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The overall goal of our research is to understand the molecular mechanisms of cell function, and to use this understanding to improve the diagnosis, treatment, and prevention of disease. A large part of our work is focused on developing cell and molecular measurement technologies that are more sensitive, quantitative, and efficient. We use optical methods, including fluorescence and Raman, to make highly multiparameter and multiplexed measurements of cells and molecules. Flow cytometry is the preferred platform for many of these measurements. Our current biological interests are the mechanisms of infection and immunity, and include the antigenic and phenotypic characterization of influenza virus, the quantification of pathogen-specific immune responses, and cross-talk between the immune and coagulation systems that occurs during infection.

Measurement Technologies

Raman Flow Cytometry

Highly multiparameter measurements are playing increasingly important roles in understanding complex cell systems. For fluorescence measurements using image and flow cytometry, the number of different probes that can be measured simultaneously is limited by the spectral width of the fluorophore emission spectra. Raman scattering spectra have much narrower spectral features, presenting the possibility of measuring many more probes within a given region of spectral space. To realize this potential, we have developed a Raman Flow Cytometer capable of measuring Raman scattering from single particles at high speeds. Nanoparticles exhibiting surface-enhanced Raman scattering (SERS) can serve as labels for antibodies or other binding probes, and offer the potential for higher levels of multiparameter and multiplexed measurements of cells and other particles than are possible with fluorescence alone.

Single Nanoparticle Analysis

Nanoscale structures mediate many cellular functions, including intracellular transport and intercellular signaling, and engineered nanoparticles are emerging as important tools for disease diagnosis and treatment. Most molecular analysis methods report bulk or average properties of nanoparticle preparations, and do not readily reveal population heterogeneity. However, because of their small size, it is difficult to make quantitative molecular measurements of single nanoparticles. To address this challenge, we are adapting flow cytometry as a sensitive and high speed method for the optical analysis of individual nanoparticles. High efficiency light collection, sensitive detectors, and extended measurement integration times enable the quantitative measurement of individual nanoparticles. We are using these approaches for the analysis of cell-derived membrane vesicles and synthetic model membrane vesicles, as well as to guide the engineering of nanoparticle probes for detection applications.

Ligands and Probes

Probes with molecular specificity are a key to molecular analysis of cells and cell systems. Antibodies are the most widely used molecular recognition molecules, but other ligands including proteins, peptides, and nucleic acids can also be used to provide molecular specificity. We use molecular evolution approaches, including phage and yeast display, to develop and optimize the affinity and specificity of peptide and protein ligands for various analytical targets. For detection, fluorescence is sensitive, quantitative, and often our first choice as a label for a binding ligand. For highly multiplexed detection applications, we are developing engineered assemblies of metal nanoparticles exhibiting SERS or other plasmonic effects for use as labels.

Biological Aims

Rapid microbe Identification and Characterization

For many infectious diseases, genetic, antigenic, and phenotypic characterization of  pathogen species and strains play an important role in surveillance, diagnosis, and treatment. Influenza virus, for example, undergoes constant evolution via mutations introduced during replication that, under selection pressures from the host immune system, can result in antigenic drift that renders existing vaccines ineffective.  Occasionally, reassortment of the RNA genome during co-infection of a host by multiple virus strains results in more dramatic changes, termed antigenic shift, that can lead to the emergence of new virus sub-types. It is the occurrence of this genetic shift in an animal reservoir that is thought to have resulted in the emergence of major pandemic strains. Both antigenic shift and drift are monitored by world health agencies to refine vaccine recommendations and to detect newly emerging strains. Because of their importance global infectious disease surveillance, there is great interest in improved methods for detecting and characterizing influenza viruses. We have developed multiplexed methods to determine influenza virus type and sub-type and, more recently, virus receptor binding preference. We are also developing multiplexed methods for assessing exposure based on analysis of serum antibodies against influenza virus, and are using these to understand the nature of vaccine-induced protection against the flu virus.

Analysis of the Host Antibody Response

he production of an effective antibody response depends on the production of enough antibodies with sufficient affinity to neutralize a pathogen. Effectiveness is often determined empirically in the form of protection, but it is desirable to understand protection in quantitive terms of antigen-specific binding affinity. Popular titer-based methods of assessing antigen specific immune responses do not discern between antibody concentration and affinity, a critical distinction. We have developed an assay and analysis formalism that is able to measure both concentration and affinity across multiple isotypes simultaneously. Combined with our high throughput analysis capabilities, this approach has the potential to revolutionize our understanding of the development of the immune response, leading to a more complete understanding of the evolution of the antibody response and improved approaches for vaccine development. 

Cell-derived microparticles in pathogenesis and immunity

The interaction between inflammation and coagulation in many diseases is well established, but molecular mechanisms and practical treatment strategies have been slow to emerge. Multiple lines of evidence implicate cell-derived membrane vesicles as key mediators of this interaction, but these very small (100 nm) circulating molecular assemblies are exceedingly difficult to study. We are developing instrumentation, including a Nanoparticle Flow Cytometer, and associated methods to enable the quantitative enumeration and analysis of cell-derived microparticles, with the aim of developing mechanism-based biomarkers of disease. These efforts aim to open a new window on plasma components that mediate interaction between the immune and hemostasis systems.

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