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Proceedings Paper

Using FRET to quantify changes in integrin structures in human leukocytes induced by chemoattractants with multi-frequency flow cytometry
Author(s): Jesus Sambrano; Yelena Smagley; Alexandre Chigaev; Larry A. Sklar; Jessica P. Houston
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Paper Abstract

Flow cytometry for single cell counting uses optical measurements to report multiple cell features such as cell morphology, cell phenotype, and microenvironmental changes. Time-resolved flow cytometry is a unique method that involves the detection of the average fluorescence lifetime as a cytometric parameter. Measuring the average fluorescence lifetime is helpful when discriminating between more than one emission signal from a single cell because of spectrally overlapping emission. In this contribution, we present preliminary measurements toward a study that advances simple time-resolved flow cytometry and introduces a technique to measure fluorescence lifetime values from single cells labeled with a Forster Resonance Energy Transfer (FRET) pair. Specifically, donor fluorophore fluorescein isothiocyanate (FITC) fluorescence lifetime is measured to identify its proximity to the acceptor fluorophore. We hypothesize that our time-resolved flow cytometry approach can resolve changes in FRET in order to study integrin structures on the surface of leukocyte cells. Our results show that FITC has an average lifetime of 4.2 +/-0.1 nsec, and an average fluorescence lifetime of 2.4 nsec +/-0.2 nsec when engaged in FRET. After the release of FRET (e.g. dequenched) the average fluorescence lifetime of FITC was measured to be 3.1 +/- 0.5 nsec. Phasor graphs reveal large distributions of fluorescence lifetimes on a per cell basis, suggesting the existence of multiple fluorescence lifetimes. These data suggest more than one integrin conformation occurs throughout the cell population. The impact of this work is the addition of quantitative information for FRET efficiency values and determination of FRET calculations using high-throughput data.

Paper Details

Date Published: 23 February 2017
PDF: 10 pages
Proc. SPIE 10065, Biophotonics and Immune Responses XII, 100650W (23 February 2017); doi: 10.1117/12.2253340
Show Author Affiliations
Jesus Sambrano, New Mexico State Univ. (United States)
Yelena Smagley, Univ. of New Mexico, School of Medicine (United States)
Alexandre Chigaev, Univ. of New Mexico, School of Medicine (United States)
Larry A. Sklar, Univ. of New Mexico, School of Medicine (United States)
Jessica P. Houston, New Mexico State Univ. (United States)


Published in SPIE Proceedings Vol. 10065:
Biophotonics and Immune Responses XII
Wei R. Chen, Editor(s)

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