1 March 2009 Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples
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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 (<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.
© (2009) Society of Photo-Optical Instrumentation Engineers (SPIE)
Dayong Jin, Dayong Jin, James A. Piper, James A. Piper, Robert C. Leif, Robert C. Leif, Sean Yang, Sean Yang, Belinda C. Ferrari, Belinda C. Ferrari, Jingli Yuan, Jingli Yuan, Guilan Wang, Guilan Wang, Lidia M. Vallarino, Lidia M. Vallarino, John W. Williams, John W. Williams, } "Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples," Journal of Biomedical Optics 14(2), 024023 (1 March 2009). https://doi.org/10.1117/1.3103770 . Submission:

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