Portal de Periódicos da CAPES

Sampling Protein Form and Function with the Atomic Force Microscope

2010; Elsevier BV; Volume: 9; Issue: 8 Linguagem: Inglês

10.1074/mcp.r110.001461

1535-9484

Marian Baclayon, Wouter H. Roos, Gijs J. L. Wuite,

Ion-surface interactions and analysis

To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments. To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments. To understand biological processes at the molecular level it is essential to identify the involved proteins and proteinaceous assemblies, to characterize their structure and function, and to unravel their interplay with other proteins and molecules (1.Heck A.J. Native mass spectrometry: a bridge between interactomics and structural biology.Nat. Methods. 2008; 5: 927-933Crossref PubMed Scopus (511) Google Scholar). Techniques like x-ray crystallography, electron microscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry have contributed massively to elucidate such protein properties. These techniques can easily sample the properties of a large ensemble of proteins; however, they require subjecting the sample to harsh treatments such as drying, crystallizing, or vaporizing in vacuum, thereby limiting the range of measurable dynamical properties of the sample. One powerful method that permits the investigation of molecules in their native physiological buffer condition is atomic force microscopy (AFM) 1The abbreviations used are:AFMatomic force microscopyCCMVcowpea chlorotic mottle virusHBVhepatitis B virusMVMminute virus of miceMDmolecular dynamics. (2.Binnig G. Quate C.F. Gerber C. Atomic force microscope.Phys. Rev. Lett. 1986; 56: 930-933Crossref PubMed Scopus (11692) Google Scholar). An atomic force microscope is a microscope and force spectrometer at the same time. The imaging resolution of the atomic force microscope is comparable with that of electron microscopes, and it has the special capability to image samples in a variety of environments such as in vacuum, air, or liquid, which therefore enables studying biological specimens in their native environments (i.e. in buffer solutions) (3.Fechner P. Boudier T. Mangenot S. Jaroslawski S. Sturgis J.N. Scheuring S. Structural information, resolution, and noise in high-resolution atomic force microscopy topographs.Biophys. J. 2009; 96: 3822-3831Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 4.Scheuring S. Sturgis J.N. Chromatic adaptation of photosynthetic membranes.Science. 2005; 309: 484-487Crossref PubMed Scopus (213) Google Scholar). In addition, its ability to "touch" the sample gives it the advantage to manipulate single particles/molecules and probe their mechanical properties (5.Henderson E. Imaging and nanodissection of individual supercoiled plasmids by atomic force microscopy.Nucleic Acids Res. 1992; 20: 445-447Crossref PubMed Google Scholar, 6.Engel A. Müller D.J. Observing single biomolecules at work with the atomic force microscope.Nat. Struct. Biol. 2000; 7: 715-718Crossref PubMed Scopus (0) Google Scholar, 7.Baclayon M. Wuite G.J. Roos W.H. Imaging and manipulation of single viruses by atomic force microscopy.Soft Matter. 2010; (doi:10.1039/b923992h)Crossref Scopus (48) Google Scholar, 8.Scheuring S. Fotiadis D. Moller C. Muller S.A. Engel A. Muller D.J. Single proteins observed by atomic force microscopy.Single Mol. 2001; 2: 59-67Crossref Google Scholar). However, AFM force spectroscopy is currently a technique with rather fast pulling and pushing speeds, thereby often operating out of equilibrium conditions. Improvements with ultrastable atomic force microscopes are underway to tackle this problem with promising results (9.King G.M. Carter A.R. Churnside A.B. Eberle L.S. Perkins T.T. Ultrastable atomic force microscopy: atomic-scale stability and registration in ambient conditions.Nano Lett. 2009; 9: 1451-1456Crossref PubMed Scopus (70) Google Scholar, 10.Junker J.P. Ziegler F. Rief M. Ligand-dependent equilibrium fluctuations of single calmodulin molecules.Science. 2009; 323: 633-637Crossref PubMed Scopus (164) Google Scholar). Furthermore, AFM is not well suited to apply and resolve forces at the single piconewton range due to large size tips and relatively stiff cantilevers. The issue of nonspecificity of the tip interaction with the sample is also of concern, especially in pulling experiments that require the capability to accurately recognize and select the appropriate molecule or point of interest. The current introduction of carbon nanotube tips can address the former issue (11.Hafner J.H. Cheung C.L. Woolley A.T. Lieber C.M. Structural and functional imaging with carbon nanotube AFM probes.Prog. Biophys. Mol. Biol. 2001; 77: 73-110Crossref PubMed Scopus (0) Google Scholar, 12.Cheung C.L. Hafner J.H. Lieber C.M. Carbon nanotube atomic force microscopy tips: direct growth by chemical vapor deposition and application to high-resolution imaging.