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Differentiating Unilamellar, Multilamellar, and Oligovesicular Vesicles Using a Fluorescent Dye F. M. Menger,* S. J. Lee, and J. S. Keiper Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received April 16, 1996
Introduction Vesicles of sufficient size to observe under the light microscope (so-called “giant vesicles”) are receiving heightened attention. A review of the subject has just recently appeared.1 Advantages of giant vesicles over the much more intensively investigated “small vesicles” or “large vesicles” (ranging from 200-2000 Å) are multifold: (a) Giant vesicles resemble cell membranes in their curvature, a parameter critical in determining membrane properties.2 (b) Morphological changes (e.g. fusion and fission) need not be deduced implicitly;3 they can be seen directly. (c) Giant vesicles can be manipulated (e.g. biomolecules can be injected inside them using standard cytological methods4 ). (d) Giant vesicles lend themselves to certain experiments (e.g. elucidating rates of wound healing4) that are almost impossible to carry out with submicroscopic vesicles. A question inevitably arises with all vesicle systems, and the giant vesicle is no exception: Are the vesicles unilamellar (one bilayer), multilamellar (like an onion), or oligovesicular (in which a few non-concentric vesicles reside inside a much larger one)? Figure 1 represents these possibilities schematically. Typically, the question is addressed with the aid of freeze-fracture electron microscopy (EM).5 This method is expensive and timeconsuming (especially when many different vesicle samples require assay). Moreover, in the specific case of giant vesicles, one is often dealing with flexible membranes that are sensitive to abuse that is unavoidable under even the most gentle of EM preparations. What is needed, clearly, is a simple, fast, and definitive method for assessing structure while undisturbed vesicles are viewed under the microscope. The present note fulfills this need via a new fluoresence-based method. It should be noted that phase-contrast light microscopy can, under ideal conditions, differentiate between unilamellar, multilamellar, and oligovesicular giant vesicles (Figure 2A-C). But conditions are often not ideal, and (as seen in most published photomicrographs) the exact nature of the giant vesicle cannot be specified. The currently proposed fluorescent dye method will, however, resolve the lamellarity issue even in cases where phase contrast gives little structural information. Our method utilizes an amphiphilic fluorescent dye, N-dodecyl-7-hydroxy-quinolinium triflate (drawn below).6 Structural information is obtained via epifluorescence
(1) Menger, F. M.; Gabrielson, K. D. Angew. Chem., Int. Ed. Engl. 1995, 34, 2091. (2) Deuling, H. J.; Helfrich, W. J. Phys. 1976, 37, 1335. (3) Roseman, M. A.; Thompson, T. E. Biochemistry 1980, 19, 439. (4) Menger, F. M.; Lee, S. J. Langmuir 1995, 11, 3685.
S0743-7463(96)00370-8 CCC: $12.00
Figure 1. Schematic diagrams of unilamellar, multilamellar, and oligovesicular vesicles.
