Phase-sensitive flow cytometry is a traditional and minimally disseminated method of capturing the average fluorescence lifetime on an event-by-event basis. That is, early incarnations of frequency-domain cytometry techniques involved a high degree of complexity with cumbersome analog hardware. Owing to the intricate nature of such techniques, the cytomics community has lacked a widespread and robust means of capturing excited-state decays. This project explores new digital methods as a means to introduce multiple lifetimes for additional separation power in cytometry. These include: (1) introduction of multiple-frequency modulation, (2) implementation of innovative approaches for capturing time-resolved signals through non-modulated digital signal processing, and (3) application of fluorescence lifetime parameters to sorting systems so as to separate cells or particles based on decay kinetic measurements. A cytometry system capable of not only average fluorescence lifetime measurements but also multi-exponential decay is appealing for several reasons which include: improving the separation of autofluorescence from exogenous signals, capturing surface enhanced Raman scattering in flow, and quantifying fluorescence protein expression during cell sorting.
The development of a multiple lifetime cytometry instrument will significantly impact the cytometry community at large. A temporal method for sorting and analysis may directly affect a wide range of staining and analysis protocols in flow cytometry, improve accuracy of low-density antigen studies, reveal possible rare events that exhibit unique excited state decays, and allow users of all kinds to implement the capability on their commercial instrument. The advancement of excited-state cytometry techniques will also broaden the participation of underrepresented undergraduate and graduate students at New Mexico State University, a Hispanic Serving Institution. Minorities working on the development of the cytometer will be exposed to high-impact bioengineering, chemical engineering, and other multidisciplinary fields needed for the project outcomes. The end goal for all or part of the described cytometry techniques is the integration into the biotechnology industry. All project outcomes can be perused at the Principle Investigator research website: http://che.nmsu.edu/JPH/index.html.
The overall goal of this research was to prove the ability to build a time-resolved flow cytometer. Time-resolved flow cytometry is an approach in which the fluorescence lifetime can be measured and introduced as a new cytometric parameter for high-throughput screening and sorting. The original objectives were to establish this idea as feasible, to produce a 1st generation prototype, and to disseminate this to the biological community. The activities for this project have to-date involved engineering, design, and experimental methods leading to the optimization of our proposed time-resolved flow cytometry instrument. At this stage we have finalized all of the aims, with most goals met and one new outcome achieved. The list below summarizes the outcomes related to the intellectual merit of the project. We have strengthened partnerships with small buisnesses We finalized a fully functioning fluorescence lifetime cell sorter Manuscripts are now published on this work for the broader scientific community Several presentations and abstracts (with graduate and undergraduate students as authors) have been made at National conferences. This project has also been essential to the broader community in that it has supported the training and promotion of chemical engineering graduate students, two of whom are underrepresented minorities in this discipline. This project has also led to one key outcome related to outreach activities and that is fostering partnerships between engineering scientists and the cell biology community.
