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

Abstract

A time‐resolved flow cytometer capable of measuring a luminescence with a decay time in the range of 10 μs 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 μs 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.