Edited by

Arturo M. Baro and Ronald G. Reifenberger

Atomic Force Microscopy in Liquid

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Edited by Arturo M. Baro and Ronald G. Reifenberger

Atomic Force Microscopy in Liquid

Biological Applications

The Editors

Prof. Arturo M. BaroInstituto de Ciencia de Materialsde Madrid (CSIC)Sor Ines de la CruzMadrid 28049Spain

Prof. Ronald G. ReifenbergerPurdue UniversityDepartment of Physics525, Northwestern AvenueWest Lafayette, IN 47907-2036USA

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V

Contents

Preface XIII

List of Contributors XV

Part I General Atomic Force Microscopy 1

1 AFM: Basic Concepts 3Fernando Moreno-Herrero and Julio Gomez-Herrero

1.1 Atomic Force Microscope: Principles 31.2 Piezoelectric Scanners 51.2.1 Piezoelectric Scanners for Imaging in Liquids 81.3 Tips and Cantilevers 81.3.1 Cantilever Calibration 101.3.2 Tips and Cantilevers for Imaging in Liquids 111.3.3 Cantilever Dynamics in Liquids 131.4 Force Detection Methods for Imaging in Liquids 151.4.1 Piezoelectric Cantilevers and Tuning Forks 151.4.2 Laser Beam Deflection Method 171.4.2.1 Liquid Cells and Beam Deflection 181.5 AFM Operation Modes: Contact, Jumping/Pulsed, Dynamic 191.5.1 Contact Mode 191.5.2 Jumping and Pulsed Force Mode 201.5.3 Dynamic Modes 221.5.3.1 Liquid Cells and Dynamic Modes 231.6 The Feedback Loop 241.7 Image Representation 251.8 Artifacts and Resolution Limits 281.8.1 Artifacts Related to the Geometry of the Tip 281.8.2 Artifacts Related to the Feedback Loop 301.8.3 Resolution Limits 31

Acknowledgments 32References 32

VI Contents

2 Carbon Nanotube Tips in Atomic Force Microscopy with Applicationsto Imaging in Liquid 35Edward D. de Asis, Jr., Joseph Leung, and Cattien V. Nguyen

2.1 Introduction 352.2 Fabrication of CNT AFM Probes 372.2.1 Mechanical Attachment 382.2.2 CNT Attachment Techniques Employing Magnetic and Electric

Fields 392.2.3 Direct Growth of CNT Tips 412.2.4 Emerging CNT Attachment Techniques 432.2.5 Postfabrication Modification of the CNT Tip 432.2.5.1 Shortening 432.2.5.2 Coating with Metal 442.3 Chemical Functionalization 442.3.1 Functionalization of the CNT Free End 452.3.2 Coating the CNT Sidewall 452.4 Mechanical Properties of CNTs in Relation to AFM Applications 462.4.1 CNT Atomic Structure 472.4.2 Mechanical Properties of CNT AFM Tips 492.5 Dynamics of CNT Tips in Liquid 502.5.1 Interaction of Microfabricated AFM Tips and Cantilevers in

Liquid 502.5.2 CNT AFM Tips in Liquid 522.5.3 Interaction of CNT with Liquids 522.5.3.1 CNT Tips at the Air–Liquid Interface During Approach 542.5.3.2 CNT Tips at the Liquid–Solid Interface 562.5.3.3 CNT Tips at the Air–Liquid Interface during Withdrawal 582.6 Performance and Resolution of CNT Tips in Liquid 582.6.1 Performance of CNT AFM Tips When Imaging in Liquid 582.6.2 Biological Imaging in Liquid Medium with CNT AFM Tips 592.6.3 Cell Membrane Penetration and Applications of Intracellular CNT

