Characterizing submicron vesicles with wavelength-resolved fluorescence in flow cytometry

Individual synthetic vesicles 0.1-1.0 μm in diameter are sized by using single-particle fluorescence emission spectra obtained in a custom sheath flow cell with an imaging spectrograph and a charge-coupled device. Data are acquired at 1 Hz, with limits of detection (3σ) less than 6.0 x 10³ and 1.0 x 10⁴ molecules of free sulforhodamine 101 and fluorescein, respectively, and with a spectral range for fluorescence emission collection from 350 to 800 nm (0.45 nm/pixel resolution). The system is used for small-particle population analysis by analyzing a suspension of submicron, unilamellar, synthetic vesicles prepared by standard procedures with phosphatidylserine and Texas red- or fluorescein head-group-conjugated dihydropalmitoylphosphatidylethanolamine. The submicron particles are individually identified, sized, and discriminated based on single-particle fluorescence emission spectra. Excellent agreement is found between fluorescence sizing data and transmission electron microscopic measurements.