Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1997 Feb;72(2 Pt 1):964–976. doi: 10.1016/s0006-3495(97)78731-9

High-Resolution Scanning Tunneling Microscopy of Fully Hydrated Ripple-Phase Bilayers

J T Woodward IV *, J A Zasadzinski #
PMCID: PMC1185620  PMID: 9017222

Abstract

A modified freeze-fracture replication technique for use with the scanning tunneling microscope (STM) has provided a quantitative, high-resolution description of the waveform and amplitude of rippled bilayers in the Pβ, phase of dimyristoylphosphatidylcholine (DMPC) in excess water. The ripples are uniaxial and asymmetrical, with a temperature-dependent amplitude of 2.4 nm near the chain melting temperature that decreases to zero at the chain crystallization temperature. The wavelength of 11 nm does not change with temperature. The observed ripple shape and the temperature-induced structural changes are not predicted by any current theory. Calibration and reproducibility of the STM/replica technique were tested with replicas of well-characterized bilayers of cadmium arachidate on mica that provide regular 5.5-nm steps. STM images were analyzed using a cross-correlation averaging program to eliminate the effects of noise and the finite size and shapes of the metal grains that make up the replica. The correlation averaging allowed us to develop a composite ripple profile averaged over hundreds of individual ripples measured on different samples with different STM tips. The STM/replica technique avoids many of the previous artifacts of biological STM imaging and can be used to examine a variety of periodic hydrated lipid and protein samples at a lateral resolution of about 1 nm and a vertical resolution of about 0.3 nm. This resolution is superior to conventional and tapping mode AFM of soft biological materials; the technique is substrate-free, and the conductive and chemically uniform replicas make image interpretation simple and direct.

Full text

PDF
964

Images in this article

968FIGURE 1
on p.968
969FIGURE 2
on p.969
970FIGURE 3
on p.970
972FIGURE 5
on p.972

