The international flow cytometry community consistently uses fluorescence activated cell sorters and counters to solve a wide range of biomedical, biophysical, and biomaterial engineering problems. Yet among the plethora of fluorescence and light scatter applications, no commercial cytometry instrument presently measures fluorescence decay kinetic properties of the materials and fluorophores tagged to cells or particles. Although fluorescence is the main cytometric parameter measured from an excited cell/particle the average time fluorescent molecules spend in the excited state, or the fluorescence lifetime, is simply ignored. This disregard is mainly due to the fact that commercial flow cytometers lack the ability to measure the fluorescence lifetime, and therefore it has never been a capability for cytometrists in general. In the past several years our laboratory has made strides toward adapting lifetime technologies onto existing commercial systems to effectively make fluorescence lifetime measurements simple and achievable for a range of cytometric architectures. The fluorescence lifetime is a unique photophysical trait that when measured with time-resolved detection hardware, can provide quantitative information for cellular and particle-based assays. Thus we have adapted cytometry hardware in order to measure the fluorescence lifetime from a range of fluorophores at the throughput of standard cytometry for sorting as well as cell counting. Examples will be provided that cover a variety of biomedical assays, each with unique reasons for obtaining fluorescence decay kinetics. For example, we show the ability to measure Förster resonance energy transfer, fluorescence lifetime shifts elicited by autofluorescence species and fluorescent proteins, and the translation of imaging tricks to flow cytometry. It is non-trivial to capture heterogeneous time-resolved information from several different excitable molecules when excitation is observed from single cells and particles in fluidic states. Therefore this contribution also discusses and summarizes new techniques now being used for high throughput multi-exponential fluorescence decay detection using flow cytometry.
Dr. Jessica P. Houston is currently an Associate Professor in the Department of Chemical and Materials Engineering at New Mexico State University (NMSU). Her research interests include biophotonics, optical imaging, flow cytometry, and fluorescence-lifetime measurements. She began research in the area of photon migration while at Texas A&M University in College Station (TAMU), TX. Dr. Houston received her Ph.D. in Chemical Engineering from TAMU in 2005 and later worked at the University of Texas M.D. Anderson Cancer Center, and Baylor College of Medicine. Dr. Houston concentrated on developing an expertise in flow cytometry while a Director’s Postdoctoral Fellow from 2006-2009 at Los Alamos National Laboratory (LANL). She joined the faculty at New Mexico State University in 2009, and has continued to focus her research on fluorescence-based high-throughput diagnostics to capture excited-state phenomena. Some of Dr. Houston’s most notable honors are the Outstanding Junior Faculty Award by the Hispanic Faculty and Staff Caucus at NMSU, the Research Achievement Award by the NMSU Vice President for Research Office, and the NSF CAREER award.