Jin Dayong, Connally Russell, Piper James
Centre for Lasers and Applications, Division of Information and Communication Sciences, Macquarie University, NSW, Australia.
Cytometry A. 2007 Oct;71(10):783-96. doi: 10.1002/cyto.a.20450.
The method of time-gated detection of long-lifetime (1-2,000 micros) luminescence-labeled microorganisms following rapid excitation pulses has proved highly efficient in suppressing nontarget autofluorescence (<0.1 micros), 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 long-lived 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-microm 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.
在快速激发脉冲之后，对长寿命（1 - 2000微秒）发光标记微生物进行时间分辨检测的方法，已被证明在抑制非目标自发荧光（<0.1微秒）、散射以及其他即时杂散光方面非常有效（赫米拉和穆卡拉，《临床实验室科学评论》2001年；38:441 - 519）。将此类技术应用于流式细胞术极具吸引力，但要对快速连续流动的样品实施脉冲操作模式以实现高细胞分析速率，存在重大挑战（莱夫·R、瓦拉里诺·L，《稀土螯合物作为细胞分离和分析中的荧光标记》，载于《细胞分离科学与技术》，美国化学学会专题系列464，1991年，第41 - 58页；孔德罗等人，《细胞计数》1994年；16:187 - 194；孔德罗等人，《细胞计数》1994年；16:195 - 205；夏皮罗·H·M，《改善标记信号：放大及其他技术》，载于《实用流式细胞术》第4版，威利出版社，纽约，2002年，第345页）。在此，我们展示了利用时间分辨发光（TGL）流式细胞术在100%检测效率下实现高细胞分析速率的实用方法。特别是，我们报告新一代紫外发光二极管是TGL流式细胞术中切实可行的光源。研究了快速流动样品流中目标荧光团长寿命发光的空间效应；从理论上分析了TGL流式细胞术中的激发和检测要求；根据流速、激发/检测光斑的大小和相对位置、标记寿命、激发脉冲持续时间/强度以及检测持续时间，考虑了两种实用方法，即触发模式和连续流段模式。详细模拟了在这样一个系统中使用发光二极管激发来检测铕染料标记目标的特定配置。在触发模式中，TGL模式限于低重复率（<1千赫兹），并且仅在激发区域存在目标粒子时才启动。在流段模式中，TGL模式以高重复率持续启动，以允许更高的细胞到达率。已证明在紫外发光二极管激发的TGL流式细胞仪中检测5.7微米的铕校准微珠是可行的，计算得出的信噪比高达11:1。