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amrein M., Stasiak A., Gross H., Stoll E., Travaglini G. Scanning tunneling microscopy of recA-DNA complexes coated with a conducting film. Science. 1988 Apr 22;240(4851):514–516. doi: 10.1126/science.3358130. [DOI] [PubMed] [Google Scholar]
  2. Bailey S. M., Chiruvolu S., Longo M. L., Zasadzinski J. A. Design and operation of a simple environmental chamber for rapid freezing fixation. J Electron Microsc Tech. 1991 Sep;19(1):118–126. doi: 10.1002/jemt.1060190112. [DOI] [PubMed] [Google Scholar]
  3. Bailey S. M., Zasadzinski J. A. Validation of convection-limited cooling of samples for freeze-fracture electron microscopy. J Microsc. 1991 Sep;163(Pt 3):307–320. doi: 10.1111/j.1365-2818.1991.tb03182.x. [DOI] [PubMed] [Google Scholar]
  4. Bellare J. R., Davis H. T., Scriven L. E., Talmon Y. Controlled environment vitrification system: an improved sample preparation technique. J Electron Microsc Tech. 1988 Sep;10(1):87–111. doi: 10.1002/jemt.1060100111. [DOI] [PubMed] [Google Scholar]
  5. Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett. 1986 Mar 3;56(9):930–933. doi: 10.1103/PhysRevLett.56.930. [DOI] [PubMed] [Google Scholar]
  6. Blackman GS, Mate CM, Philpott MR. Interaction forces of a sharp tungsten tip with molecular films on silicon surfaces. Phys Rev Lett. 1990 Oct 29;65(18):2270–2273. doi: 10.1103/PhysRevLett.65.2270. [DOI] [PubMed] [Google Scholar]
  7. Branton D. Fracture faces of frozen membranes. Proc Natl Acad Sci U S A. 1966 May;55(5):1048–1056. doi: 10.1073/pnas.55.5.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carlson JM, Sethna JP. Theory of the ripple phase in hydrated phospholipid bilayers. Phys Rev A Gen Phys. 1987 Oct 1;36(7):3359–3374. doi: 10.1103/physreva.36.3359. [DOI] [PubMed] [Google Scholar]
  9. Cevc G. Polymorphism of the bilayer membranes in the ordered phase and the molecular origin of the lipid pretransition and rippled lamellae. Biochim Biophys Acta. 1991 Feb 11;1062(1):59–69. doi: 10.1016/0005-2736(91)90335-6. [DOI] [PubMed] [Google Scholar]
  10. Chen CJ. Effects of m. Phys Rev Lett. 1992 Sep 14;69(11):1656–1659. doi: 10.1103/PhysRevLett.69.1656. [DOI] [PubMed] [Google Scholar]
  11. Ciraci S, Baratoff A, Batra IP. Tip-sample interaction effects in scanning-tunneling and atomic-force microscopy. Phys Rev B Condens Matter. 1990 Feb 15;41(5):2763–2775. doi: 10.1103/physrevb.41.2763. [DOI] [PubMed] [Google Scholar]
  12. Clemmer C. R., Beebe T. P., Jr Graphite: a mimic for DNA and other biomolecules in scanning tunneling microscope studies. Science. 1991 Feb 8;251(4994):640–642. doi: 10.1126/science.1992517. [DOI] [PubMed] [Google Scholar]
  13. Crepeau R. H., Fram E. K. Reconstruction of imperfectly ordered zinc-induced tubulin sheets using cross-correlation and real space averaging. Ultramicroscopy. 1981;6(1):7–17. doi: 10.1016/s0304-3991(81)80173-8. [DOI] [PubMed] [Google Scholar]
  14. Fetter R. D., Costello M. J. A procedure for obtaining complementary replicas of ultra-rapidly frozen sandwiched samples. J Microsc. 1986 Mar;141(Pt 3):277–290. doi: 10.1111/j.1365-2818.1986.tb02722.x. [DOI] [PubMed] [Google Scholar]
  15. Florin E. L., Moy V. T., Gaub H. E. Adhesion forces between individual ligand-receptor pairs. Science. 1994 Apr 15;264(5157):415–417. doi: 10.1126/science.8153628. [DOI] [PubMed] [Google Scholar]
  16. Foster J. S., Frommer J. E., Arnett P. C. Molecular manipulation using a tunnelling microscope. Nature. 1988 Jan 28;331(6154):324–326. doi: 10.1038/331324a0. [DOI] [PubMed] [Google Scholar]
  17. Frank J., Goldfarb W., Eisenberg D., Baker T. S. Reconstruction of glutamine synthetase using computer averaging. Ultramicroscopy. 1978;3(3):283–290. doi: 10.1016/s0304-3991(78)80038-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Frisbie C. D., Rozsnyai L. F., Noy A., Wrighton M. S., Lieber C. M. Functional group imaging by chemical force microscopy. Science. 1994 Sep 30;265(5181):2071–2074. doi: 10.1126/science.265.5181.2071. [DOI] [PubMed] [Google Scholar]
  19. Goldstein RE, Leibler S. Model for lamellar phases of interacting lipid membranes. Phys Rev Lett. 1988 Nov 7;61(19):2213–2216. doi: 10.1103/PhysRevLett.61.2213. [DOI] [PubMed] [Google Scholar]
