High intensity solid-state UV source for time-gated luminescence microscopy
High intensity solid-state UV source for time-gated luminescence microscopy
复制标题
DOI：
10.1002/cyto.a.20326
复制
发表时间：
2006-09-01
期刊：
影响因子：
3.7
通讯作者：
Piper, James
中科院分区：
文献类型：
--
作者：
Connally, Russell;Jin, Dayong;Piper, James
背景：当探针发射与天然存在的固有荧光物质（自发荧光团）竞争时，免疫荧光探针独特的鉴别能力可能会严重受损。在时域下工作的发光显微镜能够选择性地分辨长荧光寿命（τ>100微秒）的探针与短寿命荧光，从而大大提高信噪比（SNR）。本文报道了一种新型的时间门控发光显微镜设计，该设计采用紫外（UV）发光二极管（LED）激发具有长荧光寿命的铕螯合物免疫偶联物的荧光。
方法：通过配备时间门控图像增强CCD相机和高功率（100毫瓦）紫外LED，对一台商用蔡司落射荧光显微镜进行改造以用于时间门控发光（TGL）操作。在激发后的精确间隔延迟对发光的捕捉，从而抑制自发荧光。贾第虫包囊用与铕螯合物（BHHST）偶联的抗体原位标记，其荧光寿命>500微秒。
结果：BHHST标记的贾第虫包囊在紫外激发时在617纳米处发射荧光，使用常规荧光显微镜很难在荧光藻类基质中定位，探针与自发荧光背景的信噪比为0.51∶1。然而，在门控延迟为5微秒的时间门控发光模式下，信噪比提高到12.8∶1，提高了25倍。
结论：与氙气闪光灯相比，紫外LED价格低廉、易于供电且能快速熄灭。此外，LED的尖峰发射使得能够从显微镜上移除光谱滤光片，从而显著提高荧光激发和捕捉的效率。
Background: The unique discriminative ability of immunofluorescent probes can be severely compromised when probe emission competes against naturally occurring, intrinsically fluorescent substances (autofluorophores). Luminescence microscopes that operate in the time-domain can selectively resolve probes with long fluorescence lifetimes (tau > 100 mu s) against short-lived fluorescence to deliver greatly improved signal-to-noise ratio (SNR). A novel time-gated luminescence microscope design is reported that employs an ultraviolet (UV) light emitting diode (LED) to excite fluorescence from a europium chelate immunoconjugate with a long fluorescence lifetime.Methods: A commercial Zeiss epifluorescence microscope was adapted for TGL operation by fitting with a time-gated image-intensified CCD camera and a high-power (100 mW) UV LED. Capture of the luminescence was delayed for a precise interval following excitation so that autofluorescence was suppressed. Giardia cysts were labeled in situ with antibody conjugated to a europium chelate (BHHST) with a fluorescence lifetime > 500 mu s.Results: BHHST-labeled Giardia cysts emit at 617 nm when excited in the UV and were difficult to locate within the matrix of fluorescent algae using conventional fluorescence microscopy, and the SNR of probe to aurofluorescent background was 0.51:1. However in time-gated luminescence mode with a gate-delay of 5 mu s, the SNR was improved to 12.8:1, a 25-fold improvement.Conclusion: In comparison to xenon flashlamps, UV LEDs are inexpensive, easily powered, and extinguish quickly. Furthermore, the spiked emission of the LED enabled removal of spectral filters from the microscope to significantly improve efficiency of fluorescence excitation and capture.