Advanced Cytometry Laboratories at Macquarie, MQ Biofocus Research Centre, Faculty of Science, Macquarie University, NSW 2109 Australia.
Anal Chem. 2011 Mar 15;83(6):2294-300. doi: 10.1021/ac103207r. Epub 2011 Feb 23.
Application of standard immuno-fluorescence microscopy techniques for detection of rare-event microorganisms in dirty samples is severely limited by autofluorescence of nontarget organisms or other debris. Time-gated detection using gateable array detectors in combination with microsecond-lifetime luminescent bioprobes (usually lanthanide-based) is highly effective in suppression of (nanosecond-lifetime) autofluorescence background; however, the complexity and cost of the instrumentation is a major barrier to application of these techniques to routine diagnostics. We report a practical, low-cost implementation of time-gated luminescence detection in a standard epifluorescence microscope which has been modified to include a high-power pulsed UV light-emitting diode (LED) illumination source and a standard fast chopper inserted in the focal plane behind a microscope eyepiece. Synchronization of the pulsed illumination/gated detection cycle is driven from the clock signal from the chopper. To achieve time-gated luminescence intensities sufficient for direct visual observation, we use high cycle rates, up to 2.5 kHz, taking advantage of the fast switching capabilities of the LED source. We have demonstrated real-time direct-visual inspection of europium-labeled Giardia lamblia cysts in dirty samples and Cryptosporidium parvum oocysts in fruit juice concentrate. The signal-to-background ratio has been enhanced by a factor of 18 in time-gated mode. The availability of low-cost, robust time-gated microscopes will aid development of long-lifetime luminescence bioprobes and accelerate their application in routine laboratory diagnostics.
应用标准免疫荧光显微镜技术检测脏样中的稀有事件微生物，受到非目标生物或其他碎片的自发荧光的严重限制。使用可门控阵列探测器结合微秒寿命发光生物探针（通常基于镧系元素）进行时间门控检测，可有效抑制（纳秒寿命）自发荧光背景；然而，仪器的复杂性和成本是将这些技术应用于常规诊断的主要障碍。我们报告了一种在标准荧光显微镜中实现时间门控荧光检测的实用、低成本方法，该显微镜经过改装，包括高功率脉冲紫外发光二极管（LED）照明源和一个标准的快速斩波器，插入显微镜目镜焦平面后面。脉冲照明/门控检测周期的同步由斩波器的时钟信号驱动。为了实现足以进行直接目视观察的时间门控荧光强度，我们利用 LED 源的快速开关能力，采用高达 2.5 kHz 的高循环率。我们已经证明了在脏样中直接实时观察到镧系元素标记的蓝氏贾第鞭毛虫包囊和浓缩果汁中的微小隐孢子虫卵囊。在时间门控模式下，信号与背景的比值提高了 18 倍。低成本、坚固的时间门控显微镜的可用性将有助于开发长寿命发光生物探针，并加速其在常规实验室诊断中的应用。
