APPLICATIONS OF HIGH SENSITIVITY DETECTION

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。该项目的总体目标是通过开发的方法和仪器改进来扩展高灵敏度流式细胞术（HSFCM）的生物应用，从而实现多参数测量能力。流式细胞术通过提供单个细胞颗粒的快速、定量和灵敏的多参数测量，使生物医学科学取得了重大进展。个体分析产生群体异质性的信息，这是不显示的合奏分析，并允许更精确的测量个体属性比可能的测量时，在散装阶段。然而，传统流式细胞术的一个局限性是不能测量小于0.5 μ m的小颗粒或具有小于几百个荧光分子的暗淡颗粒。许多重要的生物粒子、分子和分子集合体都属于这些类别。我们的方法是通过增强的能力HSFCM访问这个测量域。我们已经确定了3个重要的生物学领域：通过基因组分析进行细菌鉴定; DNA单分子扫描在高通量基因组学中的应用;以及线粒体基因组和生化分析。在多色和光散射检测方面的特定仪器增强将使这些新的应用成为可能。该研究计划分为四个相关的具体目标：基于DNA片段大小的细菌指纹分析;单DNA分子的高分辨率扫描;单个线粒体的分析;和高灵敏度仪器开发。这项研究和开发的结果将创造一种新的高灵敏度流式细胞仪，并将在亚细胞单分子水平上激发流式细胞术的新领域。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project is to expand the biological applications of High Sensitivity Flow Cytometry (HSFCM) through methods developed and through instrumentation improvements leading to a multiparameter measurement capability. Flow cytometry has enabled major advances in the biomedical sciences by providing rapid, quantitative and sensitive multiparameter measurements of individual cellular particles. Individual analysis produces information on population heterogeneity that is not revealed by ensemble analysis and allows more precise measurement of individual attributes than is possible when measurement is done in bulk phase. However, one limitation of conventional flow cytometry is the inability to measure small particles less than 0.5 ¿m or dim particles having less than several hundred fluorescent molecules. A wide variety of important biological particles, molecules, and molecular assemblies fall into these categories. Our approach is to access this measurement domain through enhanced capability HSFCM. We have identified 3 important biological areas to focus on: bacterial identification by genome analysis; DNA single molecule scanning applications in high throughput genomics; and mitochondrial genomic and biochemical analysis. Specific instrument enhancements in multi-color and light scatter detection will enable these new applications. The research plan is divided into four related Specific Aims: DNA Fragment Size-Based Analysis of Bacterial Fingerprints; High Throughput Scanning of Single DNA Molecules; Analysis of Individual Mitochondria; and High Sensitivity Instrumentation Development. The results of this research and development will create a new class of high sensitivity flow cytometry instrumentation and will stimulate a new field of flow cytometry at the sub-cellular single molecule level.