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Indexed by:期刊论文

Date of Publication:2009-03-01

Journal:JOURNAL OF BIOMEDICAL OPTICS

Included Journals:SCIE、EI、Scopus

Volume:14

Issue:2

ISSN No.:1083-3668

Key Words:flow cytometry; true positive; rare event; europium; time-gated luminescence (TGL); Giardia; UV LED; autofluorescence

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 mu s) 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-mu m calibration beads in environmental water dirt samples. Europium-labeled 5-mu m 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. (C) 2009 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3103770]