The primary focus of our research is to develop and demonstrate the utility of ‘time-resolved flow cytometry.’ We build, modify, and advance a variety of flow cytometry systems to enable the measurement of the fluorescence lifetime, as a unique fluorescence decay-dependent parameter. Our research stemmed from work conducted by Dr. Houston when she was a Director’s Postdoctoral Fellow at the Los Alamos National Laboratory (LANL) from 2006-2009. In the late 1980s “phase-sensitive flow cytometry” was established at LANL as a new tool for counting cells based on the fluorescence lifetime parameter. During those early years establishing lifetime technologies, two ‘phase’ instruments were built by a group of LANL cytometrists & scientists (including but not limited to: J. Steinkamp, H. Crissman, L. Sklar, T. Yoshida, C. Deka, J. Keij, S. Cram, J. Jett, R. Habbersett, & J. Martin). Both lifetime cytometers (see photograph above) were used to study many new cell applications and initiated many new collaborations. Around 2002, one of the phase instruments in Los Alamos was disassembled and the other remained in tact but was not used owing to a shift in the interest of cytometry instrumentation development. New and exciting areas for cytometry at LANL were expanding (acoustic focusing, full spectral cytometry, high sensitivity systems, low-cost portable systems, etc). Therefore after the phase instrument was dormant for a few years, Dr. Houston began to introduce new techniques that would advance how lifetime measurements are made in cytometry (see picture to the right of Dr. Houston circa 2006). With Dr. James P. Freyer and Mark Naivar, Dr. Houston worked to demonstrate sorting of cells based on lifetime measurements as well as proving that digital techniques can be performed for processing of the fluorescence lifetime value.
Now parts of the original LANL phase instrument are located at NMSU. At this time we are continuing to make advances in digital, compact, and multi-lifetime approaches for cell sorting and analysis. Since August of 2009 we have transformed a number of lifetime technologies and in doing so, our laboratory has provided a mechanism for underrepresented minorities’ (and all students!) access to high impact biomedical engineering research (see photograph of Dr. Houston with Ph.D. student, Ruofan Cao and photograph below of Patrick Jenkins, M.S. CHE’12). We work with Mark Naivar (formerly of DarklingX LLC) to enable fluorescence lifetime-ready data systems, with Dr. Roger Brent (Fred Hutchinson Cancer Center) to measure fluorescence lifetime shifts in yeast cells, and with Dr. K. Houston (Chemistry&Biochemistry, NMSU) to study cancer cell applications, and Dr. Larry Sklar (Center for Molecular Discovery, UNM) on Forster Resonance Energy Transfer applications. We also have a number of other collaborators using lifetime technologies. Additionally in 2012 we contacted Dr. Giacomo Vacca (Kinetic River Corp.) to work with us on an idea we had to use a dithering laser for fluorescence lifetime measurements. Our collaboration led to a new technology area for Kinetic River to subsequently develop.
What is flow cytometry and how are fluorescence lifetime measurements made using a cytometer?
Flow cytometry is a method of counting, analyzing, and sorting individual cells and particles at the high-throughput level. Standard cytometry instruments detect individual cells (or small particles) as they pass in single file through a tightly focused laser beam in the plane of an optical detector. Fluorescence or Raleigh scatter are the primary optical signals that make up “multiparametric” measurements in cytometric analyses.
In the Houston Laboratory we are quite interested in improving the ability of flow cytometry systems to measure the fluorescence lifetime, or average time a fluorophore has spent in its excited state prior to relaxation to the ground state. This is known as fluorescence decay kinetics and is appealing to the cytometry community because it can provide additional quantitative information about the fluorophore under interrogation or molecular milieu to which the fluorophore is attached. For example, the fluorescence lifetime is independent of fluorophore concentration and can help discriminate signals that have similar emission strength and which spectrally overlap.
Therefore we are pioneering new digital lifetime techniques to make sensitive fluorescence lifetime measurements using ‘frequency-domain’ and ‘time-domain’ methods of analysis. We are specifically studying a variety of biomedical and health-related applications such as (but not limited to): (i) time-dependent methods for separating autofluorescence from exogenous signals for low-density antigen assays; (ii) sorting of cells expressing fluorescence proteins based on fluorescence lifetime; (iii) phase filtering techniques to identify the uniqueness of fluorescent tags that occur in a “dual-labeled” fashion on the surface or interior of cells; (iv) multiplexing with surface enhanced Raman scattering nanoparticles; and (v) heterodyning/multi-frequency measurements for multiexponential fluorescence decay.
May of our applications require novelty cytometry systems and instrumentation. Some examples of the new techniques we have introduced since 2006 are: (1) the measurement of multi-exponential fluorescence decay kinetics in flow with digital data acquisition techniques (see illustration above), (2) cell sorting based on fluorescence lifetimes (see photographs below), (3) phase-filtering techniques, (4) expanding modulation limits to measure ultra-fast scattering phenomena in flow, and (5) commercially translating the time-dependent technologies with small businesses.