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3809-3813Crossref PubMed Scopus (0) Google Scholar), whereas techniques in chemical functionalization can provide directed tip specificity and recognition capability (13.Ebner A. Wildling L. Zhu R. Rankl C. Haselgrubler T. Hinterdorfer P. Gruber H.J. Functionalization of probe tips and supports for single-molecule recognition force microscopy.STM and AFM Studies On (Bio)Molecular Systems: Unravelling the Nanoworld. Springer-Verlag, Berlin2008: 29-76Crossref Google Scholar, 14.Ebner A. Hinterdorfer P. Gruber H.J. Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers.Ultramicroscopy. 2007; 107: 922-927Crossref PubMed Scopus (141) Google Scholar, 15.Hinterdorfer P. Baumgartner W. Gruber H.J. Schilcher K. Schindler H. Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 3477-3481Crossref PubMed Scopus (990) Google Scholar, 16.Wong S.S. Joselevich E. Woolley A.T. Cheung C.L. Lieber C.M. Covalently functionalized nanotubes as nanometre-sized probes in chemistry and biology.Nature. 1998; 394: 52-55Crossref PubMed Scopus (0) Google Scholar, 17.Hinterdorfer P. Dufrêne Y.F. Detection and localization of single molecular recognition events using atomic force microscopy.Nat. Methods. 2006; 3: 347-355Crossref PubMed Scopus (807) Google Scholar, 18.Ebner A. Wildling L. Kamruzzahan A.S. Rankl C. Wruss J. Hahn C.D. Hölzl M. Zhu R. Kienberger F. Blaas D. Hinterdorfer P. Gruber H.J. A new, simple method for linking of antibodies to atomic force microscopy tips.Bioconjug. Chem. 2007; 18: 1176-1184Crossref PubMed Scopus (206) Google Scholar), thereby further improving and widening the applicability of AFM in the future. In addition, the coupling of the atomic force microscope to fluorescence microscopes further enhances its versatility by adding (single molecule) fluorescence imaging to the AFM imaging capability (19.Hards A. Zhou C. Seitz M. Bräuchle C. Zumbusch A. Simultaneous AFM manipulation and fluorescence imaging of single DNA strands.ChemPhysChem. 2005; 6: 534-540Crossref PubMed Scopus (33) Google Scholar, 20.Kufer S.K. Puchner E.M. Gumpp H. Liedl T. Gaub H.E. Single-molecule cut-and-paste surface assembly.Science. 2008; 319: 594-596Crossref PubMed Scopus (0) Google Scholar, 21.Kassies R. van der Werf K.O. Lenferink A. Hunter C.N. Olsen J.D. Subramaniam V. Otto C. Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology.J. Microsc. 2005; 217: 109-116Crossref PubMed Scopus (0) Google Scholar), and the development of high speed systems makes it possible for AFM to probe fast dynamics of various biological processes (22.Ando T. Kodera N. Takai E. Maruyama D. Saito K. Toda A. A high-speed atomic force microscope for studying biological macromolecules.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 12468-12472Crossref PubMed Scopus (735) Google Scholar, 23.Sulchek T. Hsieh R. Adams J.D. Minne S.C. Quate C.F. Adderton D.M. High-speed atomic force microscopy in liquid.Rev. Sci. Instrum. 2000; 71: 2097-2099Crossref Scopus (90) Google Scholar, 24.Casuso I. Kodera N. Le Grimellec C. Ando T. Scheuring S. Contact-mode high-resolution high-speed atomic force microscopy movies of the purple membrane.Biophys. J. 2009; 97: 1354-1361Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 25.Fantner G.E. Barbero R.J. Gray D.S. Belcher A.M. Kinetics of antimicrobial peptide activity measured on individual bacterial cells using high-speed atomic force microscopy.Nat. Nanotechnol. 2010; 5: 280-285Crossref PubMed Scopus (222) Google Scholar, 26.Hansma P.K. Schitter G. Fantner G.E. Prater C. Applied physics—high-speed atomic force microscopy.Science. 2006; 314: 601-602Crossref PubMed Scopus (0) Google Scholar). atomic force microscopy cowpea chlorotic mottle virus hepatitis B virus minute virus of mice molecular dynamics. The applicability of AFM in proteomics is diverse and includes the characterization of the cell surface proteome (for a recent review, see Ref. 27.Dufrêne Y.F. Atomic force microscopy: a powerful molecular toolkit in nanoproteomics.Proteomics. 2009; 9: 5400-5405Crossref PubMed Scopus (15) Google Scholar), label-free detection and counting of single proteins (28.Yu X. Xu D. Cheng Q. Label-free detection methods for protein microarrays.Proteomics. 