microscopy upon injecting the dye onto a giant vesicle as detailed in the Experimental Section. Fluorescence has, of course, been widely applied to vesicle work,7 but to our knowledge no one has used it specifically to reveal vesicle morphology. Experimental Section All vesicles were prepared with 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (Avanti Polar Lipids) containing 12% cholesterol (Sigma). A mixture of the two compounds was dissolved in CHCl3/MeOH in a roundbottom flask, and the solvent was removed under reduced pressure to create a thin film. The film was shaken briefly with deionized water and lyopholized to produce a fluffy white powder. Approximately 10 µg of the powder was smeared with a spatula inside a 14 mm i.d. O-ring cemented to a microscope slide. The O-ring was filled with deionized water and sealed with a cover slip such that no air bubbles were trapped beneath it. After hydration of the lipid for at least 10 h at 8 °C, there appeared a complex mixture of lamellar plates, tubes, and vesicles. Among the vesicles, multilamellar structures were the most common. Vesicles were examined under phase-contrast or epifluorescence illumination using a Nikon Diaphot-TMD microscope. Phase-contrast images were recorded using a Dage-MTI CCD-72 solid state camera, while fluorescence images were observed using a Dage-MTI SIT camera. The cameras were connected in sequence to a Panasonic AG1960 SVHS, a Hamamatsu Argus-10 image processor, and a Hitachi black-and-white monitor. The vesicle images, processed with the aid of Image-Pro Plus software on a PC workstation, were printed on a Tektronix Phase 440 dye sublimation printer. Micropipets, used to inject dye solution and hold vesicles (see Figure 2), were fabricated from G-1 microcapillaries on a Narishige pipet-puller. Holding pipets were polished using a Narishige Microforge. Tips of the injection pipets averaged 2 µm in diameter. A Nikon PLI-188 piconinjector, set at ca. 10 psi and 5-100 ms injection time (depending on the particular pipet and the desired volume), was used to direct dye solution at the giant vesicles. An injection generally consisted of 10-100 pL. In a typical experiment, an aqueous solution of dye (0.1 mM in deionized water) was injected at a giant vesicle held in placed by a pipet under slight suction. A Nikon V-2A fluorescence cube was used to excite and observe the dye (λex ) ca. 400 nm; λem ) ca. 500 nm). All (5) Kachar, B.; Fuller, N.; Rand, R. P. Biophys. J. 1986, 50, 779. (6) We thank Mr. Kingsley Nelson for carrying out the synthesis of the dye. He followed the procedure given by: Ranganathan, N.; Storey, B. T. J. Heterocycl. Chem. 1980, 17, 1069. (7) Niles, W. D.; Li, Q.; Cohen, F. S. Biophys. J. 1992, 63, 710. Hargreaves, W. R.; Deamer, D. W. Biochemistry 1978, 17, 3759. Armhold, J.; Wiegel, D.; Husler, O., Arnold, K. Biochim. Biophys. Acta 1994, 1191, 375. Lissi, E. A.; Gallardo, S.; Sepulveda, P. J. Colloid Interface Sci. 1992, 152, 104. Rupert, L. A. M.; Hoekstra, D.; Engberts, J. B. F. N. J. Am. Chem. Soc. 1985, 107, 2628. Moss, R. A.; Bhattacharya, S. J. Am. Chem. Soc. 1995, 117, 8688. Struck, D. K.; Pagano, R. E. J. Biol. Chem. 1980, 255, 5404.
© 1996 American Chemical Society
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Notes
Figure 2. Photomicrographs A, B, and C: Phase-contrast images of unilamellar, multilamellar, and oligovesicular vesicles, respectively, composed of POPC plus 12% cholesterol. Photomicrographs D, E, and F: Epifluorescence images of unilamellar, multilamellar, and oligovesicular vesicles, respectively, of the same composition as in A-C but which were exposed to N-dodecyl7-hydroxy-quinolinium triflate. All bars ) 50 µm.
experimental observations, as embodied in Figure 2, were obtained repeatedly in multiple runs. Results and Discussion Unilamellar, multilamellar, and oligolamellar vesicles can be readily differentiated by the fluorescent dye method (Figure 2D-F). In Figure 2D, the dye has adsorbed into or onto the phospholipid bilayer of a unilamellar vesicle. One sees a “halo” of light because the microscope, with its finite depth of field, is focused only on the equator of the vesicle. Thus, regions of the sphere behind and in front of the focal plane do not appear nearly as bright. It is important to mention that our method cannot distinguish between truly unilamellar vesicles and those that possess a few closely packed concentric bilayers. Clearly, however, lipid is confined to a thin outer shell surrounding a large empty inner space.
Multilamellar vesicles emit light from the entire circular image (Figure 2E). Apparently, the dye can flip-flop from outer leaflet to inner leaflet and jump from one bilayer to the adjacent one inside it in a few seconds. As a consequence, the microscope detects light from dyeladened lipid that fills the vesicle volume within the focal plane. Oligovesicular vesicles display non-concentric rings of light (Figure 2F). The fact that inner vesicles are well outlined by the dye again reveals the surprisingly fast invasion of the dye into the vesicles’ interior. Acknowledgment. This work was supported by the National Institutes of Health. LA960370U
