Practical time-gated luminescence flow cytometry. II

Experimental evaluation using UV LED excitation

Dayong Jin*, Russell Connally, James Piper

*Corresponding author for this work

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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. The prototype TGL flow cytometer was able to distinguish the target beads, and a maximum signal to background ratio of 38:1 was observed. Neither the environmental water concentrates nor S101 solution contributed to the background in the TGL detection phase. The counting efficiency of the TGL flow cytometer was typically >93% of values determined using conventional counting methods.

Original languageEnglish
Pages (from-to)797-808
Number of pages12
JournalCytometry Part A
Volume71
Issue number10
DOIs
Publication statusPublished - Oct 2007

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