Poe Bobby G, Navratil Marian, Arriaga Edgar A
Department of Chemistry, University of Minnesota, 207 Pleasant St SE, Minneapolis, MN 55455, USA.
J Chromatogr A. 2006 Dec 29;1137(2):249-55. doi: 10.1016/j.chroma.2006.10.011. Epub 2006 Oct 27.
Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.
流式细胞术（FCM）以及最近出现的柱后激光诱导荧光检测毛细管电泳（CE-LIF）都已用于亚细胞颗粒分析，但它们的分析性能尚未得到比较。在这项工作中，我们使用荧光标记的微球和分离的线粒体，将商用FCM与自制的CE-LIF仪器进行了比较。由各个荧光强度的相对标准偏差（RSD）证明，对于等效可溶性荧光素（MESF）分子数≥1.5×10⁶的微球，FCM的性能比CE-LIF好两倍。然而，对于MESF＜1.5×10⁶的微球，FCM的信噪比（S/N）相对较低且RSD较高。另一方面，对于所有测试的微球，CE-LIF产生的S/N比FCM高25倍以上，对于MESF＜1.5×10⁶的微球，其RSD更低。当分析用10-N-壬基吖啶橙（NAO）标记的线粒体时，两种技术的S/N比相似。这似乎是由于CE-LIF仪器的LIF检测器检测NAO标记的线粒体时发生了光漂白。两种技术在亚细胞分析中都有其优势；FCM的优势在于能快速收集数千个颗粒的数据，而CE-LIF消耗的样品量不到一纳升，并能提供单个颗粒的电泳迁移率。
