Several methods for the preparation of giant unilamellar vesicles (GUVs) using synthetic phosphatidylcholine phospholipids were evaluated. We compared the physical characteristics--in terms of lamellarity and morphology--of the whole lipid sample for each different lipid preparation using the sectioning capability of the two-photon excitation fluorescence microscope. From the evaluation of the entire lipid sample we determined that vesicle size, internal shape and shell thickness distributions depend on the vesicle's preparation method. Our results show that the preparation of giant unilamellar vesicles by the application of external electric fields offers several advantages among the other methods tested here. Using this method a high yield (approximately 95%) of giant unilamellar vesicles with a narrow size distribution was obtained. Independently of the preparation method, some lipid structures, which are held together by lipid tethers, were identified and resolved. These particular lipid structures show shell thickness and size heterogeneity. Labeling the lipid samples with 6-lauroyl-2-(N,N-dimethylamino)naphtalene (LAURDAN) and using the LAURDAN generalized polarization function we show that the lipid packing in these tethers or tubes is similar to those found in the phospholipid vesicles. The fact that both vesicles and tethers are found in the lipid preparations indicates similar stability between these structures.