2006; 6: 5493-5503Crossref PubMed Scopus (0) Google Scholar, 29.Archakov A.I. Ivanov Y.D. Lisitsa A.V. Zgoda V.G. AFM fishing nanotechnology is the way to reverse the Avogadro number in proteomics.Proteomics. 2007; 7: 4-9Crossref PubMed Scopus (0) Google Scholar), and force spectroscopy measurements of binding and unbinding events (30.Rief M. Grubmüller H. Force spectroscopy of single biomolecules.ChemPhysChem. 2002; 3: 255-261Crossref PubMed Scopus (175) Google Scholar, 31.Zeng G. Chen J. Zhong L. Wang R. Jiang L. Cai J. Yan L. Huang D. Chen C.Y. Chen Z.W. NSOM- and AFM-based nanotechnology elucidates nano-structural and atomic-force features of a Y-pestis V immunogen-containing particle vaccine capable of eliciting robust response.Proteomics. 2009; 9: 1538-1547Crossref PubMed Scopus (18) Google Scholar). In structural biology, AFM has shown to be a powerful tool for high resolution imaging of proteins in near native conditions (3.Fechner P. Boudier T. Mangenot S. Jaroslawski S. Sturgis J.N. Scheuring S. Structural information, resolution, and noise in high-resolution atomic force microscopy topographs.Biophys. J. 2009; 96: 3822-3831Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 6.Engel A. Müller D.J. Observing single biomolecules at work with the atomic force microscope.Nat. Struct. Biol. 2000; 7: 715-718Crossref PubMed Scopus (0) Google Scholar) and structural studies of supramolecular assemblies like protein filaments and viruses by nanoindentation methods (32.Roos W.H. Wuite G.L. Nanoindentation studies reveal material properties of viruses.Adv. Mater. 2009; 21: 1187-1192Crossref Scopus (54) Google Scholar, 33.Kasas S. Dietler G. Probing nanomechanical properties from biomolecules to living cells.Pflugers Arch. 2008; 456: 13-27Crossref PubMed Scopus (78) Google Scholar). These experiments show the potential of AFM to study both "form" and "function" of proteins, thereby resolving questions in proteomics and structural biology quasi-simultaneously. In the following, we will explain the principles of atomic force microscopy and its different operation modes and finally discuss examples of imaging, nanoindentation, and protein (un)binding and unfolding studies using AFM. The atomic force microscope is a member of the scanning probe microscopy techniques that utilize a probing tip that scans the surface of a sample. The measured interaction between the sample and the probe (e.g. current in scanning tunneling microscopy or force in AFM) renders a three-dimensional image of the sample surface. In AFM, the probe is a sharp tip with a typical radius of about 1–20 nm mounted on a cantilever. The interaction between the sample and the tip is recorded by the bending of the cantilever as shown in Fig. 1. The deflection of the cantilever is monitored by the change in direction of the reflected light (laser), which is recorded by a position-sensitive detector (quadrant photodiode). Soft cantilevers with spring constants of about 0.01–0.1 newton/m can sense forces as low as a few piconewtons, whereas modern piezoelectric scanners can translate the sample or tip in x-, y-, and z-directions with subnanometer resolutions. This combined functionality provides the atomic force microscope with the capability of rendering three-dimensional images of the sample with atomic resolution and manipulating them at single molecular level with nanoscale forces (34.Binnig G. Gerber C. Stoll E. Albrecht T.R. Quate C.F. Atomic resolution with atomic force microscope.Europhys. Lett. 1987; 3: 1281-1286Crossref Google Scholar, 35.Giessibl F.J. Binnig G. Investigation of the (001) cleavage plane of potassium-bromide with an atomic force microscope at 4.2-k in ultra-high vacuum.Ultramicroscopy. 1992; 42: 281-289Crossref Scopus (83) Google Scholar, 36.Parot P. Dufrêne Y.F. Hinterdorfer P. Le Grimellec C. Navajas D. Pellequer J.L. Scheuring S. Past, present and future of atomic force microscopy in life sciences and medicine.J. Mol. Recognit. 2007; 20: 418-431Crossref PubMed Scopus (0) Google Scholar, 37.Zlatanova J. Leuba S.H. Stretching and imaging single DNA molecules and chromatin.J. Muscle Res. Cell Motil. 2002; 23: 377-395Crossref PubMed Scopus (0) Google Scholar). As a microscope, there are various modes available to construct an image of the sample. Choosing the appropriate imaging mode and parameters influences the amount of contact force between the sample and the probe tip (38.Hansma H.G. Hoh J.H. Biomolecular imaging with the atomic-force microscope.Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 115-139Crossref PubMed Google Scholar, 39.Giessibl F.J. Advances in atomic force microscopy.Rev. Mod. Phys. 2003; 75: 949-983Crossref Scopus (0) Google Scholar, 40.Jalili N. Laxminarayana K. A review of atomic force microscopy imaging systems: application to molecular metrology and biological sciences.Mechatronics. 2004; 14: 907-945Crossref Scopus (0) Google Scholar). The different operation modes can be divided into static and dynamic categories (41.Morris V.J. Kirby A.R. Gunning A.P. Atomic Force Microscopy for Biologists. 2nd Ed. Imperial College Press, London2010Google Scholar, 42.Garcia R. Perez R. Dynamic atomic force microscopy methods.Surf. Sci. Rep. 2002; 47: 197-301Crossref Google Scholar, 43.Braga P.C. Ricci D. Atomic Force Microscopy: Biomedical Methods and Applications. Humana Press, Totowa, NJ2004Google Scholar). In static imaging mode, the sample is brought into physical contact with the tip until their interaction reaches a condition where it satisfies a predefined parameter setting, such as the specified z-position of the sample (constant height mode) or the amount of interaction force between the tip and sample (constant force mode). In constant height mode, the sample is moved in x-y direction while maintaining its z-position. The surface or height information of the sample is reconstructed from the deflection of the cantilever, thus the measured contact force. However, because the distance between the tip and sample is fixed, lateral dragging of the sample during imaging is quite common. In constant force mode, it is the imaging force that is set on a fixed value. The sample is brought in contact with the tip by moving the piezoelectric scanner in the z-direction until the contact force reaches the set value. The height of the sample at that position is then determined by how much the sample was moved in z. The tip is then moved to the next x-y position and using a feedback mechanism will retract or approach the sample until the set contact force is obtained. The constant force mode provides a careful and cautious way of handling the sample by setting the contact force to a minimum. However, the cantilever normally moves laterally in a continuous manner, and therefore the retraction or approach during feedback can still impart considerable dragging or damage during these lateral movements, especially in imaging soft biological samples. An alternative way of performing the constant force imaging mode is called jumping mode. This mode provides a safer scheme by retracting the tip to a defined height away from the sample before moving on to the next lateral x-y position (44.de Pablo P.J. Colchero J. Gomez-Herrero J. Baro A.M. Jumping mode scanning force microscopy.Appl. Phys. Lett. 1998; 73: 3300-3302Crossref Scopus (148) Google Scholar). With this mode, there is less probability of dragging the sample laterally because each time it moves to another position it is first retracted away from the sample surface, which could be set such that it is above the highest structural feature. In addition, because it also operates in contact force mode, the tip-sample interaction force can be preset to the lowest possible value, thereby preventing damage to the sample. In dynamic imaging mode, the tip is made to oscillate while it approaches the sample. The change in the oscillation amplitude or frequency during approach is used to construct its surface information. In this mode, the tip can be intermittently touching the sample (tapping mode) or not touching the sample at all (non-contact dynamic mode). In tapping mode, the contact between the tip and sample is minimized by oscillating the cantilever so that the tip bounces up and down and therefore only "taps" the surface as it moves around the sample (45.Zhong Q. Inniss D. Kjoller K. Elings V.B. Fractured polymer silica fiber surface studied by tapping mode atomic force microscopy.Surf. Sci. 1993; 290: L688-L692Crossref Scopus (0) Google Scholar). The cantilever is made to oscillate lower than the natural resonance frequency; but when it approaches the sample, the resonance frequency decreases because of damping effects. The result is an amplitude increase because the driving frequency is now closer to the new resonance. Finally, the oscillation amplitude decreases again when the tip hits the sample. The monitored change in the oscillation amplitude is used as the feedback signal for constructing the image of the sample surface. In non-contact dynamic mode, the tip is never in