AFM Probes 60References 61

3 Force Spectroscopy 65Arturo M. Baro

3.1 Introduction 653.2 Measurement of Force Curves 673.2.1 Analysis of Force Curves Taken in Air 683.2.2 Analysis of Force Curves in a Liquid 703.3 Measuring Surface Forces by the Surface Force Apparatus 703.4 Forces between Macroscopic Bodies 713.5 Theory of DLVO Forces between Two Surfaces 713.6 Van der Waals Forces – the Hamaker Constant 723.7 Electrostatic Force between Surfaces in a Liquid 72

Contents VII

3.8 Spatially Resolved Force Spectroscopy 763.9 Force Spectroscopy Imaging of Single DNA Molecules 783.10 Solvation Forces 793.11 Hydrophobic Forces 813.12 Steric Forces 813.13 Conclusive Remarks 83

Acknowledgments 83References 83

4 Dynamic-Mode AFM in Liquid 87Takeshi Fukuma and Michael J. Higgins

4.1 Introduction 874.2 Operation Principles 884.2.1 Amplitude and Phase Modulation AFM (AM- and PM-AFM) 884.2.2 Frequency-Modulation AFM (FM-AFM) 894.3 Instrumentation 904.3.1 Cantilever Excitation 904.3.2 Cantilever Deflection Measurement 914.3.3 Operating Conditions 934.3.4 AM-AFM 934.3.4.1 FM-AFM 954.3.4.2 PM-AFM 964.4 Quantitative Force Measurements 974.4.1 Calibration of Spring Constant 984.4.2 Conservative and dissipative forces 1014.4.3 Solvation Force Measurements 1034.4.3.1 Inorganic Solids in Nonpolar Liquids 1044.4.3.2 Measurements in Pure Water 1064.4.3.3 Solvation Forces in Biological Systems 1064.4.4 Single-Molecule Force Spectroscopy 1084.4.4.1 Unfolding and ‘‘Stretching’’ of Biomolecules 1084.4.4.2 Ligand–Receptor Interactions 1104.5 High-Resolution Imaging 1104.5.1 Solid Crystals 1124.5.2 Biomolecular Assemblies 1134.5.3 Water Distribution 1144.6 Summary and Future Prospects 116

References 117

5 Fundamentals of AFM Cantilever Dynamics in LiquidEnvironments 121Daniel Kiracofe, John Melcher, and Arvind Raman

5.1 Introduction 1215.2 Review of Fundamentals of Cantilever Oscillation 1225.3 Hydrodynamics of Cantilevers in Liquids 123

VIII Contents

5.4 Methods of Dynamic Excitation 1265.4.1 Review of Cantilever Excitation Methods 1285.4.2 Theory 1305.4.2.1 Direct Forcing 1305.4.2.2 Ideal Piezo/Acoustic 1325.4.2.3 Thermal 1325.4.2.4 Comparison of Excitation Methods 1335.4.3 Practical Considerations for Acoustic Method 1355.4.4 Photothermal Method 1375.4.5 Frequency Modulation Considerations in Liquids 1405.5 Dynamics of Cantilevers Interacting with Samples in Liquids 1405.5.1 Experimental Observations of Oscillating Probes Interacting with

Samples in Liquids 1415.5.2 Modeling and Numerical Simulations of Oscillating Probes

Interacting with Samples in Liquids 1425.5.3 Compositional Mapping in Liquids 1455.5.4 Implications for Force Spectroscopy in Liquids 1485.6 Outlook 150

References 150

6 Single-Molecule Force Spectroscopy 157Albert Galera-Prat, Rodolfo Hermans, Ruben Hervas,Angel Gomez-Sicilia, and Mariano Carrion-Vazquez

