Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1999 Aug;77(2):1150–1158. doi: 10.1016/S0006-3495(99)76966-3

Tapping-mode atomic force microscopy produces faithful high-resolution images of protein surfaces.

C Möller 1, M Allen 1, V Elings 1, A Engel 1, D J Müller 1
PMCID: PMC1300406  PMID: 10423460

Abstract

Compared to contact-mode atomic force microscopy (CMAFM), tapping-mode atomic force microscopy (TMAFM) has the advantage of allowing imaging surfaces of macromolecules, even when they are only weakly attached to the support. In this study, TMAFM is applied to two different regular protein layers whose structures are known to great detail, the purple membrane from Halobacterium salinarum and the hexagonally packed intermediate (HPI) layer from Deinococcus radiodurans, to assess the faithfulness of high-resolution TMAFM images. Topographs exhibited a lateral resolution between 1.1 and 1. 5 nm and a vertical resolution of approximately 0.1 nm. For all protein surfaces, TMAFM and CMAFM topographs were in excellent agreement. TMAFM was capable of imaging the fragile polypeptide loop connecting the transmembrane alpha-helices E and F of bacteriorhodopsin in its native extended conformation. The standard deviation (SD) of averages calculated from TMAFM topographs exhibited an enhanced minimum (between 0.1 and 0.9 nm) that can be assigned to the higher noise of the raw data. However, the SD difference, indicating the flexibility of protein subunits, exhibited an excellent agreement between the two imaging modes. This demonstrates that the recently invented imaging-mode TMAFM has the ability to faithfully record high-resolution images and has sufficient sensitivity to contour individual peptide loops without detectable deformations.

Full Text

The Full Text of this article is available as a PDF (324.2 KB).

