BODIPY phosphatidylinositol probes incorporation into the membrane of giant unilamellar vesicles grown in carbohydrate and physiological buffer solutions

The use of Giant Unilamellar Vesicles (GUVs) composed of fluorescently labelled lipid analogues has become an increasingly popular model to study both structural and complex biophysical properties of bilayers. However, there is a common assumption that the number of probes incorporated into the membrane of the GUVs is proportional to the mole fraction (%) of these lipid molecules in the original solvent solution. To test this assumption, a commercial confocal laser scanning microscope (Nikon C1) was used to obtain single point fluorescence correlation spectroscopy (FCS) data. We measured the diffusion coefficient and number of molecules incorporated into the membrane of the GUVs for several BODIPY labelled lipid i.e. BODIPY TMR-phosphatidylinositol (4,5) bisphosphate, BODIPY TR- phosphatidylinositol (4,5) bisphosphate and BODIPY (530/550) hexadecanoyl-sn-glycero-3-phosphocholine. We investigated the effect of various mole fraction of these lipids and compared the results with (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC18] when grown in carbohydrate and physiological buffer solutions. We show that the number of DiIC18 molecules incorporated into the membrane of the GUVs (formed by the electroformation method) is in agreement with the expected number of molecules calculated from the mole fraction of the organic stock solution. However, we find that the actual proportion of β-BODIPY-HPC, TR-PI(4,5)P2, and TMR-PI(4,5)P2 incorporated into the bilayer is highly variable and appear significantly less than the proportion of these lipids in the organic solvent stock solution. This apparently low incorporation is observed regardless of the solutions (carbohydrate or physiological buffer) used when growing the GUVs. These variations in incorporation can be explained by the formation of a blue fluorescent species which is probably due to the formation of dimers of the BODIPY labelled lipids.