Time-gated flow cytometry

An ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

Dayong Jin*, James A. Piper, Robert C. Leif, Sean Yang, Belinda C. Ferrari, Jingli Yuan, Guilan Wang, Lidia M. Vallarino, John W. Williams

*Corresponding author for this work

Research output: Contribution to journalArticle

33 Citations (Scopus)
6 Downloads (Pure)

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 (>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.

Original languageEnglish
Article number024023
Pages (from-to)1-10
Number of pages10
JournalJournal of Biomedical Optics
Volume14
Issue number2
DOIs
Publication statusPublished - 2009

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