6.1 Introduction 1576.1.1 Why Single-Molecule Force Spectroscopy? 1576.1.2 SMFS in Biology 1586.1.3 SMFS Techniques and Ranges 1586.2 AFM-SMFS Principles 1596.2.1 Length-Clamp Mode 1606.2.2 Force-Clamp Mode 1636.3 Dynamics of Adhesion Bonds 1656.3.1 Bond Dissociation Dynamics in Length Clamp 1656.3.2 General Considerations 1676.3.3 Bond Dissociation Dynamics in Force Clamp 1686.3.3.1 The Need for Robust Statistics 1696.4 Specific versus Other Interactions 1696.4.1 Intramolecular Single-Molecule Markers 1706.4.1.1 The Wormlike Chain: an Elasticity Model 1706.4.1.2 Proteins 1716.4.1.3 DNA and Polysaccharides 1746.4.2 Intermolecular Single-Molecule Markers 1746.5 Steered Molecular Dynamics Simulations 1766.6 Biological Findings Using AFM–SMFS 1776.6.1 Titin as an Adjustable Molecular Spring in the Muscle

Sarcomere 177

Contents IX

6.6.2 Monitoring the Folding Process by Force-Clamp Spectroscopy 1806.6.3 Intermolecular Binding Forces and Energies in Pairs of

Biomolecules 1806.6.4 New Insights in Catalysis Revealed at the Single-Molecule Level 1816.7 Concluding Remarks 182

Acknowledgments 182Disclaimer 182References 182

7 High-Speed AFM for Observing Dynamic Processes in Liquid 189Toshio Ando, Takayuki Uchihashi, Noriyuki Kodera, Mikihiro Shibata,Daisuke Yamamoto, and Hayato Yamashita

7.1 Introduction 1897.2 Theoretical Derivation of Imaging Rate and Feedback Bandwidth 1907.2.1 Imaging Time and Feedback Bandwidth 1907.2.2 Time Delays 1917.3 Techniques Realizing High-Speed Bio-AFM 1927.3.1 Small Cantilevers 1927.3.2 Fast Amplitude Detector 1947.3.3 High-Speed Scanner 1947.3.4 Active Damping Techniques 1967.3.5 Suppression of Parachuting 1987.3.6 Fast Phase Detector 1997.4 Substrate Surfaces 2007.4.1 Supported Planar Lipid Bilayers 2007.4.1.1 Choice of Alkyl Chains 2017.4.1.2 Choice of Head Groups 2017.4.2 Streptavidin 2D Crystal Surface 2017.5 Imaging of Dynamic Molecular Processes 2037.5.1 Bacteriorhodopsin Crystal Edge 2037.5.2 Photoactivation of Bacteriorhodopsin 2047.6 Future Prospects of High-Speed AFM 2067.6.1 Imaging Rate and Low Invasiveness 2067.6.2 High-Speed AFM Combined with Fluorescence Microscope 2067.7 Conclusion 207

References 207

8 Integration of AFM with Optical Microscopy Techniques 211Zhe Sun, Andreea Trache, Kenith Meissner, and Gerald A. Meininger

8.1 Introduction 2118.1.1 Combining AFM with Fluorescence Microscopy 2148.1.1.1 Epifluorescence Microscopy 2148.1.2 Examples of Applications 2158.1.2.1 Ca2+ Fluorescence Microscopy 2158.1.2.2 AFM – Epifluorescence Microscopy 217

X Contents

8.2 Combining AFM with IRM and TIRF microscopy 2178.2.1 Interference Reflection Microscopy 2178.2.1.1 Optical Setup 2188.2.2 Total Internal Reflection Fluorescence Microscopy 2188.2.2.1 Optical Setup 2188.2.2.2 Applications of Combined AFM–TIRF and AFM–IRM

Microscopy 2208.3 Combining AFM and FRET 2218.3.1 FRET 2218.3.2 FRET and Near-Field Scanning Optical Microscopy (NSOM) 2228.4 FRET-AFM 2228.5 Sample Preparation and Experiment Setup 2238.5.1 Cell Culture, Transfection, and Fura-Loading 2238.5.2 Cantilever Preparation 2248.5.3 Typical Experimental Procedure 225

