Quantitative Analysis of Structure and Dynamics in AFM Images of Lipid Membranes

  • Simon D. ConnellEmail author
  • George R. Heath
  • James A. Goodchild
Part of the Methods in Molecular Biology book series (MIMB, volume 1886)


AFM is now established as a powerful and direct technique for studying lipid membranes, and is highly complementary with other techniques. It is the only method for direct imaging and mechanical probing of lipid phase structure in a liquid environment down to the nanometer level. In order to understand the structure, function, and interactions of membranes at this level, we must be able to reliably and quantitatively measure the AFM images. Here we describe the methods used to process and analyze AFM images of phase-separated supported lipid bilayers . This initially takes a static approach, where we simply quantify the % of domain area, number of domains, and morphology, and quantify how many images must be taken to obtain reliable statistics. We then look at dynamics, describing the methods we use to study the nanometer scale motion of the domain perimeter as observed using Fast Scan AFM, and hence extract a quantitative line tension.

Key words

AFM Lipid membrane Bilayer Line tension Phase separation Domains Fast scan 



This work was supported by the EPSRC Programme Grant “CAPITALS” EP/J017566/1.


  1. 1.
    Meyer E, Howald L, Overney RM, Heinzelmann H, Frommer J, Guntherodt HJ, Wagner T, Schier H, Roth S (1991) Molecular-resolution images of Langmuir–Blodgett films using atomic force microscopy. Nature 349:398–400CrossRefGoogle Scholar
  2. 2.
    Zasadzinski JAN, Jelm CA, Longo ML, Weisenhorn AL, Gould SAC, Hansma PK (1991) Atomic force microscopy of hydrated phosphatidylethanolamine bilayers. Biophys J 59:755–760CrossRefGoogle Scholar
  3. 3.
    Mennicke U, Salditt T (2002) Preparation of solid-supported lipid bilayers by spin coating. Langmuir 18:8172–8177CrossRefGoogle Scholar
  4. 4.
    Reviakine I, Brisson AR (2000) Formation of supported phospholipid bilayers from unilamellar vesicles investigated by atomic force microscopy. Langmuir 16:1806–1815CrossRefGoogle Scholar
  5. 5.
    Richter RP, Brisson AR (2005) Following the formation of supported lipid bilayers on mica: A study combining AFM, QCM-D, and ellipsometry. Biophys J 88:3422–3433CrossRefGoogle Scholar
  6. 6.
    Connell SD, Smith DA (2006) The atomic force microscope as a tool for studying phase separation in lipid membranes. Molec Mem Biol 23:17–28CrossRefGoogle Scholar
  7. 7.
    Goksu EI, Vanegas JM, Blanchette CD, Lin W-C, Longo ML (2009) AFM for structure and dynamics of biomembranes. BBA Biomem 1788:254–266CrossRefGoogle Scholar
  8. 8.
    Unsay JD, Cosentino K, Garcia-Saez AJ (2015) Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers. J Vis Exp 101:e52867Google Scholar
  9. 9.
    Connell SD, Heath G, Olmsted PD, Kisil A (2013) Critical Point Fluctuations in Supported Lipid Membranes. Faraday Discuss 151:91–111CrossRefGoogle Scholar
  10. 10.
    Khadka NK, Ho CS, Pan J (2015) Macroscopic and Nanoscopic Heterogeneous Structures in a Three-Component Lipid Bilayer Mixtures Determined by Atomic Force Microscopy. Langmuir 31:12417–12425CrossRefGoogle Scholar
  11. 11.
    Cromey DW (2010) Avoiding twisted pixels: ethical guidelines for the appropriate use and manipulation of scientific digital images. Sci Eng Ethics 16:639–667CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Simon D. Connell
    • 1
    • 2
    Email author
  • George R. Heath
    • 1
    • 3
  • James A. Goodchild
    • 1
  1. 1.Molecular and Nanoscale Physics Group, School of Physics and AstronomyUniversity of LeedsLeedsUK
  2. 2.Astbury Centre for Structural Molecular BiologyUniversity of LeedsLeedsUK
  3. 3.Faculty of Biological SciencesUniversity of LeedsLeedsUK

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