Flow cytometric characterization and classification of multiple dual-color fluorescent microspheres using fluorescence lifetime.

FlowMetrix (Luminex, Austin, TX) microspheres were recently introduced as a platform for bead-based assays involving antibodies, enzymes, toxins, and nucleic acids. The procedure involves classification of the microspheres by their orange and red fluorescence and quantitation of the BODIPY-tagged biological probes by their green fluorescence. In an attempt to increase the number of fluorochromes available for the biological probes, we explored the possibility of using excited singlet state lifetime as an alternative to one of the fluorochromes. For a set of 20 dual-color microspheres the excited singlet state lifetimes were measured using the total emissions (>515 nm), the orange emissions (515-600 nm), and the red emissions (>665 nm). The microspheres could not all be resolved in bivariates of fluorescence intensity versus excited singlet state lifetime. However, 13 of the microspheres could be resolved using the total emissions and lifetime. Although this result required both fluorochromes, the merits and limitations of this approach to other systems are briefly discussed.