）。我们的实验室都位于 同一栋建筑，使核心成为一个共同的会议场所，来自不同实验室的团队成员 聚集，不仅是为了使用设施，而且是为了互动和交流思想。执行联合国的使命 核心，我们提出以下具体目标。在目标1中，我们将与项目1和项目4一起执行克隆 基于它们的多色荧光蛋白的表达的造血细胞的分析。我们将扩大 我们的体内流式细胞仪的多色能力，这是我们实验室开发的一种实时 检测和定量活体动物循环中的荧光细胞，而无需抽取 血液样本，以实现外周循环中克隆动力学的非侵入性监测 白细胞群在目标2中，我们将与项目2合作，评估骨髓（BM）血管系统如何 心血管疾病（CVD）改变了它。我们将测量功能参数，如血流量， 血管渗透性、血管反应性和跨内皮迁移， BM血管成像和细胞外基质二次谐波成像 成分我们还将与项目3合作，以表征造血干细胞（HSC）的定位， 使用基因编辑的HSC在MI和动脉粥样硬化背景下的BM动力学 基因组工程核心。在目标3中，我们提出了一种新的方法，使图像引导激光 显微切割和从BM中提取活细胞用于单细胞分子谱分析。我们将初步 专注于捕获BM血管内皮细胞，因为它们形成HSC生态位的关键组成部分， 还调节白细胞运输和大分子转运。该技术将连接现有的 分为单细胞分析，一方面，它提供分子但没有空间信息， 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。的 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。

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.