References 225

Part II Biological Applications 231

9 AFM Imaging in Liquid of DNA and Protein–DNA Complexes 233Yuri L. Lyubchenko

9.1 Overview: the Study of DNA at Nanoscale Resolution 2339.2 Sample Preparation for AFM Imaging of DNA and Protein–DNA

Complexes 2349.3 AFM of DNA in Aqueous Solutions 2369.3.1 Elevated Resolution in Aqueous Solutions 2369.3.2 Segmental Mobility of DNA 2379.4 AFM Imaging of Alternative DNA Conformations 2399.4.1 Cruciforms in DNA 2399.4.2 Intramolecular Triple Helices 2449.4.3 Four-Way DNA Junctions and DNA Recombination 2459.5 Dynamics of Protein–DNA Interactions 2479.5.1 Site-Specific Protein–DNA Complexes 2479.5.2 Chromatin Dynamics Time-Lapse AFM 2519.6 DNA Condensation 2539.7 Conclusions 254

Acknowledgments 254References 255

10 Stability of Lipid Bilayers as Model Membranes: Atomic ForceMicroscopy and Spectroscopy Approach 259Lorena Redondo-Morata, Marina Ines Giannotti, and Fausto Sanz

10.1 Biological Membranes 25910.1.1 Cell Membrane 25910.1.2 Supported Lipid Bilayers 259

Contents XI

10.2 Mechanical Characterization of Lipid Membranes 26310.2.1 Breakthrough Force as a Molecular Fingerprint 26310.2.2 AFM Tip-Lipid Bilayer Interaction 26510.2.3 Effect of Chemical Composition on the Mechanical Stability of Lipid

Bilayers 26710.2.4 Effect of Ionic Strength on the Mechanical Stability of Lipid

Bilayers 26810.2.5 Effect of Different Cations on the Mechanical Stability of Lipid

Bilayers 27110.2.6 Effect of Temperature on the Mechanical Stability of Lipid

Bilayers 27310.2.7 The Case of Phase-Segregated Lipid Bilayers 27410.3 Future Perspectives 279

References 279

11 Single-Molecule Atomic Force Microscopy of Cellular Sensors 285Jurgen J. Heinisch and Yves F. Dufrene

11.1 Introduction 28511.1.1 Mechanosensors in Living Cells 28511.1.2 Yeast Cell Wall Integrity Sensors: a Valuable Model for

Mechanosensing 28611.2 Methods 28811.2.1 Atomic Force Microscopy of Live Cells 28811.2.2 AFM Detection of Single Sensors 29011.2.3 Bringing Yeast Sensors to the Surface 29111.3 Probing Single Yeast Sensors in Live Cells 29211.3.1 Measuring Sensor Spring Properties 29211.3.2 Imaging Sensor Clustering 29511.3.3 Using Sensors as Molecular Rulers 29811.4 Conclusions 302

Acknowledgments 303References 303

12 AFM-Based Single-Cell Force Spectroscopy 307Clemens M. Franz and Anna Taubenberger

12.1 Introduction 30712.2 Cantilever Choice 31012.3 Cantilever Functionalization 31012.4 Cantilever Calibration 31112.5 Cell Attachment to the AFM Cantilever 31112.6 Recording a Force–Distance Curve 31312.7 Processing F–D Curves 31512.8 Quantifying Overall Cell Adhesion by SCFS 31712.9 SFCS with Single-Molecule Resolution 32012.10 Dynamic Force Spectroscopy 321

XII Contents

12.11 Measuring Cell–Cell Adhesion 32512.12 Conclusions and Outlook 326

References 327

13 Nanosurgical Manipulation of Living Cells with the AFM 331Atsushi Ikai, Rehana Afrin, Takahiro Watanabe-Nakayama,and Shin-ichi Machida

13.1 Introduction: Mechanical Manipulation of Living Cells 33113.2 Basic Mechanical Properties of Proteins and Cells 33113.3 Hole Formation on the Cell Membrane 33213.4 Extraction of mRNA from Living Cells 33413.5 DNA Delivery and Gene Expression 33513.6 Mechanical Manipulation of Intracellular Stress Fibers 33813.6.1 AFM Used as a Lateral Force Microscope 33813.6.2 Force Curves and Fluorescence Images under Lateral Force

