Goodwin P M, Ambrose W P, Martin J C, Keller R A
Chemical Science and Technology Division, Center for Human Genome Studies, Los Alamos National Laboratory, New Mexico, USA.
Cytometry. 1995 Oct 1;21(2):133-44. doi: 10.1002/cyto.990210205.
The sensitive flow cytometric detection of fluorescent species in liquid sample streams requires efficient collection of light from small [approximately 1 picoliter (pl)] sample volumes. This is often accomplished with high numerical aperture (NA) imaging collection optics used in combination with a spatial filter. A method to measure the spatial variation of the optical collection efficiency within the sample volume, using a submicrometer light source, is described. Measurements of the relative optical collection efficiency are presented for two optical collection systems used in our laboratory for single molecule detection. The measurement are in qualitative agreement with relative optical collection efficiency calculations using a simple geometrical optics model. Absolute measurements of the peak collection efficiencies for the two collection systems are also presented. These absolute collection efficiency measurements are in good quantitative agreement with ideal collection efficiencies calculated using measured transmissions and rated NAs of the collection optics. The utility of this information for the characterization and optimization of sensitive fluorescence detection apparatus is discussed.
对液体样本流中荧光物质进行灵敏的流式细胞术检测，需要从小[约1皮升（pl）]样本体积中高效收集光。这通常通过与空间滤波器结合使用的高数值孔径（NA）成像收集光学器件来实现。本文描述了一种使用亚微米光源测量样本体积内光学收集效率空间变化的方法。给出了我们实验室用于单分子检测的两种光学收集系统的相对光学收集效率测量结果。这些测量结果与使用简单几何光学模型计算的相对光学收集效率在定性上一致。还给出了两种收集系统的峰值收集效率的绝对测量结果。这些绝对收集效率测量结果与使用收集光学器件的测量透射率和额定NA计算的理想收集效率在定量上吻合良好。讨论了该信息在灵敏荧光检测装置表征和优化方面的实用性。