contact with the sample. To implement this, the cantilever is made to oscillate at a frequency higher than resonance. As it approaches the sample, its amplitude of oscillation decreases because of damping: the closer it is to the sample, the lower its amplitude of oscillation. The tip is prevented from coming too close to the sample by setting a limit on the lowest possible oscillation amplitude. As with tapping mode, this parameter is used as the feedback signal for image construction. The dynamic modes provide the option of little physical contact with the sample during imaging. In force spectroscopy, the atomic force microscope is operating in the force-distance measurement mode (46.Cappella B. Dietler G. Force-distance curves by atomic force microscopy.Surf. Sci. Rep. 1999; 34: 1-104Crossref Google Scholar, 47.Butt H.J. Cappella B. Kappl M. Force measurements with the atomic force microscope: technique, interpretation and applications.Surf. Sci. Rep. 2005; 59: 1-152Crossref Scopus (2438) Google Scholar). In this mode, the force is now specifically monitored and recorded as the tip is brought into contact (e.g. pushing/indentation experiments) or out of contact from the sample (e.g. unbinding and stretching experiments) while simultaneously recording the amount of distance of approach or retraction of the tip. In this mode, no lateral movement is performed during the approach and retraction cycles. Typical applications of the spectroscopy mode in protein studies are in pushing or nanoindentation of viruses and microtubules to probe their mechanical properties (32.Roos W.H. Wuite G.L. Nanoindentation studies reveal material properties of viruses.Adv. Mater. 2009; 21: 1187-1192Crossref Scopus (54) Google Scholar, 33.Kasas S. Dietler G. Probing nanomechanical properties from biomolecules to living cells.Pflugers Arch. 2008; 456: 13-27Crossref PubMed Scopus (78) Google Scholar, 48.de Pablo P.J. Schaap I.A. MacKintosh F.C. Schmidt C.F. Deformation and collapse of microtubules on the nanometer scale.Phys. Rev. Lett. 2003; 91 (098101)Crossref Scopus (195) Google Scholar) and in pulling or stretching experiments to measure the (un)binding strength of two protein molecules (49.Chilkoti A. Boland T. Ratner B.D. Stayton P.S. The relationship between ligand-binding thermodynamics and protein-ligand interaction forces measured by atomic force microscopy.Biophys. J. 1995; 69: 2125-2130Abstract Full Text PDF PubMed Google Scholar, 50.Takano H. Kenseth J.R. Wong S.S. O'Brien J.C. Porter M.D. Chemical and biochemical analysis using scanning force microscopy.Chem. Rev. 1999; 99: 2845-2890Crossref PubMed Google Scholar, 51.Leckband D. Measuring the forces that control protein interactions.Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 1-26Crossref PubMed Scopus (0) Google Scholar, 52.Zlatanova J. Lindsay S.M. Leuba S.H. Single molecule force spectroscopy in biology using the atomic force microscope.Prog. Biophys. Mol. Biol. 2000; 74: 37-61Crossref PubMed Scopus (310) Google Scholar) or to measure the interactions of the different structural elements comprising large molecules such as muscle protein titin (53.Oberhauser A.F. Hansma P.K. Carrion-Vazquez M. Fernandez J.M. Stepwise unfolding of titin under force-clamp atomic force microscopy.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 468-472Crossref PubMed Google Scholar, 54.Linke W.A. Grützner A. Pulling single molecules of titin by AFM—recent advances and physiological implications.Pflugers Arch. 2008; 456: 101-115Crossref PubMed Scopus (0) Google Scholar), ubiquitin (55.Fernandez J.M. Li H.B. Force-clamp spectroscopy monitors the folding trajectory of a single protein.Science. 2004; 303: 1674-1678Crossref PubMed Scopus (441) Google Scholar, 56.Schlierf M. Li H. Fernandez J.M. The unfolding kinetics of ubiquitin captured with single-molecule force-clamp techniques.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7299-7304Crossref PubMed Scopus (275) Google Scholar), and membrane protein bacteriorhodopsin (57.Oesterhelt F. Oesterhelt D. Pfeiffer M. Engel A. Gaub H.E. Müller D.J. Unfolding pathways of individual bacteriorhodopsins.Science. 2000; 288: 143-146Crossref PubMed Scopus (571) Google Scholar, 58.Kedrov A. Janovjak H. Sapra K.T. Müller D.J. Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy.Annu. Rev. Biophys. Biomol. Struct. 