Application 34013.6.2.1 Case 1 34013.6.2.2 Case 2 34013.7 Cellular Adaptation to Local Stresses 34313.8 Application of Carbon Nanotube Needles 34413.9 Use of Fabricated AFM Probes with a Hooking Function 34613.9.1 Result for a Semi-Intact Cell 34813.9.2 Result for a Living Cell 34813.10 Membrane Protein Extraction 34813.11 Future Prospects 350

Acknowledgments 350References 350

Index 355

XIII

Preface

The atomic force microscope (AFM) is a member of the broad family of scanningprobe microscopes and arguably has become the most widely used scanning probeinstrument in the world. The high image resolution coupled with a new class ofspectroscopic tools has enabled AFMs to perform real-time dynamic studies as wellas controlled nanomanipulation, resulting in significant breakthroughs in manydifferent realms of science and engineering.

The book Atomic Force Microscopy in Liquid: Biological Applications broadly focuseson phenomena relevant to AFM studies at a solid–liquid interface, with an emphasison biological applications ranging from small biomolecules to living cells. As faras we know, there are no books closely related to this one. The ability of an AFMto study samples in a liquid environment provides a significant advantage whencompared to other microscopies such as SEM and TEM. This unique capabilityallows measurements of native biological samples in aqueous environments underphysiologically relevant conditions. The weakness of van der Waals interactionsand the absence of capillary forces in liquid drastically reduce the tip–sampleinteraction, resulting in little damage to soft biological samples. The highly localcharacter of AFM that directly results from probe proximity to the sample coupledwith tip sharpness not only allows high-resolution images but also permits laterallyresolved spectroscopic measurements capable of reaching the level of a singlemolecule, as, for example, in single molecule force spectroscopy applications.

This book provides a thorough description of AFM operation in liquid environ-ments and will serve as a useful reference for all AFM groups. It is organized intotwo sections in an effort to be especially useful for new researchers who desire tostart bio-related studies. The first part of the book is focused on the study of featuresunique to AFM including instrumentation, force spectroscopic analysis, generalimaging and spectroscopy considerations, single molecule force spectroscopy, oper-ational modes, electrostatic forces in liquids containing ions, high-speed imaging,nanomanipulation, and lithography. Historically, there have been a number ofAFM studies on biological systems in liquid by contact-mode AFM. Recently, dy-namic force spectroscopy (DFS) experiments have appeared that utilize noncontactimaging, further reducing sample damage. Therefore, DFS is discussed in twochapters, one connected with experimental work and a second that deals with thetheory of dynamic AFM. We also include a chapter on the combination of AFM

XIV Preface

with more traditional optical techniques such as fluorescence. A growing trend willbe the simultaneous utilization of one or more auxiliary techniques with AFM toexploit the many advantages when complementary techniques are combined.

The second part of the book deals with applications of AFM to the study of bio-logical materials ranging from the smallest biomolecules (phospholipids, proteins,DNA, RNA, and protein complexes) to subcellular structures (e.g., membranes),and finally culminating with studies of living microbial cells. Single cell forcespectroscopy and the manipulation of biological material with an AFM are alsoincluded. The goal is to feature recent advances that emphasize in vivo experiments.

This book is timely and up-to-date. It is aimed at a mixed audience thatincludes starting graduate students, young researchers, and established scientists.Physicists need to learn how to handle/prepare biological samples; biologists needto understand the important issues related to imaging of complex samples inliquid. We hope the book is useful, especially for those who enjoy breaking groundin a new and interdisciplinary field.

We would like to thank all the distinguished scientists and their coauthorsfor their timely and well-referenced contributions. Grateful acknowledgments areoffered to the Wiley-VCH editorial staff, in particular Lesley Belfit, Project Editor,and Publisher Dr. Gudrun Walter.

