Characterizing submicron vesicles with wavelength-resolved fluorescence in flow cytometry.
Individual synthetic vesicles 0.1-1.0 micron 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 (3 sigma) less than 6.0 x 10(3) and 1.0 x 10(4) 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.
Department of Chemistry and Beckman Institute, University of Illinois, Urbana, USA.
R R
RR
Fuller
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JV
Sweedler
D000818:Animals; D001048:Aplysia; D002138:Calibration; D005434:Flow Cytometry / Q000379:methods; D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D008854:Microscopy, Electron; D010316:Particle Size; D010714:Phosphatidylethanolamines; D013050:Spectrometry, Fluorescence / Q000379:methods; D014966:Xanthenes
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010714:Phosphatidylethanolamines; D014966:Xanthenes; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine; C034657:Texas red; D019793:Fluorescein