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Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: I. Concept and theoretical evaluation


Condrau, M A; Schwendener, R; Niederer, P; Anliker, M (1994). Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: I. Concept and theoretical evaluation. Cytometry Part A, 16(3):187-194.

Abstract

The concept of a flow cytometer suited for the time-resolved measurement of lanthanide chelate luminescence with a decay time on the order of 10 microseconds to 2 ms is presented and evaluated. The instrument proposed encompasses a continuous-wave laser for fluorescence excitation and an optical switch for the elimination of cellular autofluorescence decaying within 1 ns to 1 microseconds during the luminescence detection period. The slowly decaying fluorescence is to be quantified by a photon-counting system, whereas light scatter and prompt fluorescence parameters are acquired by a conventional detection system. The detection limit of the method, in terms of the smallest detectable number of fluorescing chelates per cell, is examined. It was found to be nearly 30,000 complexes of a europium chelate with a decay time of 1.6 ms and a quantum efficiency of 17%, independent of fast decaying cellular autofluorescence or prompt dye emission intensity. The probability of cells passing through the instrument without being detected while the laser beam is turned off was estimated, and the implications for cell throughput and sorting performance of the instrument were assessed. At typical fluorescence detection intervals of 500 microseconds to 1 ms, cell flow rates of 100-200 particles per second lead to detection probabilities of more than 90% and sorting purities comparable to those found in conventional fluorescence-activated cell sorting.

Abstract

The concept of a flow cytometer suited for the time-resolved measurement of lanthanide chelate luminescence with a decay time on the order of 10 microseconds to 2 ms is presented and evaluated. The instrument proposed encompasses a continuous-wave laser for fluorescence excitation and an optical switch for the elimination of cellular autofluorescence decaying within 1 ns to 1 microseconds during the luminescence detection period. The slowly decaying fluorescence is to be quantified by a photon-counting system, whereas light scatter and prompt fluorescence parameters are acquired by a conventional detection system. The detection limit of the method, in terms of the smallest detectable number of fluorescing chelates per cell, is examined. It was found to be nearly 30,000 complexes of a europium chelate with a decay time of 1.6 ms and a quantum efficiency of 17%, independent of fast decaying cellular autofluorescence or prompt dye emission intensity. The probability of cells passing through the instrument without being detected while the laser beam is turned off was estimated, and the implications for cell throughput and sorting performance of the instrument were assessed. At typical fluorescence detection intervals of 500 microseconds to 1 ms, cell flow rates of 100-200 particles per second lead to detection probabilities of more than 90% and sorting purities comparable to those found in conventional fluorescence-activated cell sorting.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:1994
Deposited On:20 Oct 2009 13:13
Last Modified:06 Dec 2017 21:25
Publisher:Wiley-Blackwell
ISSN:0196-4763
Additional Information:The definitive version is available at www.blackwell-synergy.com
Publisher DOI:https://doi.org/10.1002/cyto.990160302
PubMed ID:7924687

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