TIME-RESOLVED FLOW-CYTOMETRY FOR THE MEASUREMENT OF LANTHANIDE CHELATE FLUORESCENCE .2. INSTRUMENT DESIGN AND EXPERIMENTAL RESULTS

A time-resolved flow cytometer capable of measuring a luminescence wit h a decay time in the range of 10 mu s to 2 ms, typical for some lanth anide chelates, is presented. The instrument permits acquisition of co nventional light scatter and prompt fluorescence signals as well as de tection of slowly decaying luminescence by a photon counting unit for a selectable time period of 1 mu s to 1 ms. During photon counting, th e laser beam is turned off by an acoustooptic deflector. The design of a flow chamber with an average geometrical light collection efficienc y of 35% over a distance of 1.7 mm is presented and analyzed by ray tr acing. A pulse processing system featuring digital integration of the conventional signals and a transputer system for the acquisition and t he transfer of the measured parameter values to a host computer is des cribed. Instrument function is verified with lyophilized human lymphoc ytes stained for the CD8 antigen with dye-loaded liposomes. Quantitati on of cell-associated europium chelate fluorescence, displaying a deca y time of 1.6 ms, is demonstrated. Elimination of fast decaying backgr ound emission generated by DNA-associated ethidium bromide is shown. T he background generated by instrument components in the time-gated mea surement channel is characterized, and measures for its complete elimi nation are discussed. (C) 1994 Wiley-Liss, Inc.