For several decades, high-throughput single cell counting, discovery of important intracellular functions, and an understanding of single cell responses to external stimuli have been possible with the aid of devices called flow cytometers. Flow cytometers are used for research as well as in clinical settings where there is a need to obtain an accurate account of cells collected from blood to provide prognoses for diseases such as HIV. Cytometers work by causing proteins and other molecular species in or on individual cells to reach elevated energy levels as the cells travel through fluidic chambers and traverse laser beams over microsecond transit times. Despite the prevalence of cytometry systems commercially, available cytometry devices do not capture "photodynamic" properties from individual cells. That is, no current cytometry instrument measures the fluorescence decay and average fluorescence lifetime from molecules in or on cells. This capability is important because time-dependent information is very valuable in quantitative cell counting, cell sorting, and improvement of signal-to-noise among cellular assays. The lack of fluorescence lifetime-dependent cytometry is mainly due to the difficulties and complexities required for measuring excited state kinetics; this dearth has in turn resulted in a lack of assays and fluorescence decay-dependent applications. Thus, this CAREER development plan involves the discovery of new approaches for time-dependent flow cytometry and introduces new ways to easily integrate fluorescence dynamic-based measurements into commercial cytometry systems. Moreover, this research advances cellular applications for time-resolved flow cytometry. Specific objectives of this work are (i) to provide an understanding of autofluorescence lifetime changes of intrinsic cellular proteins with differences in cell cycle and cell viability, (ii) to explore fluorescent protein lifetime changes associated with intracellular protein transport, protein-protein interactions and protein complex formation detectable by Forster resonance energy transfer (FRET) and loss of FRET; and (iii) to identify ultra-rapid excited state decay times based on microsphere-bound nanoparticles that exhibit surface-enhanced Raman scattering for multiplex bead assays.
New Mexico State University (NMSU) is home to many underrepresented minority (URM) students who remain within the state's borders for financial and cultural reasons. The training opportunities at NMSU in the discipline of bioengineering are limited despite high student interest and the historical excellence of biotechnology and bioscience industry in New Mexico. In an effort to increase educational opportunities for NMSU students as well as the surrounding communities that feed into the state's Land Grant Institution, Hispanic NMSU undergraduates, graduate students, 5th grade students, and K-8 educators will be enlisted in an educational plan that (1) implements learning-through-research activities and (2) cultivates K-8 educational outreach. Direct interaction with Hispanic students at NMSU as well as a culturally marginalized science-magnet elementary school will be initiated to help build integrated science curricula using research activities in flow cytometry. New Mexico has a rich history in flow cytometry, where it was in part invented. This educational plan incorporates real-world solutions to be conveyed to science students. It also emphasizes the historical significance and provides a context on who they are as science learners and what they, as native New Mexicans, can achieve. All project outcomes can be seen at the Principal Investigator's research website: http://che.nmsu.edu/JPH/index.html.
