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Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: II. Instrument design and experimental results


Condrau, M A; Schwendener, R; Zimmermann, M; Muser, M H; Graf, U; Niederer, P; Anliker, M (1994). Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: II. Instrument design and experimental results. Cytometry Part A, 16(3):195-205.

Abstract

A time-resolved flow cytometer capable of measuring a luminescence with a decay time in the range of 10 microseconds to 2 ms, typical for some lanthanide chelates, is presented. The instrument permits acquisition of conventional light scatter and prompt fluorescence signals as well as detection of slowly decaying luminescence by a photon counting unit for a selectable time period of 1 microsecond to 1 ms. During photon counting, the laser beam is turned off by an acoustooptic deflector. The design of a flow chamber with an average geometrical light collection efficiency of 35% over a distance of 1.7 mm is presented and analyzed by ray tracing. A pulse processing system featuring digital integration of the conventional signals and a transputer system for the acquisition and the transfer of the measured parameter values to a host computer is described. Instrument function is verified with lyophilized human lymphocytes stained for the CD8 antigen with dye-loaded liposomes. Quantitation of cell-associated europium chelate fluorescence, displaying a decay time of 1.6 ms, is demonstrated. Elimination of fast decaying background emission generated by DNA-associated ethidium bromide is shown. The background generated by instrument components in the time-gated measurement channel is characterized, and measures for its complete elimination are discussed.

Abstract

A time-resolved flow cytometer capable of measuring a luminescence with a decay time in the range of 10 microseconds to 2 ms, typical for some lanthanide chelates, is presented. The instrument permits acquisition of conventional light scatter and prompt fluorescence signals as well as detection of slowly decaying luminescence by a photon counting unit for a selectable time period of 1 microsecond to 1 ms. During photon counting, the laser beam is turned off by an acoustooptic deflector. The design of a flow chamber with an average geometrical light collection efficiency of 35% over a distance of 1.7 mm is presented and analyzed by ray tracing. A pulse processing system featuring digital integration of the conventional signals and a transputer system for the acquisition and the transfer of the measured parameter values to a host computer is described. Instrument function is verified with lyophilized human lymphocytes stained for the CD8 antigen with dye-loaded liposomes. Quantitation of cell-associated europium chelate fluorescence, displaying a decay time of 1.6 ms, is demonstrated. Elimination of fast decaying background emission generated by DNA-associated ethidium bromide is shown. The background generated by instrument components in the time-gated measurement channel is characterized, and measures for its complete elimination are discussed.

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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 12:21
Last Modified:05 Apr 2016 13:30
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.990160303
PubMed ID:7924688

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