2007; 36: 233-260Crossref PubMed Scopus (103) Google Scholar, 59.Fisher T.E. Marszalek P.E. Fernandez J.M. Stretching single molecules into novel conformations using the atomic force microscope.Nat. Struct. Biol. 2000; 7: 719-724Crossref PubMed Scopus (273) Google Scholar). In these experiments, the plot of the measured force against the deformation of the sample (force-distance curve) provides information on the material properties such as the spring constants, Young moduli, and the binding and unbinding, stretching and breaking forces of the probed structures. A typical force-distance curve generated by nanoindenting a virus particle is depicted in Fig. 2. In the beginning, the tip is far from the sample; thus, the measured interaction force between them is zero. At the instance the tip touches the sample surface, the cantilever starts to bend, and the force-distance curve starts to rise as the tip pushes further on the sample. When the tip is retracted, the retraction curve may simply trace back the curve during approach (in the case of a hard surface), or it retraces a completely different curve depending on the properties of the sample (37.Zlatanova J. Leuba S.H. Stretching and imaging single DNA molecules and chromatin.J. Muscle Res. Cell Motil. 2002; 23: 377-395Crossref PubMed Scopus (0) Google Scholar, 47.Butt H.J. Cappella B. Kappl M. Force measurements with the atomic force microscope: technique, interpretation and applications.Surf. Sci. Rep. 2005; 59: 1-152Crossref Scopus (2438) Google Scholar, 60.Vinckier A. Gervasoni P. Zaugg F. Ziegler U. Lindner P. Groscurth P. Plückthun A. Semenza G. Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins.Biophys. J. 1998; 74: 3256-3263Abstract Full Text Full Text PDF PubMed Google Scholar, 61.Willemsen O.H. Snel M.M. Cambi A. Greve J. De Grooth B.G. Figdor C.G. Biomolecular interactions measured by atomic force microscopy.Biophys. J. 2000; 79: 3267-3281Abstract Full Text Full Text PDF PubMed Google Scholar). As illustrated in Fig. 2, the virus particle has exceeded its elastic limits during approach at about 0.8 nanonewton and thereafter suffered irreversible deformation, maybe breakage. During retraction, the tip did not interact anymore with an intact virus particle, and the retraction curve resembled that of the glass curve. To ensure that the measured parameters are accurate and precise, the piezoelectric scanner, spring constant of the cantilever, and photodiode signal must be calibrated. The scanner can be calibrated by imaging surfaces or microgrids with known dimensions. For the cantilever calibration, there are three different methods in measuring its spring constant: dimensional, static, and dynamic methods. Dimensional methods calculate the spring constant based on the physical dimensions and material properties of the cantilever (62.Senden T.J. Ducker W.A. Experimental determination of spring constants in atomic force microscopy.Langmuir. 1994; 10: 1003-1004Crossref Google Scholar, 63.Clifford C.A. Seah M.P. The determination of atomic force microscope cantilever spring constants via dimensional methods for nanomechanical analysis.Nanotechnology. 2005; 16: 1666-1680Crossref Scopus (146) Google Scholar), and the static method is based on the deflection of the cantilever in response to a known force (64.Chen G.Y. Warmack R.J. Thundat T. Allison D.P. Huang A. Resonance response of scanning force microscopy cantilevers.Rev. Sci. Instrum. 1994; 65: 2532-2537Crossref Scopus (0) Google Scholar, 65.Albrecht T.R. Akamine S. Carver T.E. Quate C.F. Microfabrication of cantilever styli for the atomic force microscope.J. Vac. Sci. Technol. A. 1990; 8: 3386-3396Crossref Google Scholar), whereas the dynamic method measures the resonance frequency and Q-factor by oscillating the cantilever (66.Hutter J.L. Bechhoefer J. Calibration of atomic-force microscope tips.Rev. Sci. Instrum. 1993; 64: 1868-1873Crossref Scopus (3098) Google Scholar, 67.Sader J.E. Larson I. Mulvaney P. White L.R. Method for the calibration of atomic force microscope cantilevers.Rev. Sci. Instrum. 1995; 66: 3789-3798Crossref Scopus (753) Google Scholar, 68.Sader J.E. Chon J.W. Mulvaney P. Calibration of rectangular atomic force microscope cantilevers.Rev. Sci. Instrum. 1999; 70: 3967-3969Crossref Google Scholar). Most atomic force microscopes use the beam bounce method to measure the cantilever deflection using a photodiode. Thus, an a priori calibration procedure is important to convert the photodiode signal into the actual force applied by the tip. Force calibration is usually implemented by pushing the tip on a hard surface such as a mica or glass substrate. This curve measures the deflection due to the cantilever alone, referred to as the glass curve in Fig. 2.