  20. Guckenberger R., Heim M., Cevc G., Knapp H. F., Wiegräbe W., Hillebrand A. Scanning tunneling microscopy of insulators and biological specimens based on lateral conductivity of ultrathin water films. Science. 1994 Dec 2;266(5190):1538–1540. doi: 10.1126/science.7985024. [DOI] [PubMed] [Google Scholar]
  21. Gulik-Krzywicki T., Costello M. J. The use of low temperature X-ray diffraction to evaluate freezing methods used in freeze-fracture electron microscopy. J Microsc. 1978 Jan;112(1):103–113. doi: 10.1111/j.1365-2818.1978.tb01158.x. [DOI] [PubMed] [Google Scholar]
  22. Hallmark VM, Chiang S, Rabolt JF, Swalen JD, Wilson RJ. Observation of atomic corrugation on Au(111) by scanning tunneling microscopy. Phys Rev Lett. 1987 Dec 21;59(25):2879–2882. doi: 10.1103/PhysRevLett.59.2879. [DOI] [PubMed] [Google Scholar]
  23. Hansma P. K., Elings V. B., Marti O., Bracker C. E. Scanning tunneling microscopy and atomic force microscopy: application to biology and technology. Science. 1988 Oct 14;242(4876):209–216. doi: 10.1126/science.3051380. [DOI] [PubMed] [Google Scholar]
  24. Hentschel MP, Rustichelli F. Structure of the Ripple Phase P beta ' in Hydrated Phosphatidylcholine Multimembranes. Phys Rev Lett. 1991 Feb 18;66(7):903–906. doi: 10.1103/PhysRevLett.66.903. [DOI] [PubMed] [Google Scholar]
  25. Hui S. W., Viswanathan R., Zasadzinski J. A., Israelachvili J. N. The structure and stability of phospholipid bilayers by atomic force microscopy. Biophys J. 1995 Jan;68(1):171–178. doi: 10.1016/S0006-3495(95)80172-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ihn K. J., Zasadzinski J. A., Pindak R., Slaney A. J., Goodby J. Observations of the liquid-crystal analog of the abrikosov phase. Science. 1992 Oct 9;258(5080):275–278. doi: 10.1126/science.258.5080.275. [DOI] [PubMed] [Google Scholar]
  27. Jacobson K., Sheets E. D., Simson R. Revisiting the fluid mosaic model of membranes. Science. 1995 Jun 9;268(5216):1441–1442. doi: 10.1126/science.7770769. [DOI] [PubMed] [Google Scholar]
  28. Janiak M. J., Small D. M., Shipley G. G. Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin. J Biol Chem. 1979 Jul 10;254(13):6068–6078. [PubMed] [Google Scholar]
  29. Katsaras J, Raghunathan VA. Molecular chirality and the "ripple" phase of phosphatidylcholine multibilayers. Phys Rev Lett. 1995 Mar 13;74(11):2022–2025. doi: 10.1103/PhysRevLett.74.2022. [DOI] [PubMed] [Google Scholar]
  30. Keller S. L., Bezrukov S. M., Gruner S. M., Tate M. W., Vodyanoy I., Parsegian V. A. Probability of alamethicin conductance states varies with nonlamellar tendency of bilayer phospholipids. Biophys J. 1993 Jul;65(1):23–27. doi: 10.1016/S0006-3495(93)81040-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Krbecek R., Gebhardt C., Gruler H., Sackmann E. Three dimensional microscopic surface profiles of membranes reconstructed from freeze etching electrol micrographs. Biochim Biophys Acta. 1979 Jun 13;554(1):1–22. doi: 10.1016/0005-2736(79)90002-6. [DOI] [PubMed] [Google Scholar]
  32. Longo M. L., Bisagno A. M., Zasadzinski J. A., Bruni R., Waring A. J. A function of lung surfactant protein SP-B. Science. 1993 Jul 23;261(5120):453–456. doi: 10.1126/science.8332910. [DOI] [PubMed] [Google Scholar]
  33. Lubensky TC, MacKintosh FC. Theory of "Ripple" Phases of Lipid Bilayers. Phys Rev Lett. 1993 Sep 6;71(10):1565–1568. doi: 10.1103/PhysRevLett.71.1565. [DOI] [PubMed] [Google Scholar]
  34. Luna E. J., McConnell H. M. The intermediate monoclinic phase of phosphatidylcholines. Biochim Biophys Acta. 1977 May 2;466(3):381–392. doi: 10.1016/0005-2736(77)90331-5. [DOI] [PubMed] [Google Scholar]
  35. Mamin HJ, Ganz E, Abraham DW, Thomson RE, Clarke J. Contamination-mediated deformation of graphite by the scanning tunneling microscope. Phys Rev B Condens Matter. 1986 Dec 15;34(12):9015–9018. doi: 10.1103/physrevb.34.9015. [DOI] [PubMed] [Google Scholar]
  36. Matuoka S., Kato S., Hatta I. Temperature change of the ripple structure in fully hydrated dimyristoylphosphatidylcholine/cholesterol multibilayers. Biophys J. 1994 Aug;67(2):728–736. doi: 10.1016/S0006-3495(94)80533-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McCullough WS, Scott HL. Statistical-Mechanical Theory of the Ripple Phase of Lipid Bilayers. Phys Rev Lett. 1990 Aug 13;65(7):931–934. doi: 10.1103/PhysRevLett.65.931. [DOI] [PubMed] [Google Scholar]