UAM, Madrid Arturo M. BaroPurdue University Ronald G. Reifenberger

XV

List of Contributors

Rehana AfrinTokyo Institute of TechnologyInnovation Laboratory4259 NagatsutaYokohama 226-8501Japan

Toshio AndoKanazawa UniversityDepartment of PhysicsKakuma-machiKanazawa 920-1192Japan

Arturo M. BaroInstituto de Ciencia de Materialesde Madrid (CSIC)Sor Inos de la Cruz28049 MadridSpain

Mariano Carrion-VazquezInstituto CajalConsejo Superior deInvestigacionesCientıficas, IMDEANanociencia and Centro deInvestigacion Biomedica en Redsobre EnfermedadesNeurodegenerativas (CIBERNED)Avenida Doctor Arce 3728002 MadridSpain

Edward D. de Asis Jr.Santa Clara UniversityDepartments of ElectricalEngineering and BioengineeringSchool of Engineering500 El camin RealSanta ClaraCA 95053USA

Yves F. DufreneUniversite catholique de LouvainInstitute of Condensed Matterand NanosciencesCroix du Sud 2/181348 Louvain-la-NeuveBelgium

Clemens M. FranzKarlsruhe Institute of TechnologyDFG-Center for FunctionalNanostructuresWolfgang-Gaede-Str. 1a76131 KarlsruheGermany

Takeshi FukumaKanazawa UniversityFrontier Science OrganizationKakuma-machiKanazawa 920-1192Japan

XVI List of Contributors

Albert Galera-PratInstituto CajalConsejo Superior deInvestigaciones Cientıficas,IMDEA Nanociencia and Centrode Investigacion Biomedica enRed sobre EnfermedadesNeurodegenerativas (CIBERNED)Avenida Doctor Arce 3728002 MadridSpain

Marina Ines GiannottiCIBER de BioingenierıaBiomateriales y Nanomedicina(CIBER-BBN)Campus Rıo Ebro, Edificio I+D,Poeta Mariano Esquillor s/n,50018 ZaragozaSpain

and

University of Barcelona (UB)Physical Chemistry Department1-3 Martı i Franques08028 BarcelonaSpain

and

Institute for Bioengineering ofCatalonia (IBEC)15-21 Baldiri I Reixac08028 BarcelonaSpain

Julio Gomez-HerreroUniversidad Autonoma deMadrid (UAM)Departamento de Fısica de laMateria Condensada28049 MadridSpain

Angel Gomez-SiciliaInstituto CajalConsejo Superior deInvestigaciones Cientıficas,IMDEA Nanociencia and Centrode Investigacion Biomedica enRed sobre EnfermedadesNeurodegenerativas (CIBERNED)Avenida Doctor Arce 3728002 MadridSpain

Jurgen J. HeinischFachbereich Biologie/ChemieUniversitat OsnabruckAG GenetikBarbarastr. 1149076 OsnabruckGermany

Rodolfo HermansUniversity College LondonLondon Centre forNanotechnology17-19 Gordon StreetLondon WC1H 0AHUK

Ruben HervasInstituto CajalConsejo Superior deInvestigaciones Cientıficas,IMDEA Nanociencia and Centrode Investigacion Biomedica enRed sobre EnfermedadesNeurodegenerativas (CIBERNED)Avenida Doctor Arce 3728002 MadridSpain

List of Contributors XVII

Michael J. HigginsUniversity of WollongongAIIM FacilityARC Centre of Excellence forElectromaterials ScienceIntelligent Polymer ResearchInstituteNew South Wares 2522Australia

Atsushi IkaiTokyo Institute of TechnologyInnovation Laboratory4259 NagatsutaYokohama 226-8501Japan

Daniel KiracofePurdue UniversitySchool of MechanicalEngineering and the BirckNanotechnology Center1205 W. State StreetWest LafayetteIN 47906USA

Noriyuki KoderaDepartment of PhysicsKanazawa UniversityKakuma-machiKanazawa 920-1192Japan

Joseph LeungNASA Ames Research CenterMoffett FieldCA 94035-1000USA

Yuri L. LyubchenkoDepartment of PharmaceuticalSciencesUniversity of NebraskaMedical Center4350 Dewey AvenueOhama, NE 68198USA

