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

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.