Compensation Free Highly Multiplexed Flow Cytometer

Project Summary Flow cytometry is a workhorse technique in research and development as well as in clinical laboratories for diagnosis and monitoring of disease It is particularly useful in distinguishing between populations of immune cells based on their expressed cell surface antigens Standard flow cytometers use fluorescent tags often conjugated to monoclonal antibodies to give qualitative and quantitative information about specific molecules in the cell This molecular specificity coupled with the fact that information is obtained on a cell by cell basis and that very high throughput is possible cells per second can be analyzed make this a powerful technique The ability to multiplex measure a variety of different molecular species in a single cell further adds to its utility and to the complexity of the scientific questions that can be addressed using this technique However the level of multiplexing currently has limitations Typically flow cytometry analysis relies solely on spectral information of the fluorescent tags and is thus limited by the spectral overlap of fluorophore emissions Currently employing even moderate levels of multiplexing for the simultaneous interrogation of multiple parameters within a cell requires high levels of complexity in instrumentation and analysis and careful design and execution of experiments The related andquot compensation problemandquot compensating for spillover of signal from a fluorophore into multiple channels due to the broad spectrum of most fluorophores also causes significant instrument complexity cumbersome workflow and inaccurate results These factors put severe limits on the range of scientific questions that can be addressed using current technologies deter novices in the technique from attempting more complex yet scientifically relevant experiments and collectively are widely regarded as the major current bottleneck in flow cytometry To overcome this limitation we have developed an innovative approach that uses fluorescence lifetime as a separate additional discriminating measurement parameter Our scheme for using fluorescent lifetime for multiplexing is simple scalable and supported by preliminary data from our prototype instrument The proposed project will establish the feasibility of lifetime based multiplexing by modifying our experimental platform with key hardware and algorithm improvements challenging the resulting prototype with a comprehensive set of verification and validation tests of increasing complexity and culminating with a comparison of our technology to existing technology in a standard four color cell based assay A successful outcome will lay the foundation for our planned development of commercial instruments both analyzers and sorters that offer two major benefits to end users a simple turnkey compensation free operation for instruments with low to medium levels of multiplexing and b high end instruments with two to three times the current maximum multiplexing capability Project Narrative Flow cytometry is a widely used cell analysis technique in research and clinical diagnostics e g drug development cancer AIDS and other diseases Current flow cytometers use information from fluorescent markers in a way that makes experiments complicated to perform and also limits the scope of the experiments that a researcher can carry out The current proposal aims to develop an innovative technique that will relieve these limitations by extracting additional information from the fluorescent markers and by using the information to simplify the operation and increase the performance of flow cytometers