Multiparametric high-resolution imaging of native proteins by force-distance curve–based AFM

Journal name:
Nature Protocols
Year published:
Published online


A current challenge in the life sciences is to understand how the properties of individual molecular machines adjust in order to meet the functional requirements of the cell. Recent developments in force-distance (FD) curve–based atomic force microscopy (FD-based AFM) enable researchers to combine sub-nanometer imaging with quantitative mapping of physical, chemical and biological properties. Here we present a protocol to apply FD-based AFM to the multiparametric imaging of native proteins under physiological conditions. We describe procedures for experimental FD-based AFM setup, high-resolution imaging of proteins in the native unperturbed state with simultaneous quantitative mapping of multiple parameters, and data interpretation and analysis. The protocol, which can be completed in 1–3 d, enables researchers to image proteins and protein complexes in the native unperturbed state and to simultaneously map their biophysical and biochemical properties at sub-nanometer resolution.

At a glance


  1. Principles of FD curve–based AFM for imaging and mapping multiple properties of biological samples.
    Figure 1: Principles of FD curve–based AFM for imaging and mapping multiple properties of biological samples.

    (a) In FD-based AFM, an AFM stylus is made to approach to and retract from a biological sample in a pixel-by-pixel manner to record FD curves. The high precision of the AFM enables the user to detect pixel sizes <1 nm2, with a positional accuracy of ∼0.2 nm and forces at piconewton (10−12 N) sensitivity. The height of every pixel of the final sample topography is determined by the stylus-sample distance, measured at a preset imaging force Fi. (b) Approach (red) and retraction (black) FD curves. Zero distance indicates the contact point of the tip and the sample. Analyzing the FD curves provides information such as the sample height, deformation, elasticity (Young's or DMT modulus), energy dissipation and adhesion. Cartoons depict the cantilever approaching to and retracting from the sample as follows: (1) noncontact, (2) initial contact and (3) repulsive contact regimes of cantilever stylus and sample detected in the approach FD curve. (4) Adhesion and (5) noncontact regimes recorded upon retracting the stylus and sample. (c) Information on the height and deformation of the biological sample can be extracted from the approach FD curve. The sample deformation DDef is determined in this example as the stylus-sample distance DFi reached at the imaging force Fi (here 150 pN) minus the distance DFLow reached at a much lower force FLow (here 45 pN). (d) Elastic modulus, adhesion force and energy dissipation can be extracted from the retraction FD curve. The adhesion force FAdh is the minimum of the retraction FD curve. Energy dissipation W represents the blue shaded area between the approach and retraction FD curve. Stiffness k of the sample can be determined by the pink-colored slope (F = Fi − FMod)/DModulus. (e) Formulas suitable for extracting parameters described in c and d from FD curves. The sample elasticity E* is estimated by using the DMT model143, 144, 145, with the imaging force Fi, the adhesion force FAdh, the stylus-sample contact area R and the stiffness k = (F = FiFMod)/DModulus of the biological sample.

  2. High-resolution FD-based AFM images of membrane proteins and fibrillated water-soluble proteins.
    Figure 2: High-resolution FD-based AFM images of membrane proteins and fibrillated water-soluble proteins.

    (a) Cytoplasmic surface of purple membrane showing individual bacteriorhodopsin trimers87. (b) Densely packed assembly of OmpF porin trimers reconstituted into the lipid bilayer. Adjacent OmpF trimers either expose their extracellular or periplasmic surfaces. Highly protruding OmpF trimers (brighter) expose the extracellular surface, whereas the low-protruding OmpF trimers (darker) expose their periplasmic pores89. (c) Ferric hydroxamate uptake receptor (FhuA) from E. coli reconstituted into the lipid bilayer88. The high protrusions (brighter) represent single FhuA exposing their extracellular side, whereas lower donut-shaped features (darker) are FhuA exposing their periplasmic pores. (d) Amyloid-like fibrils assembled from full-length human tau92. (e) Fibrillating core fragment (hIAPP20–29) of the human islet amyloid polypeptide91. (f) α-synuclein fibrils (E46K mutant form). Images adapted with permission from refs. 87,88,90,91,92, with copyrights from the American Chemical Society (refs. 89,90), from Elsevier (ref. 88), from Wiley and Sons (ref. 87) and from the National Academy of Sciences (USA) refs. 91,92.

  3. Approach and retraction FD curves recorded on supporting surfaces.
    Figure 3: Approach and retraction FD curves recorded on supporting surfaces.

    (a) FD curves recorded in buffer solution on a clean and mechanically stiff support. The sharp transition close to the contact area (0 nm) indicates a clean AFM stylus (here, Si3N4) approaching a clean support (here, mica), and it indicates that both materials are mechanically stiff. The good agreement between approach and retraction FD curves shows no hysteresis and thus also indicates that the AFM system has been set up properly and that the AFM stylus and sample are not contaminated. (b) FD curves recorded in buffer solution on a clean, mechanically flexible sample. The relatively smooth transition around the contact area and the missing hysteresis indicate that a clean AFM stylus (here, Si3N4) approaches a clean support (here, purple membrane) and that, in this case, the support is mechanically softer than the stylus. (c) FD curves recorded in buffer solution using a contaminated AFM stylus. The discontinuous transition and the hysteresis between approach and retraction FD curves indicate a contaminated AFM stylus and/or mica surface. FD curves were recorded in buffer solution (150 mM KCl, 10 mM Tris-HCl, pH 7.6), by applying an imaging force of 150 pN, a cantilever oscillation amplitude of 50 nm and a frequency of 2 kHz, as well as 20.5 data points per nm.

  4. FD-based AFM images of purple membrane adsorbed onto mica.
    Figure 4: FD-based AFM images of purple membrane adsorbed onto mica.

    (ae) AFM topography (a), deformation map (b), adhesion map (c), DMT modulus map (d) and force error map (e). (ae) Vertical scales correspond to 10 nm (a), 0.5 nm (b), 40 pN (c), 8–90 MPa (d) and from −50 pN to 50 pN (e). The red lines in the AFM images (ae) indicate where the vertical profile shown below each image has been extracted. Gray lines in panels be show the topographic profile recorded in a. The topography shows densely packed patches of bacteriorhodopsin surrounded by thin rims of a lipid bilayer. Data were recorded in buffer solution (150 mM KCl, 10 mM Tris-HCl, pH 7.8), by applying an imaging force of 140 pN, a cantilever amplitude of 40 nm, a frequency of 2 kHz and a scanning frequency of 1 Hz per line. Note that a soft cantilever was chosen to measure the Young's modulus of purple membrane, which gives a wrong estimate of the Young's (DMT) modulus of mica (see 'Critical cantilever selection').

  5. High-resolution FD-based AFM of the extracellular purple membrane surface reveals sub-structural details of bacteriorhodopsin trimers.
    Figure 5: High-resolution FD-based AFM of the extracellular purple membrane surface reveals sub-structural details of bacteriorhodopsin trimers.

    (a,b) Raw data (a) and average (b) AFM topography of bacteriorhodopsin trimers. (c,d) Raw data (c) and average (d) DMT modulus map. (e,f) Raw data (e) and average (f) deformation map. Averages were calculated from unit cells extracted at the positions from which the bacteriorhodopsin trimers were observed in the topograph87. (af) Vertical scales of 0–1.0 nm (a,b), 5–18 MPa (c,d) and 0.2–1.2 nm (e,f). The red lines in the AFM images indicate where the vertical profile shown below each image has been extracted. Gray lines in cf show the topographic profile recorded in a and b. Data were recorded in buffer solution (150 mM KCl, 10 mM Tris-HCl, pH 7.8), by applying an imaging force of 45 pN, a cantilever amplitude of 14 nm, a frequency of 2 kHz and a scanning frequency of 0.77 Hz per line.


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  1. Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

    • Moritz Pfreundschuh,
    • David Martinez-Martin,
    • Estefania Mulvihill &
    • Daniel J Muller
  2. Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.

    • Susanne Wegmann


M.P., D.M.-M., S.W. and D.J.M. designed the AFM protocol. M.P., E.M. and S.W. performed and optimized the experimental procedure. M.P., D.M.-M., S.W. and D.J.M. wrote the manuscript.

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