期刊
JOURNAL OF BIOMEDICAL OPTICS
卷 14, 期 2, 页码 -出版社
SPIE-SOC PHOTOPTICAL INSTRUMENTATION ENGINEERS
DOI: 10.1117/1.3103770
关键词
flow cytometry; true positive; rare event; europium; time-gated luminescence (TGL); Giardia; UV LED; autofluorescence
资金
- Australian ARC/NHMRC Fluorescence Applications in Biotechnology and Life Sciences (FABLS) network
- International Society for Analytical Cytology (ISAC) scholar program
- National Natural Science Foundation of China [20575069]
- Newport Instruments' Internal Development Funds
- Lidia Vallarino's Gift Fund
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]
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