Time-resolved measurement used in flow cytometry
R. Cao, P. Jenkins, and J. P. Houston
Department of Chemical Engineering
New Mexico State University
Las Cruces, NM 88003
Time-resolved measurements are widely used in fluorescence spectroscopy, particularly for studies of biological macromolecules and increasingly for cellular imaging. Time-resolved measurements contain more information than is available from the steady-state data. For example, with time-dependent information the kinetic phenomena of biological macromolecules can be obtained as well as interactions of macromolecules with each other (i.e. measurement of Forster resonance energy transfer). In this contribution we present frequency-domain fluorescence measurements from a home-built time-resolved cytometry system (TRCS). This device is capable of measuring decay-kinetic phenomena from fluorescent species in flowing cells or particles, which pass a laser excitation source at microsecond transit times. A quantum dot (QDot) experiment was performed that validated the resolution limits and multiple-lifetime capabilities of the TRCS. QDots were selected because of their favorable fluorescence decay, extremely long Stoke’s shift and high photostability. The QDots were bound to the membrane of Chinese Hamster Ovary (CHO-K1) cell populations as well as the surface of polystyrene microspheres. Three primary measurements were made: (1) the capture of average fluorescence lifetimes at different laser modulation frequencies, (2) phase filtering of the QDot fluorescence from other background fluorescence when the QDot was quenched with a heavy metal or non-quenched, and (3) multiple fluorescence decay of the QDot with other organic fluorophores with a novel multi-frequency flow cytometry capability. Many challenges remain before our TRCS can be translated for commercial exploitation. By implementing straightforward analysis of QDots we not only assess and optimize our device such that it becomes simple, stable and efficient, but also delve into more quantitative biological studies that leverage the fluorescence lifetime for true concentration, fraction of molecules in specific environments, monitoring of single cells for kinetic processes, and the elimination of background autofluorescence.
See more of this Group/Topical: Topical 3: 2011 Annual Meeting of the American Electrophoresis Society (AES)