Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: II. Instrument design and experimental results.
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.
Condrau
M A
MA
Institute of Biomedical Engineering, The University, Zurich, Switzerland.
Schwendener
R A
RA
Zimmermann
M
M
Muser
M H
MH
Graf
U
U
Niederer
P
P
Anliker
M
M
D002614:Chelating Agents; D004996:Ethidium; D005434:Flow Cytometry / Q000295:instrumentation*; D005456:Fluorescent Dyes; D006801:Humans; D007811:Lanthanum / Q000737:chemistry; D008081:Liposomes; D008214:Lymphocytes / Q000166:cytology; D008674:Metals, Rare Earth / Q000737:chemistry*
D002614:Chelating Agents; D005456:Fluorescent Dyes; D008081:Liposomes; D008674:Metals, Rare Earth; D007811:Lanthanum; D004996:Ethidium