Selected References

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

  1. Baumeister W., Barth M., Hegerl R., Guckenberger R., Hahn M., Saxton W. O. Three-dimensional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans. J Mol Biol. 1986 Jan 20;187(2):241–250. doi: 10.1016/0022-2836(86)90231-7. [DOI] [PubMed] [Google Scholar]
  2. Baumeister W., Karrenberg F., Rachel R., Engel A., ten Heggeler B., Saxton W. O. The major cell envelope protein of Micrococcus radiodurans (R1). Structural and chemical characterization. Eur J Biochem. 1982 Jul;125(3):535–544. doi: 10.1111/j.1432-1033.1982.tb06715.x. [DOI] [PubMed] [Google Scholar]
  3. Bezanilla M., Drake B., Nudler E., Kashlev M., Hansma P. K., Hansma H. G. Motion and enzymatic degradation of DNA in the atomic force microscope. Biophys J. 1994 Dec;67(6):2454–2459. doi: 10.1016/S0006-3495(94)80733-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bremer A., Henn C., Engel A., Baumeister W., Aebi U. Has negative staining still a place in biomacromolecular electron microscopy? Ultramicroscopy. 1992 Oct;46(1-4):85–111. doi: 10.1016/0304-3991(92)90008-8. [DOI] [PubMed] [Google Scholar]
  6. Butt H. J. Measuring local surface charge densities in electrolyte solutions with a scanning force microscope. Biophys J. 1992 Aug;63(2):578–582. doi: 10.1016/S0006-3495(92)81601-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Czajkowsky D. M., Allen M. J., Elings V., Shao Z. Direct visualization of surface charge in aqueous solution. Ultramicroscopy. 1998 Jul;74(1-2):1–5. doi: 10.1016/s0304-3991(98)00024-2. [DOI] [PubMed] [Google Scholar]
  8. Czajkowsky D. M., Sheng S., Shao Z. Staphylococcal alpha-hemolysin can form hexamers in phospholipid bilayers. J Mol Biol. 1998 Feb 20;276(2):325–330. doi: 10.1006/jmbi.1997.1535. [DOI] [PubMed] [Google Scholar]
  9. Drake B., Prater C. B., Weisenhorn A. L., Gould S. A., Albrecht T. R., Quate C. F., Cannell D. S., Hansma H. G., Hansma P. K. Imaging crystals, polymers, and processes in water with the atomic force microscope. Science. 1989 Mar 24;243(4898):1586–1589. doi: 10.1126/science.2928794. [DOI] [PubMed] [Google Scholar]
  10. Dunlap D. D., Maggi A., Soria M. R., Monaco L. Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acids Res. 1997 Aug 1;25(15):3095–3101. doi: 10.1093/nar/25.15.3095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engel A., Baumeister W., Saxton W. O. Mass mapping of a protein complex with the scanning transmission electron microscope. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4050–4054. doi: 10.1073/pnas.79.13.4050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Engel A., Lyubchenko Y., Müller D. Atomic force microscopy: a powerful tool to observe biomolecules at work. Trends Cell Biol. 1999 Feb;9(2):77–80. doi: 10.1016/s0962-8924(98)01415-9. [DOI] [PubMed] [Google Scholar]
  13. Essen L., Siegert R., Lehmann W. D., Oesterhelt D. Lipid patches in membrane protein oligomers: crystal structure of the bacteriorhodopsin-lipid complex. Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11673–11678. doi: 10.1073/pnas.95.20.11673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grigorieff N., Ceska T. A., Downing K. H., Baldwin J. M., Henderson R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J Mol Biol. 1996 Jun 14;259(3):393–421. doi: 10.1006/jmbi.1996.0328. [DOI] [PubMed] [Google Scholar]
  15. Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899–929. doi: 10.1016/S0022-2836(05)80271-2. [DOI] [PubMed] [Google Scholar]
  16. Hoh J. H., Sosinsky G. E., Revel J. P., Hansma P. K. Structure of the extracellular surface of the gap junction by atomic force microscopy. Biophys J. 1993 Jul;65(1):149–163. doi: 10.1016/S0006-3495(93)81074-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jubb J. S., Worcester D. L., Crespi H. L., Zaccaï G. Retinal location in purple membrane of Halobacterium halobium: a neutron diffraction study of membranes labelled in vivo with deuterated retinal. EMBO J. 1984 Jul;3(7):1455–1461. doi: 10.1002/j.1460-2075.1984.tb01996.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Karrasch S., Dolder M., Schabert F., Ramsden J., Engel A. Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution. Biophys J. 1993 Dec;65(6):2437–2446. doi: 10.1016/S0006-3495(93)81327-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Karrasch S., Hegerl R., Hoh J. H., Baumeister W., Engel A. Atomic force microscopy produces faithful high-resolution images of protein surfaces in an aqueous environment. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):836–838. doi: 10.1073/pnas.91.3.836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kasas S., Thomson N. H., Smith B. L., Hansma H. G., Zhu X., Guthold M., Bustamante C., Kool E. T., Kashlev M., Hansma P. K. Escherichia coli RNA polymerase activity observed using atomic force microscopy. Biochemistry. 1997 Jan 21;36(3):461–468. doi: 10.1021/bi9624402. [DOI] [PubMed] [Google Scholar]
  21. Kimura Y., Vassylyev D. G., Miyazawa A., Kidera A., Matsushima M., Mitsuoka K., Murata K., Hirai T., Fujiyoshi Y. Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature. 1997 Sep 11;389(6647):206–211. doi: 10.1038/38323. [DOI] [PubMed] [Google Scholar]
  22. Luecke H., Richter H. T., Lanyi J. K. Proton transfer pathways in bacteriorhodopsin at 2.3 angstrom resolution. Science. 1998 Jun 19;280(5371):1934–1937. doi: 10.1126/science.280.5371.1934. [DOI] [PubMed] [Google Scholar]
  23. Lyubchenko Y. L., Shlyakhtenko L. S. Visualization of supercoiled DNA with atomic force microscopy in situ. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):496–501. doi: 10.1073/pnas.94.2.496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Martin L. D., Vesenka J. P., Henderson E., Dobbs D. L. Visualization of nucleosomal substructure in native chromatin by atomic force microscopy. Biochemistry. 1995 Apr 11;34(14):4610–4616. doi: 10.1021/bi00014a014. [DOI] [PubMed] [Google Scholar]
  25. Müller D. J., Amrein M., Engel A. Adsorption of biological molecules to a solid support for scanning probe microscopy. J Struct Biol. 1997 Jul;119(2):172–188. doi: 10.1006/jsbi.1997.3875. [DOI] [PubMed] [Google Scholar]
