Direct observation of the distribution of fluorescent probes in phosphatidylcholine/cholesterol vesicles using flow microfluorometry

Abstract

The technique of flow microfluorometry has been extended to the study of small lipid complexes to assess either the lipid (hydrophobic) or aqueous (hydrophilic) compartments of selected natural or model membrane systems. sn-1-Palmitoyl- sn-2-oleoyl-phosphatidylcholine/cholesterol unilamellar vesicles, averaging 268 nm in diameter and containing varying concentrations of the synthetic lipophile probe, sn-1-palmitoyl- sn-2-12-[ N-4-nitrobenzo-2-oxa-1,3-diazole]-aminocaproyl-phosphatidylcholine (NBD-PC), were analyzed using an Ortho Series 50-H Cytofluorograf and an Ortho 2150 computer system. NBD-labeled vesicles were analyzed for green fluorescence and the intensity of scattered light, the later being analyzed both at low angle (2–5°) and at 90° to the incident beam. At the high amplification required for vesicle detection, background signals from the sheath buffer, nonspecific laser light, and electronic noise were observed. However, this background noise signal was removed by appropriately setting a discriminator window. Profiles of signals falling within this region were then constructed. For the settings selected, more than 98% of data recorded could be attributed to observations on vesicles. Size information from the intensity of scattered light was obtained by comparison of the sample with fluorescent microspheres after correcting for the particle-scattering function difference between hollow and solid spheres and for refractive index differences. Additionally, cytograms and profiles were constructed for vesicles containing 5 m m 6-carboxyfluorescein, 3′,6′-dihydroxy-3-oxospiro(isobenzofuran-1 (3 H),9′-(9 H)xanthen)-6-carboxylic acid, trapped in the aqueous core. Thus, the utility of flow microfluorometry has been extended to much smaller particle populations than studied previously by this technique. It has significant potential for studying several important properties of selected populations of vesicles and lipoproteins including (i) the size and fluorescence distribution of particles, (ii) the equilibrium distribution of probes among different size populations and among different domains within populations, (iii) the time dependence of probe transfer from a specific labeled population to a specific unlabeled population, (iv) the time dependence of vesicle fusion (combining aqueous compartments), and (v) sorting particles which are labeled differently.