Shin-ichi MachidaInnovation LaboratoryTokyo Institute of Technology4259 NagatsutaYokohama 226-8501Japan

Gerald A. MeiningerDalton CardiovascularResearch CenterDepartment of MedicalPharmacology and PhysiologyUniversity of Missouri-Columbia134 Research Park DriveColumbiaMO 65211USA

Kenith MeissnerTexas A&M UniversityDepartment of BiomedicalEngineeringCollege StationTX 77843USA

John MelcherPurdue UniversitySchool of MechanicalEngineering and the BirckNanotechnology Center1205 W. State StreetWest LafayetteIN 47906USA

XVIII List of Contributors

Fernando Moreno-HerreroDepartamento de Estructura deMacromoleculasCentro Nacional de BiotecnologıaConsejo Superior deInvestigaciones Cientıficas (CSIC)Darwin 328049 MadridSpain

Takahiro Watanabe-NakayamaTokyo Institute of TechnologyInnovation Laboratory4259 NagatsutaYokohama 226-8501Japan

Cattien V. NguyenEloret CorporationNASA Ames Research CenterM/S 229-1Moffett FieldCA 94035-1000USA

Arvind RamanPurdue UniversitySchool of MechanicalEngineering and the BirckNanotechnology Center1205 W. State StreetWest LafayetteIN 47906USA

Lorena Redondo-MorataInstitute for Bioengineering ofCatalonia (IBEC)15-21 Baldiri I Reixac08028 BarcelonaSpain

and

University of Barcelona (UB)Physical Chemistry Department1-3 Martı i Franques08028 BarcelonaSpain

and

CIBER de BioingenierıaBiomateriales y Nanomedicina(CIBER-BBN)Campus Rıo Ebro, Edificio I+DPoeta Mariano Esquillor s/n50018 ZaragozaSpain

Fausto SanzInstitute for Bioengineering ofCatalonia (IBEC)15-21 Baldiri I Reixac08028 BarcelonaSpain

and

CIBER de BioingenierıaBiomateriales y Nanomedicina(CIBER-BBN)50018 ZaragozaSpain

and

University of Barcelona (UB)Physical Chemistry Department1-3 Martı i Franques08028 BarcelonaSpain

List of Contributors XIX

Mikihiro ShibataKanazawa UniversityDepartment of PhysicsKakuma-machiKanazawa 920-1192Japan

Zhe SunUniversity of Missouri-ColumbiaDalton Cardiovascular ResearchCenter134 Research Park DriveColumbiaMO 65211USA

Anna TaubenbergerQueensland University ofTechnologyInstitute of Health andBiomedical Innovation60 Musk AvenueKelvin GroveQLD 4059Australia

Andreea TracheTexas A&M UniversityDepartment of Systems Biology &Translational MedicineTexas A&M Health ScienceCenterand Department of BiomedicalEngineering336 Reynolds Medical BldgCollege StationTX 77843USA

Takayuki UchihashiKanazawa UniversityDepartment of PhysicsKakuma-machiKanazawa 920-1192Japan

Daisuke YamamotoKanazawa UniversityDepartment of PhysicsKakuma-machiKanazawa 920-1192Japan

Hayato YamashitaKanazawa UniversityDepartment of PhysicsKakuma-machiKanazawa 920-1192Japan

1

Part IGeneral Atomic Force Microscopy

Atomic Force Microscopy in Liquid: Biological Applications, First Edition.Edited by Arturo M. Baro and Ronald G. Reifenberger.© 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

3

1AFM: Basic ConceptsFernando Moreno-Herrero and Julio Gomez-Herrero

1.1Atomic Force Microscope: Principles

A conceptually new family of microscopes emerged after the invention of thescanning tunneling microscope (STM) by Binnig and Rohrer in 1982 [1]. Thisfamily of instruments called scanning probe microscopes (SPMs) is based on thestrong distance-dependent interaction between a sharp probe or tip and a sample.The atomic force microscope therefore uses the force existing between the probeand the sample to build an image of an object [2, 3]. AFMs can operate in almostany environment including aqueous solution, and that opened myriad uses inbiology [4, 5]. When thinking about how an AFM works, all notions of conventionalmicroscope design need to be disregarded, since there are no lenses through whichthe operator looks at the sample. In AFM, images are obtained by sensing with theprobe rather than by seeing.

