Practical time‐gated luminescence flow cytometry. I: Concepts

Abstract

The method of time-gated detection of long-lifetime (1-2,000 ls) luminescence-labeled microorganisms following rapid excitation pulses has proved highly efficient in suppressing nontarget autofluorescence (<0.1 ls), scatterings, and other prompt stray light (Hemmila and Mukkala, Crit Rev Clin Lab Sci 2001;38:441-519). The application of such techniques to flow cytometry is highly attractive but there are significant challenges in implementing pulsed operation mode to rapid continuous flowing sample to achieve high cell analysis rates (Leif R, Vallarino L, Rare-earth chelates as fluorescent markers in cell separation and analysis, In: Cell Separation Science and Technology, ACS Symposium Series 464, American Chemical Society, 1991, pp 41-58; Condrau et al., Cytometry 1994;16:187-194; Condrau et al., Cytometry 1994;16:195-205; Shapiro HM, Improving signals from labels: Amplification and other techniques, In: Practical Flow Cytometry, 4th ed., Wiley, New York, 2002, p 345). We present here practical approaches for achieving high cell analysis rates at 100% detection efficiency, using time-gated luminescence (TGL) flow cytometry. In particular, we report that new-generation UV LEDs are practical sources in TGL flow cytometry. Spatial effects of longlived luminescence from the target fluorophore in a fast-flowing sample stream have been investigated; excitation and detection requirements in TGL flow cytometry were theoretically analyzed; two practical approaches, a triggered model and a continuous flow-section model, were considered as a function of flow speed, sizes and relative positions of the excitation/detection spots, label lifetime, excitation pulse duration/intensity, and detection duration. A particular configuration using LED excitation to detect europium dye-labeled targets in such a system has been modeled in detail. In the triggered model, TGL mode is confined to a low repetition rate (<1 kHz) and engaged only while a target particle is present in the excitation zone. In the flow-section model, TGL mode is engaged continuously at high repetition rates to permit much higher cell arrival rates. The detection of 5.7-lm europium calibration beads in a UV LED-excited TGL flow cytometer has been shown to be feasible with a calculated signal-to-background ratio up to 11:1. ' 2007 International Society for Analytical Cytology

UV LED excited time-gated luminescence flow cytometry: evaluation for rare-event particle counting

“…According to the microscopy confirmation (10±1 counts) on the exact number of beads each sample contained, the recovery rate for rare-event counting of ~10-bead sample was calculated as 100%±20% [(12+10+10+11+9+11)/10*6=100%]. approve the rare-event counting capability offered by the TGL flow cytometry technique, which resulted in 13,11,9,11,9, and 8 events detected respectively. The water concentrates did not increase the background level during the time-gated detection phase.…”

“…We previously reported a practical flow cytometry method based on time-gated detection of long-lifetime luminescence labeled targets 13,14 . In these studies, time-gated detection of target events in temporal, spectral and spatial 3-D domains rendered both the scattering and autofluorescence backgrounds invisible.…”

“…In this report, a prototype lot of 5µm europium-labelled beads which display long-lived (>~340µs) fluorescence were utilized to evaluate the performance of TGL flow cytometry, specifically focusing on rare-event recovery capabilities in environmental samples. The details on both concepts and prototype operation of TGL flow cytometer were stated previously 13,14 . Briefly, Figure 1 shows the basics of geometric layout for the current development of TGL flow cytometer: The targets of interest are fluorescently labeled with a long-lived fluorescent probe, typically lanthanide complexes, and then excited with a pulsed LED (pulse duration of up to 100 µs) as they flow in single cell profile through the hydrodynamically focused laminar stream ( figure 1A).…”

“…This enhanced the detection likelihood for the low concentration of micron-size targets. Our early development of time-gated luminescence (TGL) flow cytometry technique focused on a continuous-flow-section approach using LED excitation 13,14 . This technique showed great potential for the detection of trace amounts of micronsized targets by flow cytometry.…”

Cyto 2011—Overview of the XXVI ISAC Congress Proceedings Issue of Cytometry

“…The detection of lu-minescence-labeled microorganisms by time-gated long-lifetime signatures using excitation conditions that facilitate the suppression of nontarget emission has proved a highly attractive option for exploration in flow cytometry (7). In a previous review in Cytometry, the chemical and physical properties of lanthanide ion complexes and of other narrow-emitting species have been highlighted with particular reference to their utility as biolabels (8).…”