Advanced Cytometry Laboratories, MQ Biofocus Research Centre, Macquarie University, Sydney, NSW 2109, Australia.
Sci Rep. 2012;2:837. doi: 10.1038/srep00837. Epub 2012 Nov 12.
We report a new development of orthogonal scanning automated microscopy (OSAM) incorporating time-gated detection to locate rare-event organisms regardless of autofluorescent background. The necessity of using long-lifetime (hundreds of microseconds) luminescent biolabels for time-gated detection implies long integration (dwell) time, resulting in slow scan speed. However, here we achieve high scan speed using a new 2-step orthogonal scanning strategy to realise on-the-fly time-gated detection and precise location of 1-μm lanthanide-doped microspheres with signal-to-background ratio of 8.9. This enables analysis of a 15 mm × 15 mm slide area in only 3.3 minutes. We demonstrate that detection of only a few hundred photoelectrons within 100 μs is sufficient to distinguish a target event in a prototype system using ultraviolet LED excitation. Cytometric analysis of lanthanide labelled Giardia cysts achieved a signal-to-background ratio of two orders of magnitude. Results suggest that time-gated OSAM represents a new opportunity for high-throughput background-free biosensing applications.
我们报告了一种新的正交扫描自动显微镜（OSAM）的发展，该技术结合了时间门控检测，可以定位罕见事件的生物体，而不受自发荧光背景的影响。为了进行时间门控检测，需要使用长寿命（数百微秒）的发光生物标记物，这意味着需要长时间的积分（停留）时间，从而导致扫描速度缓慢。然而，在这里，我们使用一种新的两步正交扫描策略实现了高速扫描，从而实现了实时时间门控检测，并精确定位了信号与背景比为 8.9 的 1 微米镧系掺杂微球。这使得在仅 3.3 分钟内即可分析 15 毫米×15 毫米的载玻片区域。我们证明，在原型系统中，使用紫外线 LED 激发，仅在 100 微秒内检测到几百个光电子就足以区分目标事件。对镧系标记的贾第虫包囊的细胞计量分析实现了两个数量级的信号与背景比。结果表明，时间门控 OSAM 为高通量无背景生物传感应用提供了新的机会。
