Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry

• Published in: Journal of chromatography Vol. 1137; no. 2; pp. 249 - 255
• Main Authors: POE, Bobby G, NAVRATIL, Marian, ARRIAGA, Edgar A
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 29.12.2006
• Subjects: Aminoacridines; Analytical chemistry; Cell Fractionation - methods; Cell Line, Tumor; Chemistry; Chromatographic methods and physical methods associated with chromatography; Electrophoresis, Capillary - methods; Exact sciences and technology; Flow Cytometry - methods; Fluorescence; Humans; Lasers; Microspheres; Mitochondria - ultrastructure; Other chromatographic methods; Photobleaching; Sensitivity and Specificity; Subcellular Fractions - chemistry; Flow cytometry; Fluorescence detector; Chemical analysis; Microsphere; Capillary electrophoresis; Laser induced fluorescence; Signal-to-noise ratio; Laser-induced fluorescence; Mitochondria; Fluorescent labelling; Electrophoretic mobility; Fluorescein; Spherical particle; Comparative study; Signal to noise ratio
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2006.10.011

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.