Practical time‐gated luminescence flow cytometry. II: Experimental evaluation using UV LED excitation

In the previous article [Part 1 (8)], we have modelled alternative approaches to design of practical time‐gated luminescence (TGL) flow cytometry and examined the feasibility of employing a UV LED as the excitation source for the gated detection of europium dye labelled target in rapid flow stream. The continuous flow‐section approach is well suited for rare‐event cell counting in applications with a large number of nontarget autofluorescent particles. This article presents details of construction, operation and evaluation of a TGL flow cytometer using a UV LED excitation and a gated high‐gain channel photomultiplier tube (CPMT) for detection. The compact prototype TGL flow cytometer was constructed and optimised to operate at a TGL cycle rate of 6 kHz, with each cycle consisting of 100 μs LED pulsed excitation and ∼60 μs delay‐gated detection. The performance of the TGL flow cytometer was evaluated by enumerating 5.7 μm Eu3+ luminescence beads (having comparable intensity to europium‐chelate‐labeled Giardia cysts) in both autofluorescence‐rich environmental water concentrates and Sulforhodamine 101 (S101) solutions (broadband red fluorescence covering the spectral band of target signals), respectively.