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Journal of Biomedical Optics

Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples
Author(s): Dayong Jin; James A. Piper; Robert C. Leif; Sean Yang; Belinda C. Ferrari; Jingli Yuan; Guilan Wang; Lidia M. Vallarino; John W. Williams
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Paper Abstract

A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (<1 μs) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-µm calibration beads in environmental water dirt samples. Europium-labeled 5-µm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (<100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%±30% and 91%±3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.

Paper Details

Date Published: 1 March 2009
PDF: 10 pages
J. Biomed. Opt. 14(2) 024023 doi: 10.1117/1.3103770
Published in: Journal of Biomedical Optics Volume 14, Issue 2
Show Author Affiliations
Dayong Jin, Macquarie Univ. (Australia)
James A. Piper, Macquarie Univ. (Australia)
Robert C. Leif, Newport Instruments (United States)
Sean Yang, Newport Instruments (United States)
Belinda C. Ferrari, Macquarie Univ. (Australia)
Jingli Yuan, Dalian Univ. of Technology (China)
Guilan Wang, Dalian Univ. of Technology (China)
Lidia M. Vallarino, Virginia Commonwealth Univ. (United States)
John W. Williams, Virginia Commonwealth Univ. (United States)

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