  38. Meyer E, Overney R, Brodbeck D, Howald L, Lüthi R, Frommer J, Güntherodt H. Friction and wear of Langmuir-Blodgett films observed by friction force microscopy. Phys Rev Lett. 1992 Sep 21;69(12):1777–1780. doi: 10.1103/PhysRevLett.69.1777. [DOI] [PubMed] [Google Scholar]
  39. Mortensen K., Pfeiffer W., Sackmann E., Knoll W. Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram. Biochim Biophys Acta. 1988 Nov 22;945(2):221–245. doi: 10.1016/0005-2736(88)90485-3. [DOI] [PubMed] [Google Scholar]
  40. Mou J., Yang J., Shao Z. Tris(hydroxymethyl)aminomethane (C4H11NO3) induced a ripple phase in supported unilamellar phospholipid bilayers. Biochemistry. 1994 Apr 19;33(15):4439–4443. doi: 10.1021/bi00181a001. [DOI] [PubMed] [Google Scholar]
  41. Müller-Reichert T., Butt H. J., Gross H. STM of metal embedded and coated DNA and DNA-protein complexes. J Microsc. 1996 Jun;182(Pt 3):169–176. doi: 10.1046/j.1365-2818.1996.62426.x. [DOI] [PubMed] [Google Scholar]
  42. Nagle J. F., Zhang R., Tristram-Nagle S., Sun W., Petrache H. I., Suter R. M. X-ray structure determination of fully hydrated L alpha phase dipalmitoylphosphatidylcholine bilayers. Biophys J. 1996 Mar;70(3):1419–1431. doi: 10.1016/S0006-3495(96)79701-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Radmacher M., Fritz M., Hansma H. G., Hansma P. K. Direct observation of enzyme activity with the atomic force microscope. Science. 1994 Sep 9;265(5178):1577–1579. doi: 10.1126/science.8079171. [DOI] [PubMed] [Google Scholar]
  44. Ruben G. C. Ultrathin (1 nm) vertically shadowed platinum-carbon replicas for imaging individual molecules in freeze-etched biological DNA and material science metal and plastic specimens. J Electron Microsc Tech. 1989 Dec;13(4):335–354. doi: 10.1002/jemt.1060130407. [DOI] [PubMed] [Google Scholar]
  45. STEERE R. L. Electron microscopy of structural detail in frozen biological specimens. J Biophys Biochem Cytol. 1957 Jan 25;3(1):45–60. doi: 10.1083/jcb.3.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sammon MJ, Zasadzinski JA, Kuzma MR. Electron-microscope observation of the smectic-nematic transition in a lyotropic liquid crystal. Phys Rev Lett. 1986 Dec 1;57(22):2834–2837. doi: 10.1103/PhysRevLett.57.2834. [DOI] [PubMed] [Google Scholar]
  47. Saxton W. O., Baumeister W. The correlation averaging of a regularly arranged bacterial cell envelope protein. J Microsc. 1982 Aug;127(Pt 2):127–138. doi: 10.1111/j.1365-2818.1982.tb00405.x. [DOI] [PubMed] [Google Scholar]
  48. Schneider M. B., Chan W. K., Webb W. W. Fast diffusion along defects and corrugations in phospholipid P beta, liquid crystals. Biophys J. 1983 Aug;43(2):157–165. doi: 10.1016/S0006-3495(83)84336-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schwartz D. K., Garnaes J., Viswanathan R., Zasadzinski J. A. Surface order and stability of langmuir-blodgett films. Science. 1992 Jul 24;257(5069):508–511. doi: 10.1126/science.257.5069.508. [DOI] [PubMed] [Google Scholar]
  50. Shao Z., Yang J. Progress in high resolution atomic force microscopy in biology. Q Rev Biophys. 1995 May;28(2):195–251. doi: 10.1017/s0033583500003061. [DOI] [PubMed] [Google Scholar]
  51. Singer S. J., Nicolson G. L. The fluid mosaic model of the structure of cell membranes. Science. 1972 Feb 18;175(4023):720–731. doi: 10.1126/science.175.4023.720. [DOI] [PubMed] [Google Scholar]
  52. Sirota E. B., Smith G. S., Safinya C. R., Plano R. J., Clark N. A. X-ray Scattering Studies of Aligned, Stacked Surfactant Membranes. Science. 1988 Dec 9;242(4884):1406–1409. doi: 10.1126/science.242.4884.1406. [DOI] [PubMed] [Google Scholar]
  53. Soler JM, Baro AM, Garcia N, Rohrer H. Interatomic forces in scanning tunneling microscopy: Giant corrugations of the graphite surface. Phys Rev Lett. 1986 Jul 28;57(4):444–447. doi: 10.1103/PhysRevLett.57.444. [DOI] [PubMed] [Google Scholar]
  54. Sonnenfeld R., Hansma P. K. Atomic-resolution microscopy in water. Science. 1986 Apr 11;232(4747):211–213. doi: 10.1126/science.232.4747.211. [DOI] [PubMed] [Google Scholar]
