APPLICATIONS OF HIGH SENSITIVITY DETECTION

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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.