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 ≥1.5 × 106 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 × 106 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 × 106 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.
Bibliographical noteFunding Information:
This work was supported by the National Institute of Health (R01-AG20866). B.P. acknowledges support from NIH-Chemistry/Biology Interface Training Grant (GM08700). E.A. is supported by an NIH Career Award (1K02-AG21453). The authors thank the Flow Cytometry Core Facility of the University of Minnesota Cancer Center, supported by the National Cancer Institute (P30 CA77598).
- Capillary electrophoresis
- Flow cytometry
- Laser-induced fluorescence
- Signal-to-noise ratio