Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry.
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.
Poe
Bobby G
BG
Department of Chemistry, University of Minnesota, 207 Pleasant St SE, Minneapolis, MN 55455, USA.
Navratil
Marian
M
Arriaga
Edgar A
EA
D000609:Aminoacridines; D002458:Cell Fractionation / Q000379:methods; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D007834:Lasers; D008863:Microspheres; D008928:Mitochondria / Q000648:ultrastructure; D038761:Photobleaching; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000737:chemistry*
D000609:Aminoacridines; C041691:10-N-nonylacridinium orange