Research Papers

Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

[+] Author Affiliations
Dayong Jin, James A. Piper

Macquarie University, Centre of MQ Photonics, New South Wales 2109 Australia

Robert C. Leif, Sean Yang

Newport Instruments, 5648 Toyon Road, San Diego, California 92115-1022

Belinda C. Ferrari

University of New South Wales, School of Biotechnology and Biomolecular Sciences, New South Wales 2052 Australia

Jingli Yuan, Guilan Wang

Dalian University of Technology, State Key Laboratory of Fine Chemicals, Dalian 116012, China

Lidia M. Vallarino, John W. Williams

Virginia Commonwealth University, Department of Chemistry, Richmond, Virginia 23284-2006

J. Biomed. Opt. 14(2), 024023 (April 14, 2009). doi:10.1117/1.3103770
History: Received July 05, 2008; Revised November 19, 2008; Accepted January 27, 2009; Published April 14, 2009
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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 (>1μs) 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.

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© 2009 Society of Photo-Optical Instrumentation Engineers

Citation

Dayong Jin ; James A. Piper ; Robert C. Leif ; Sean Yang ; Belinda C. Ferrari, et al.
"Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples", J. Biomed. Opt. 14(2), 024023 (April 14, 2009). ; http://dx.doi.org/10.1117/1.3103770


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