Optimization of confocal epifluorescence microscopy for microchip-based miniaturized total analysis systems
Optimization of confocal epifluorescence microscopy for microchip-based miniaturized total analysis systems
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DOI：
10.1039/a800153g
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发表时间：
1998-07-01
期刊：
影响因子：
4.2
通讯作者：
Harrison, DJ
中科院分区：
文献类型：
--
作者：
Ocvirk, G;Tang, T;Harrison, DJ
描述了一种共焦落射荧光检测方案，该方案经过优化，可在蚀刻至 30 μm 深度的平面玻璃基板内进行亚皮摩尔检测。使用工作距离为 3.7 mm 的 x 40、0.6 数值孔径 (N.A.) 透镜来创建直径约为 12 pm 的聚焦激光光斑，通过对透镜孔径进行底部填充，为激光束提供 0.22 的有效测量 N.A.。各种针孔的切片功率（光轴视场）和对应的探测器探针体积（激发和观察体积的重叠）分别为：（针孔直径、切片功率、探针体积）：100μm、18μm、0.1pl； 200μm、20μm、0.4pl； 400μm、26μm、1.7pl；和600μm、36μm、2.4pl。使用最佳 400 pm 针孔以连续流模式测量的荧光强度与荧光素浓度的双对数图显示相关系数为 0.996，斜率为 0.85。在此模式下，300 fM 荧光素在背景上发出 34.6 +/- 8.1 mV 的信号，信噪比为 6,1，代表芯片上报告的最低测量荧光素染料浓度。 1 pM 荧光素的毛细管区带电泳导致平均 S/N 为 5.8。注射塞估计约有 5470 个分子，平均对应于 570 个检测到的分子。简要介绍了用于将管道、熔融石英毛细管或加压系统连接到平面玻璃芯片上蚀刻的微流体通道的快速安装、无法兰配件的设计和使用。
A confocal epifluorescence detection scheme, optimized to give sub-picomolar detection within planar glass substrates etched to a 30 mu m depth, is described. A x 40, 0.6 numerical aperture (N.A.) lens with a 3.7 mm working distance was used to create a focused laser spot about 12 pm in diameter, by under-filling the lens aperture to give an effective, measured N.A. of 0.22 for the laser beam. The sectioning power (optical axis field of view) of various pinholes and the corresponding detector probe volumes (overlap of the excitation and observation volumes) were: (pinhole diameter, sectioning power, probe volume): 100 mu m, 18 mu m, 0.1 pl; 200 mu m, 20 mu m, 0.4 pl; 400 mu m, 26 mu m, 1.7 pl; and 600 mu m, 36 mu m, 2.4 pl. A log-log plot of fluorescence intensity versus fluorescein concentration, measured in continuous-flow mode using the optimum 400 pm pinhole, showed a correlation coefficient of 0.996 and, a slope of 0.85. In this mode, 300 fM fluorescein gave a signal of 34.6 +/- 8.1 mV over background with an S/N of 6,1, representing the lowest measured fluorescein dye concentration reported on-chip. Capillary zone electrophoresis of 1 pM fluorescein resulted in a mean S/N of 5.8. The injection plug, estimated to be about 5470 molecules, corresponds to 570 detected molecules on average. The design and use of quick-fit, flangeless fittings for interfacing tubing, fused-silica capillaries or pressurized systems to microfluidic channels etched in planar glass chips is briefly presented.