  26. Müller D. J., Baumeister W., Engel A. Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans monitored by atomic force microscopy. J Bacteriol. 1996 Jun;178(11):3025–3030. doi: 10.1128/jb.178.11.3025-3030.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Müller D. J., Büldt G., Engel A. Force-induced conformational change of bacteriorhodopsin. J Mol Biol. 1995 Jun 2;249(2):239–243. doi: 10.1006/jmbi.1995.0292. [DOI] [PubMed] [Google Scholar]
  28. Müller D. J., Engel A. The height of biomolecules measured with the atomic force microscope depends on electrostatic interactions. Biophys J. 1997 Sep;73(3):1633–1644. doi: 10.1016/S0006-3495(97)78195-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Müller D. J., Engel A. Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy. J Mol Biol. 1999 Jan 29;285(4):1347–1351. doi: 10.1006/jmbi.1998.2359. [DOI] [PubMed] [Google Scholar]
  30. Müller D. J., Fotiadis D., Engel A. Mapping flexible protein domains at subnanometer resolution with the atomic force microscope. FEBS Lett. 1998 Jun 23;430(1-2):105–111. doi: 10.1016/s0014-5793(98)00623-1. [DOI] [PubMed] [Google Scholar]
  31. Müller D. J., Fotiadis D., Scheuring S., Müller S. A., Engel A. Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope. Biophys J. 1999 Feb;76(2):1101–1111. doi: 10.1016/S0006-3495(99)77275-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Müller D. J., Sass H. J., Müller S. A., Büldt G., Engel A. Surface structures of native bacteriorhodopsin depend on the molecular packing arrangement in the membrane. J Mol Biol. 1999 Feb 5;285(5):1903–1909. doi: 10.1006/jmbi.1998.2441. [DOI] [PubMed] [Google Scholar]
  33. Müller D. J., Schabert F. A., Büldt G., Engel A. Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy. Biophys J. 1995 May;68(5):1681–1686. doi: 10.1016/S0006-3495(95)80345-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Müller D. J., Schoenenberger C. A., Büldt G., Engel A. Immuno-atomic force microscopy of purple membrane. Biophys J. 1996 Apr;70(4):1796–1802. doi: 10.1016/S0006-3495(96)79743-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Müller D. J., Schoenenberger C. A., Schabert F., Engel A. Structural changes in native membrane proteins monitored at subnanometer resolution with the atomic force microscope: a review. J Struct Biol. 1997 Jul;119(2):149–157. doi: 10.1006/jsbi.1997.3878. [DOI] [PubMed] [Google Scholar]
  36. Oesterhelt D., Stoeckenius W. Functions of a new photoreceptor membrane. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2853–2857. doi: 10.1073/pnas.70.10.2853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Oesterhelt D., Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974;31:667–678. doi: 10.1016/0076-6879(74)31072-5. [DOI] [PubMed] [Google Scholar]
  38. Oesterhelt D. The structure and mechanism of the family of retinal proteins from halophilic archaea. Curr Opin Struct Biol. 1998 Aug;8(4):489–500. doi: 10.1016/s0959-440x(98)80128-0. [DOI] [PubMed] [Google Scholar]
  39. Pebay-Peyroula E., Rummel G., Rosenbusch J. P., Landau E. M. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science. 1997 Sep 12;277(5332):1676–1681. doi: 10.1126/science.277.5332.1676. [DOI] [PubMed] [Google Scholar]
  40. Peters J., Peters M., Lottspeich F., Schäfer W., Baumeister W. Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies. J Bacteriol. 1987 Nov;169(11):5216–5223. doi: 10.1128/jb.169.11.5216-5223.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Radmacher M., Cleveland J. P., Fritz M., Hansma H. G., Hansma P. K. Mapping interaction forces with the atomic force microscope. Biophys J. 1994 Jun;66(6):2159–2165. doi: 10.1016/S0006-3495(94)81011-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Schabert F. A., Engel A. Reproducible acquisition of Escherichia coli porin surface topographs by atomic force microscopy. Biophys J. 1994 Dec;67(6):2394–2403. doi: 10.1016/S0006-3495(94)80726-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schabert F. A., Henn C., Engel A. Native Escherichia coli OmpF porin surfaces probed by atomic force microscopy. Science. 1995 Apr 7;268(5207):92–94. doi: 10.1126/science.7701347. [DOI] [PubMed] [Google Scholar]
  46. Schabert F. A., Rabe J. P. Vertical dimension of hydrated biological samples in tapping mode scanning force microscopy. Biophys J. 1996 Mar;70(3):1514–1520. doi: 10.1016/S0006-3495(96)79713-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Scheuring S., Muller D. J., Ringler P., Heymann J. B., Engel A. Imaging streptavidin 2D crystals on biotinylated lipid monolayers at high resolution with the atomic force microscope. J Microsc. 1999 Jan;193(1):28–35. doi: 10.1046/j.1365-2818.1999.00434.x. [DOI] [PubMed] [Google Scholar]
  48. Shlyakhtenko L. S., Potaman V. N., Sinden R. R., Lyubchenko Y. L. Structure and dynamics of supercoil-stabilized DNA cruciforms. J Mol Biol. 1998 Jul 3;280(1):61–72. doi: 10.1006/jmbi.1998.1855. [DOI] [PubMed] [Google Scholar]
  49. Stemmer A., Reichelt R., Wyss R., Engel A. Biological structures imaged in a hybrid scanning transmission electron microscope and scanning tunneling microscope. Ultramicroscopy. 1991 Jun;35(3-4):255–264. doi: 10.1016/0304-3991(91)90077-j. [DOI] [PubMed] [Google Scholar]
  50. Unser M., Trus B. L., Frank J., Steven A. C. The spectral signal-to-noise ratio resolution criterion: computational efficiency and statistical precision. Ultramicroscopy. 1989 Jul-Aug;30(3):429–433. doi: 10.1016/0304-3991(89)90074-0. [DOI] [PubMed] [Google Scholar]
  51. Wagner P. Immobilization strategies for biological scanning probe microscopy. FEBS Lett. 1998 Jun 23;430(1-2):112–115. doi: 10.1016/s0014-5793(98)00614-0. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Yang J., Mou J., Shao Z. Molecular resolution atomic force microscopy of soluble proteins in solution. Biochim Biophys Acta. 1994 Mar 2;1199(2):105–114. doi: 10.1016/0304-4165(94)90104-x. [DOI] [PubMed] [Google Scholar]
  54. Yang J., Tamm L. K., Tillack T. W., Shao Z. New approach for atomic force microscopy of membrane proteins. The imaging of cholera toxin. J Mol Biol. 1993 Jan 20;229(2):286–290. doi: 10.1006/jmbi.1993.1033. [DOI] [PubMed] [Google Scholar]

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

RESOURCES