UV LED excited time-gated luminescence flow cytometry: Concepts and experimental evaluation

This paper presents experimental and theoretical studies of time-gated discrimination of long-lived luminescence (lifetime: 12000 Vs) labelled target-organisms against non-target autofluorescence background (lifetime: < 100 ns) in flow cytometry. A theoretical model of such a TGL flow cytometer is developed which takes account of flow speed, illumination and detection apertures, fluorescence label lifetime, and pulsed illumination and gated detection timing sequences. Ultraviolet LED and channel photomultiplier were found to be practical as pulsed excitation sources and gated detector for TGL flow cytometry. The prototype cytometer was constructed and optimized to operate at 6 k Hz repetition rate of TGL cycles consisting of 100 Vs LED excitation and similar to 60 mu s gated detection. The spatial counting efficiency was evaluated by enumerating 5.5 mu m diameter europium microspheres resulting in a counting accuracy approaching 100%.