The following paper appeared in Proceedings of the fifty-fourth Annual Meeting, Microscopy Society of America; G. W. Bailey, J. M. Corbett, R. V. W. Dimlich, J. R. Michael and N. J., Zaluzec (Eds.). San Francisco Press, San Francisco, CA, pp. 898-899 (1996).
James F. Hainfeld
Department of Biology, Brookhaven National Laboratory, Upton, NY 11973
Lipids are an important class of molecules, being found in membranes, HDL, LDL, and other natural structures, serving essential roles in structure and with varied functions such as compartmentalization and transport. Synthetic liposomes are also widely used as delivery and release vehicles for drugs, cosmetics, and other chemicals; soap is made from lipids. Lipids may form bilayer or multilamellar vesicles, micelles, sheets, tubes, and other structures. Lipid molecules may be linked to proteins, carbohydrates or other moities. EM study of this essential ingredient to life has lagged, due to lack of direct methods to visualize lipids without extensive alteration. OsO4 reacts with double bonds in membrane phospholipids, forming crossbridges. This has been the method od choice to both fix and stain membranes, so far. An earlier work described the use of tungtstate clusters (W11) attached to lipid moieties to form lipid structures and lipid probes.1
With the development of gold clusters, it is now possible to covalently and specifically link a dense gold sphere2 to a lipid molecule; for example, reacting a Mono-N-hydroxysuccinimide Nanogold cluster with the amino group on phosphatidyl ethanolamine. Examples of a gold-fatty acid and a gold-phospholipid are shown in Fig. 1.
Dipalmitoyl phosphatidyl ethanolamine-Nanogold (DPPE-Nanogold, Fig. 1B), which has C16 hydrophobic tail chains, was obtained from Nanoprobes, Inc.,3 and made into vesicles. Dried DPPE-Nanogold (10 nmoles) was resuspended in 0.5 mL methanol. 20 microliters was transferred to a test tube and driedwith a nitrogen stream. 50 ml water was added and the tube sonicatedfor 10 min in a bath unit at room temperature. A drop was applied to thin carbon supported by a holey film on an EM grid. After 1 min, the solution was wicked, rinsed with 20 mM ammonium acetate several times, wicked, andquick frozen in liquid nitrogen slush. The sample was then freeze dried overnight, brought to room temperature, transferred to the EM under vacuum, and inserted into the specimen stage which was cooled to -130° C. The samples were observed in the high resolution Brookhaven Scanning Transmission EM (STEM) operating in darkfield mode.4
Several interesting lipid forms were observed, including small micelles (Fig. 2), and bilayer vesicles that had apparently broken so that single and doublelayers were displayed (Fig. 3). Since no other unlabeled lipid was used, and the labeling was virtually quantitative, nearly every lipid molecule appeared with a gold cluster. Hexagonal closepacking was observed in these regular arrays, with spacings of ~ 2.5 nm. This is approximately the diameter of the gold cluster, whose core is 1.4 nm, with an organic shell of total diameter 2.7 nm. However, this is somewhat more than the typical spacing between phospholipids in native bilayers, which is ~ 1.3 nm.5
These methods provide a way of producing regular gold sphere monolayers, orother lipid structures. For biology, they might be intercalated into membranes to follow membrane movement, or used in reconstituted membrane-protein structures to study lipid distribution. They also provide a way to directly visualize liposomes, even at the light microscopelevel, via silver enhancement.6
- J. S. Wall, J. F. Hainfeld, J. J. Lipka and F. E. Quaite, J. Histochem. Cytochem., 38 (1990) 1793.
- J. F. Hainfeld and F. R. Furuya, J. Histochem. Cytochem., 40 (1992) 177.
- Nanoprobes, 25 East Loop Road, Suite 113, Stony Brook, NY 11790.
- J. S. Wall, J. F. Hainfeld and M. N. Simon, EMSA Bulletin, 21 (1991) 81.
- L. Stryer, in Biochemistry (1975).
- J. F. Hainfeld and F. R. Furuya, in Immunogold silver staining:principles, methods and applications, M. A. Hayat, ed., CRC Press, New York (1995) 71.
- The author would like to thank M. Simon, B. Lin, and F.Kito of the BNL STEM facility, and F. Furuya for synthesis and helpful discussions. This work was supported by NIH Grant RR01777 and US Dept of Energy, OHER.
Fig. 1 (On L). Schematic drawing of gold-lipids. A. Fatty acid (palmitoyl, C16)-gold; B. Phospholipid (DPPE, C16)-gold.
Fig.2. Darkfield STEM micrograph of unstained gold-lipid micelles (arrows), made from DPPE-gold, with diameters of ~ 14 nm.
Fig. 3. Darkfield STEM micrograph of unstained gold-lipid bilayer, made from DPPE-gold; arrow points to single lipid layer region.
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