  55. Stemmer A., Hefti A., Aebi U., Engel A. Scanning tunneling and transmission electron microscopy on identical areas of biological specimens. Ultramicroscopy. 1989 Jul-Aug;30(3):263–280. doi: 10.1016/0304-3991(89)90056-9. [DOI] [PubMed] [Google Scholar]
  56. Sun W. J., Tristram-Nagle S., Suter R. M., Nagle J. F. Structure of the ripple phase in lecithin bilayers. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7008–7012. doi: 10.1073/pnas.93.14.7008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tardieu A., Luzzati V., Reman F. C. Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases. J Mol Biol. 1973 Apr 25;75(4):711–733. doi: 10.1016/0022-2836(73)90303-3. [DOI] [PubMed] [Google Scholar]
  58. Tersoff J, Lang ND. Tip-dependent corrugation of graphite in scanning tunneling microscopy. Phys Rev Lett. 1990 Aug 27;65(9):1132–1135. doi: 10.1103/PhysRevLett.65.1132. [DOI] [PubMed] [Google Scholar]
  59. Wack DC, Webb WW. Synchrotron x-ray study of the modulated lamellar phase P beta ' in the lecithin-water system. Phys Rev A Gen Phys. 1989 Sep 1;40(5):2712–2730. doi: 10.1103/physreva.40.2712. [DOI] [PubMed] [Google Scholar]
  60. Wang Z. H., Hartmann T., Baumeister W., Guckenberger R. Thickness determination of biological samples with a zeta-calibrated scanning tunneling microscope. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9343–9347. doi: 10.1073/pnas.87.23.9343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Weisenhorn AL, Maivald P, Butt H, Hansma PK. Measuring adhesion, attraction, and repulsion between surfaces in liquids with an atomic-force microscope. Phys Rev B Condens Matter. 1992 May 15;45(19):11226–11232. doi: 10.1103/physrevb.45.11226. [DOI] [PubMed] [Google Scholar]
  62. Wepf R., Amrein M., Bürkli U., Gross H. Platinum/iridium/carbon: a high-resolution shadowing material for TEM, STM and SEM of biological macromolecular structures. J Microsc. 1991 Jul;163(Pt 1):51–64. doi: 10.1111/j.1365-2818.1991.tb03159.x. [DOI] [PubMed] [Google Scholar]
  63. Wiegräbe W., Nonnenmacher M., Guckenberger R., Wolter O. Atomic force microscopy of a hydrated bacterial surface protein. J Microsc. 1991 Jul;163(Pt 1):79–84. doi: 10.1111/j.1365-2818.1991.tb03161.x. [DOI] [PubMed] [Google Scholar]
  64. Woodward J. T., Kono C., Madsen L. L., Zasadzinski J. A. Inherent bias in correlation averaged images. J Microsc. 1995 Apr;178(Pt 1):86–92. doi: 10.1111/j.1365-2818.1995.tb03583.x. [DOI] [PubMed] [Google Scholar]
  65. Woodward JT, IV, Zasadzinski JA. Amplitude, wave form, and temperature dependence of bilayer ripples in the P beta ' phase. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 1996 Apr;53(4):R3044–R3047. doi: 10.1103/physreve.53.r3044. [DOI] [PubMed] [Google Scholar]
  66. Yuan J. Y., Shao Z. F. Simple model of image formation by scanning tunneling microscopy of non-conducting materials. Ultramicroscopy. 1990 Dec;34(3):223–226. doi: 10.1016/0304-3991(90)90074-v. [DOI] [PubMed] [Google Scholar]
  67. Yuan JY, Shao Z, Gao C. Alternative method of imaging surface topologies of nonconducting bulk specimens by scanning tunneling microscopy. Phys Rev Lett. 1991 Aug 12;67(7):863–866. doi: 10.1103/PhysRevLett.67.863. [DOI] [PubMed] [Google Scholar]
  68. Zasadzinski J. A., Bailey S. M. Applications of freeze-fracture replication to problems in materials and colloid science. J Electron Microsc Tech. 1989 Dec;13(4):309–334. doi: 10.1002/jemt.1060130406. [DOI] [PubMed] [Google Scholar]
  69. Zasadzinski J. A. Effect of stereoconfiguration on ripple phases (P beta') of dipalmitoylphosphatidylcholine. Biochim Biophys Acta. 1988 Dec 22;946(2):235–243. doi: 10.1016/0005-2736(88)90398-7. [DOI] [PubMed] [Google Scholar]
  70. Zasadzinski J. A., Viswanathan R., Madsen L., Garnaes J., Schwartz D. K. Langmuir-Blodgett films. Science. 1994 Mar 25;263(5154):1726–1733. doi: 10.1126/science.8134836. [DOI] [PubMed] [Google Scholar]
  71. Zasadzinski JA, Meiboom S, Sammon MJ, Berreman DW. Freeze-fracture electron-microscope observations of the blue phase III. Phys Rev Lett. 1986 Jul 21;57(3):364–367. doi: 10.1103/PhysRevLett.57.364. [DOI] [PubMed] [Google Scholar]
  72. Zasadzinski JA, Meyer RB. Molecular imaging of tobacco mosaic virus lyotropic nematic phases. Phys Rev Lett. 1986 Feb 10;56(6):636–638. doi: 10.1103/PhysRevLett.56.636. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES