Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.
At a glance
- Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986).
This paper first described the principles of AFM.
- How the doors to the nanoworld were opened. Nat. Nanotech. 1, 3–5 (2006). &
- Atomic resolution with atomic force microscope. Europhys. Lett. 3, 1281–1286 (1987). , , , &
- Imaging crystals, polymers, and processes in water with the atomic force microscope. Science 243, 1586–1589 (1989). et al.
- Imaging viscoelasticity by force modulation with the atomic force microscope. Biophys. J. 64, 735–742 (1993). , &
- Scanning probe evolution in biology. Science 302, 1002–1005 (2003). &
- In touch with atoms. Rev. Mod. Phys. 71, S324 (1999). &
- Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nat. Nanotech. 3, 261–269 (2008). &
- Atomic force microscopy: a nanoscopic window on the cell surface. Trends Cell Biol. 21, 461–469 (2011). &
- Biomolecular imaging with the atomic force microscope. Annu. Rev. Biophys. Biomol. Struct. 23, 115–139 (1994). &
- Detection and localization of single molecular recognition events using atomic force microscopy. Nat. Methods 3, 347–355 (2006). &
- High-speed AFM and applications to biomolecular systems. Ann. Rev. Biophys. 42, 393–414 (2013). , &
- Multiparametric imaging of biological systems by force-distance curve–based AFM. Nat. Methods 10, 847–854 (2013). , , , &
- Nanomechancial mapping of soft matter by bimodal force microscopy. Eur. Polym. J. 49, 1897–1906 (2013). &
- From molecules to cells: imaging soft samples with the atomic force microscope. Science 257, 1900–1905 (1992). , , &
- Actin filament dynamics in living glial cells imaged by atomic force microscopy. Science 257, 1944–1946 (1992). , &
- Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy. J. Cell Sci. 107, 1105–1114 (1994). &
- Atomic force microscopy and dissection of gap junctions. Science 253, 1405–1408 (1991). , , , &
- Atomic force microscopy of cholera toxin B-oligomers bound to bilayers of biologically relevant lipids. J. Mol. Biol. 248, 507–512 (1995). , &
- Native Escherichia coli OmpF porin surfaces probed by atomic force microscopy. Science 268, 92–94 (1995). , &
- Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope. Science 256, 1180–1184 (1992). et al.
- Wet lipid protein membranes imaged at submolecular resolution by atomic force microscopy. J. Struct. Biol. 103, 89–94 (1990). et al.
- Langmuir-Blodgett films. Science 263, 1726–1733 (1994). , , , &
- Structure and stability of pertussis toxin studied by in situ atomic force microscopy. FEBS Lett. 338, 89–92 (1994). , &
- Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy. Biophys. J. 68, 1681–1686 (1995). , , &
- The bacteriophage phi29 head–tail connector imaged at high resolution with atomic force microscopy in buffer solution. EMBO J. 16, 2547–2553 (1997). , , &
- Staphylococcal alpha-hemolysin can form hexamers in phospholipid bilayers. J. Mol. Biol. 276, 325–330 (1998). , &
- Proton powered turbine of a plant motor. Nature 405, 418–419 (2000). et al.
- High-resolution AFM topographs of Rubrivivax gelatinosus light- harvesting complex LH2. EMBO J. 20, 3029–3035 (2001). , , , &
- Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature 421, 127–128 (2003). et al.
- Watching amyloid fibrils grow by time-lapse atomic force microscopy. J. Mol. Biol. 285, 33–39 (1999). , , , &
- Motion and enzymatic degradation of DNA in the atomic force microscope. Biophys. J. 67, 2454–2459 (1994). et al.
- Atomic force microscope imaging of phospholipid bilayer degradation by phospholipase A2. Biophys. J. 74, 2398–2404 (1998). , &
- Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy. J. Mol. Biol. 285, 1347–1351 (1999). &
- Conformational changes in surface structures of isolated connexin 26 gap junctions. EMBO J. 21, 3598–3607 (2002).
This paper reports using AFM to image animal communication channels at work with high-resolution.
, , &
- Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy. J. Mol. Biol. 287, 741–752 (1999). , , &
- Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane. EMBO J. 23, 3206–3215 (2004). , , &
- Chromatic adaptation of photosynthetic membranes. Science 309, 484–487 (2005). &
- Observing single biomolecules at work with the atomic force microscope. Nat. Struct. Biol. 7, 715–718 (2000). &
- Frequency-modulation detection using high-Q cantilevers for enhanced force microscope sensitivity. J. Appl. Phys. 69, 668–673 (1991). , , &
- Tapping mode atomic-force microscopy in liquid. Appl. Phys. Lett. 64, 2454–2456 (1994). , , , &
- The emergence of multifrequency force microscopy. Nat. Nanotech. 7, 217–226 (2012).
A review describing recent progress in multifrequency force microscopy, and discussing its potential for studying proteins and cells.
- Tapping mode atomic-force microscopy in liquids. Appl. Phys. Lett. 64, 1738–1740 (1994). et al.
- Human Tau isoforms assemble into ribbon-like fibrils that display polymorphic structure and stability. J. Biol. Chem. 285, 27302–27313 (2010). et al.
- Beyond the helix pitch: direct visualization of native DNA in aqueous solution. ACS Nano 7, 1817–1822 (2013). et al.
- Immunoactive two-dimensional self-assembly of monoclonal antibodies in aqueous solution revealed by atomic force microscopy. Nat. Mater. 13, 264–270 (2014). et al.
- Tapping mode atomic force microscopy produces faithful high-resolution images of protein surfaces. Biophys. J. 77, 1050–1058 (1999). , , , &
- From images to interactions: high-resolution phase imaging in tapping-mode atomic force microscopy. Biophys. J. 80, 3009–3018 (2001). , , &
- A method for anchoring round shaped cells for atomic force microscope imaging. Biophys. J. 68, 1678–1680 (1995). &
- Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells. Nat. Commun. 1, 27 (2010). et al.
- The scanning ion-conductance microscope. Science 243, 641–643 (1989). , , , &
- Nanoscale live-cell imaging using hopping probe ion conductance microscopy. Nat. Methods 6, 279–281 (2009). et al.
- A new ion sensing deep atomic force microscope. Rev. Sci. Instrum. 85, 083706 (2014). , , &
- Physical virology. Nat. Phys. 6, 733–743 (2010). , &
- Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000). et al.
- Single-molecule cut-and-paste surface assembly. Science 319, 594–596 (2008). , , , &
- Molecular printing. Nat. Chem. 1, 353–358 (2009). , &
- Mechanical control of mitotic progression in single animal cells. Proc. Natl Acad. Sci. USA 112, 11258–11263 (2015). et al.
- Force measurements with the atomic force microscope: technique, interpretation and applications. Surf. Sci. Rep. 59, 1–152 (2005). , &
- Determination of protein structural flexibility by microsecond force spectroscopy. Nat. Nanotech. 4, 514–517 (2009). , &
- Noninvasive protein structural flexibility mapping by bimodal dynamic force microscopy. Phys. Rev. Lett. 106, 198101 (2011). , , , &
- Fast nanomechanical spectroscopy of soft matter. Nat. Commun. 5, 3126 (2014). , &
- High-speed AFM images of thermal motion provide stiffness map of interfacial membrane protein moieties. Nano Lett. 15, 759–763 (2015). et al.
- Mapping interaction forces with the atomic force microscope. Biophys. J. 66, 2159–2165 (1994). , , , &
- Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope. Trends Biotechnol. 17, 143–150 (1999). &
- Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study. Biophys. J. 78, 520–535 (2000). &
- Direct, high-resolution measurement of furrow stiffening during division of adherent cells. Nat. Cell Biol. 3, 607–610 (2001). , &
- The nanomechanical signature of breast cancer. Nat. Nanotech. 7, 757–765 (2012). et al.
- Comparison of the viscoelastic properties of cells from different kidney cancer phenotypes measured with atomic force microscopy. Nanotechnology 24, 055102 (2013). , , &
- Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy. Langmuir 19, 4539–4543 (2003). , &
- Fast imaging and fast force spectroscopy of single biopolymers with a new atomic force microscope designed for small cantilevers. Rev. Sci. Instrum. 70, 4300–4303 (1999).
This paper reports the invention of small cantilevers for fast AFM imaging and force spectroscopy.
- Small cantilevers for force spectroscopy of single molecules. J. Appl. Phys. 86, 2258–2262 (1999). et al.
- A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl Acad. Sci. USA 98, 12468–12472 (2001). et al.
- Correction of microrheological measurements of soft samples with atomic force microscopy for the hydrodynamic drag on the cantilever. Langmuir 18, 716–721 (2002). et al.
- An atomic force microscope tip designed to measure time-varying nanomechanical forces. Nat. Nanotech. 2, 507–514 (2007). , , , &
- Imaging and quantifying chemical and physical properties of native proteins at molecular resolution by force-volume AFM. Angew. Chem. Int. Ed. 50, 12103–12108 (2011). , &
- Direct correlation of structures and nanomechanical properties of multicomponent lipid bilayers. Langmuir 25, 7471–7477 (2009). , &
- Glyphosate-induced stiffening of HaCaT keratinocytes, a Peak Force Tapping study on living cells. J. Struct. Biol. 178, 1–7 (2012). , , &
- Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG. Proc. Natl Acad. Sci. USA 113, 410–415 (2016). , , , &
- Multiparametric atomic force microscopy imaging of single bacteriophages extruding from living bacteria. Nat. Commun. 4, 2926 (2013). , , &
- Built-in mechanical stress in viral shells. Biophys. J. 100, 1100–1108 (2011). et al.
- Mechanical properties of the icosahedral shell of southern bean mosaic virus: a molecular dynamics study. Biophys. J. 96, 1350–1363 (2009). &
- DNA-mediated anisotropic mechanical reinforcement of a virus. Proc. Natl Acad. Sci. USA 103, 13706–13711 (2006). et al.
- Force-induced formation and propagation of adhesion nanodomains in living fungal cells. Proc. Natl Acad. Sci. USA 107, 20744–20749 (2010).
This paper reports using recognition imaging to demonstrate that microbial cell adhesion proteins form nanoclusters under mechanical force.
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- Quantitative imaging of the electrostatic field and potential generated by a transmembrane protein pore at subnanometer resolution. Nano Lett. 13, 5585–5593 (2013). , &
- Forces and bond dynamics in cell adhesion. Science 316, 1148–1153 (2007). &
- Nanomechanical properties of proteins and membranes depend on loading rate and electrostatic interactions. ACS Nano 7, 2642–2650 (2013). &
- Functional group imaging by chemical force microscopy. Science 265, 2071–2074 (1994). , , , &
- Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. Proc. Natl Acad. Sci. USA 93, 3477–3481 (1996). , , , &
- Atomic force microscope imaging contrast based on molecular recognition. Biophys. J. 72, 445–448 (1997). , &
- Affinity imaging of red blood cells using an atomic force microscope. J. Histochem. Cytochem. 48, 719–724 (2000). , , &
- Adhesion forces between individual ligand-receptor pairs. Science 264, 415–417 (1994). , &
- Direct measurement of the forces between complementary strands of DNA. Science 266, 771–773 (1994). , &
- Recognition force spectroscopy studies of the NTA-His6 bond. Single Mol. 1, 59–65 (2000). et al.
- Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces. Hum. Reprod. 13, 3211–3219 (1998). et al.
- Quantification of cell adhesion force with AFM: distribution of vitronectin receptors on a living MC3T3-E1 cell. Ultramicroscopy 97, 359–363 (2003). , , &
- Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM. Ultramicroscopy 106, 652–662 (2006). et al.
- Elastic membrane heterogeneity of living cells revealed by stiff nanoscale membrane domains. Biophys. J. 94, 1521–1532 (2008). et al.
- Imaging G protein–coupled receptors while quantifying their ligand-binding free-energy landscape. Nat. Methods 12, 845–851 (2015).
This paper showed that attaching a ligand to the AFM stylus allows it to image and map its binding to human G protein–coupled receptors and to reconstruct the ligand-binding free-energy landscape.
- Fluorescence and atomic force microscopy imaging of wall teichoic acids in Lactobacillus plantarum. ACS Chem. Biol. 6, 366–376 (2011). et al.
- Nanoscale mapping and functional analysis of individual adhesins on living bacteria. Nat. Methods 2, 515–520 (2005).
This paper reports that AFM tips labelled with bioligands can map the distribution of single adhesion proteins on bacterial pathogens and reveal their assembly into nanodomains.
- Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM. Nat. Commun. 6, 8857 (2015). et al.
- Antibody recognition imaging by force microscopy. Nat. Biotechnol. 17, 901–905 (1999). et al.
- Single-molecule recognition imaging microscopy. Proc. Natl Acad. Sci. USA 101, 12503–12507 (2004). et al.
- Nano-scale dynamic recognition imaging on vascular endothelial cells. Biophys. J. 93, L11–L13 (2007). , , , &
- Quantitative biomolecular imaging by dynamic nanomechanical mapping. Chem. Soc. Rev. 43, 7412–7429 (2014). , , &
- Nanomechanical coupling enables detection and imaging of 5 nm superparamagnetic particles in liquid. Nanotechnology 22, 125708 (2011). , , &
- Three-dimensional quantitative force maps in liquid with 10 piconewton, angstrom and sub-minute resolutions. Nanoscale 5, 2678–2685 (2013). , , &
- Direct imaging of individual intrinsic hydration layers on lipid bilayers at Angstrom resolution. Biophys. J. 92, 3603–3609 (2007). , &
- Mapping in vitro local material properties of intact and disrupted virions at high resolution using multi-harmonic atomic force microscopy. Nanoscale 5, 4729–4736 (2013). , , , &
- Fast, multi-frequency, and quantitative nanomechanical mapping of live cells using the atomic force microscope. Sci. Rep. 5, 11692 (2015). , , &
- Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy. Nat. Nanotech. 10, 264–269 (2015). &
- Nanoscale imaging of buried structures via scanning near-field ultrasound holography. Science 310, 89–92 (2005). &
- Imaging nanoparticles in cells by nanomechanical holography. Nat. Nanotech. 3, 501–505 (2008). et al.
- Beating beats mixing in heterodyne detection schemes. Nat. Commun. 6, 6444 (2015). &
- Rigid design of fast scanning probe microscopes using finite element analysis. Ultramicroscopy 100, 259–265 (2004). , , &
- High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes. Prog. Surf. Sci. 83, 337–437 (2008). , &
- Active damping of the scanner for high-speed atomic force microscopy. Rev. Sci. Instrum. 76, 053708 (2005). , &
- Dynamic proportional-integral-differential controller for high-speed atomic force microscopy. Rev. Sci. Instrum. 77, 083704 (2006). , &
- Probing protein–protein interactions in real time. Nat. Struct. Biol. 7, 644–647 (2000). et al.
- A high-speed atomic force microscope for studying biological macromolecules in action. ChemPhysChem 4, 1196–1202 (2003). et al.
- High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin. Nat. Nanotech. 5, 208–212 (2010). , , , &
- Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010).
This paper showed that high-speed AFM can be used to watch proteins functioning in real-time.
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- High-speed atomic force microscopy reveals rotary catalysis of rotorless F(1)-ATPase. Science 333, 755–758 (2011). , , &
- Relaxation of loaded ESCRT-III spiral springs drives membrane deformation. Cell 163, 866–879 (2015). et al.
- Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy. Nat. Nanotech. 11, 719–723 (2016). , , &
- Kinetics of antimicrobial peptide activity measured on individual bacterial cells using high-speed atomic force microscopy. Nat. Nanotech. 5, 280–285 (2010). , , &
- Single-molecule imaging on living bacterial cell surface by high-speed AFM. J. Mol. Biol. 422, 300–309 (2012). et al.
- Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells. Sci. Rep. 5, 8724 (2015). , , &
- Functional extension of high-speed AFM for wider biological applications. Ultramicroscopy 160, 182–196 (2016). , , , &
- Nanoscale imaging of the Candida — macrophage interaction using correlated fluorescence-atomic force microscopy. ACS Nano 6, 10792–10799 (2012). &
- Actin microridges characterized by laser scanning confocal and atomic force microscopy. FEBS Lett. 579, 2001–2009 (2005). , &
- PeakForce Tapping resolves individual microvilli on living cells. J. Mol. Recognit. 29, 95–101 (2016). , , , &
- Discrete interactions in cell adhesion measured by single-molecule force spectroscopy. Nat. Cell Biol. 2, 313–317 (2000). , , &
- Tensile forces govern germ-layer organization in zebrafish. Nat. Cell Biol. 10, 429–436 (2008). et al.
- Effect of thrombin and bradykinin on endothelial cell mechanical properties monitored through membrane deformation. J. Mol. Recognit. 22, 389–396 (2009). , , , &
- Mechanical dynamics of single cells during early apoptosis. Cell Motil. Cytoskel. 66, 409–422 (2009). , , , &
- Cdk1-dependent mitotic enrichment of cortical myosin II promotes cell rounding against confinement. Nat. Cell Biol. 17, 148–159 (2015). et al.
- Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature 469, 226–230 (2011). et al.
- Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging. Nanotechnology 21, 115504 (2010). et al.
- Strengthening relationships: amyloids create adhesion nanodomains in yeasts. Trends Microbiol. 20, 59–65 (2012). et al.
- Nanomechanical mapping of first binding steps of a virus to animal cells. Nat. Nanotech. 12, 177–183 (2017).
This paper showed that attaching a rabies virus to the AFM stylus allows living animal cells to be imaged with confocal microscopy and AFM, to simultaneously localize virus-binding, and to quantify the virus-binding process and free-energy landscape.
- Ultrastable atomic force microscopy: improved force and positional stability. FEBS Lett. 588, 3621–3630 (2014). &
- Ultrastable atomic force microscopy: atomic-scale stability and registration in ambient conditions. Nano Lett. 9, 1451–1456 (2009). , , , &
- Analysing focal adhesion structure by AFM. J. Cell Sci. 118, 5315–5323 (2005). &
- Crystallization of Brome mosaic virus and T = 1 Brome mosaic virus particles following a structural transition. Virology 286, 290–303 (2001). , , &
- Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM. J. Mol. Biol. 372, 594–607 (2007). , , &
- Gating of the MlotiK1 potassium channel involves large rearrangements of the cyclic nucleotide-binding domains. Proc. Natl Acad. Sci. USA 108, 20802–20807 (2011). et al.
- Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes. Nat. Nanotech. 10, 60–64 (2015). et al.