The central part of an AFM is therefore the tip that literally feels the sample. Ananometer-sharp AFM tip made by microfabricating technology is grown at thefree end of a flexible cantilever that is used as the transductor of the interactionbetween the tip and sample. The reflection of a laser beam focused at the backside of the cantilever is frequently used by most AFMs to amplify and measure themovement of the cantilever, although other detection methods may also be used(Section 1.4). The reflected beam is directed to a photodiode that provides a voltagedepending on the position of the laser beam. For imaging, the tip is scanned overthe sample, or as in some designs, it is the sample that moves with respect to thefixed tip, which is only allowed to move in the vertical direction. In both cases, thefine movements of the tip and sample are provided by piezoelectric materials thatcan move with subnanometer precision. At each position, the cantilever deflectionis measured, from which a topography map can be constructed. This scanningtechnique in which the tip is brought into mechanical contact with the samplesurface is known as contact mode, and it was first described by Binnig and coworkers[2]. Both the tip and scanner are the key features in any AFM setup.

Atomic Force Microscopy in Liquid: Biological Applications, First Edition.Edited by Arturo M. Baro and Ronald G. Reifenberger.© 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

4 1 AFM: Basic Concepts

Computersoftware3.

XYZ(Low voltage)

FNFL Σ

FNFL ΣFNFL Σ

2. High voltageelectronics

DSP

XYZ

XYZ (High voltage)

Photodiode

Laser

MechanicsAFM head

Piezotube

Sample

1.

Tip

Cantilever

10 nm10 nm

Figure 1.1 Components of a standardatomic force microscope. 1. The AFM headand the piezoelectric stage. The cantileverand its detection system as well as the sam-ple movement are the main parts of thiscomponent. From the photodiode, the sig-nals related to the normal and lateral forces(FN and FL) as well as the total intensity oflight (�) are obtained and transferred to the

high voltage electronics. 2. The high volt-age electronics. This component amplifiesvoltages from the digital signal processorto perform the movement of the piezotube(XYZ voltages). It also collects signals fromthe photodiode (FN, FL, and �) and trans-fers them to the DSP. 3. The computer,DSP, and software that controls the AFMsetup.

One of the most common models of AFM is schematically depicted in Figure 1.1.In this model, the sample is scanned over the tip, but the opposite is also possible.The latter are the so-called stand-alone AFMs that are commonly used combinedwith an inverted optical microscope to image biological samples in liquid. Ineither case, a standard AFM setup consists of three main components. (i) theAFM head and base stage: The AFM head contains the tip holder, the laser, andthe photodiode. It also includes positioning mechanisms for focusing the laserbeam on the back side of the cantilever and photodiode and small electronics forprocessing the signals coming from the photodiode. From this, the vertical (FN)and lateral (FL) deflections of the laser beam and its total intensity (�) are obtained.The AFM head is placed over a base stage that holds the piezoelectric scanner thatmoves the sample, and also a coarse, micrometer-ranged approaching mechanism,usually based on step motors.1) (ii) The high voltage (HV) electronics: It amplifiesthe signals coming from the digital signal processor (DSP) (XYZ low voltage)to drive the piezoelectric scanner with voltages of about 100 V (XYZ HV). Theelectronics also transfers the analog voltage signals from the photodiode (FN, FL, �)

1) On a stand-alone AFM, the tip is scannedover the sample and the approachingmechanisms (step motors) are placed

on the AFM head rather than on themechanical stage.

1.2 Piezoelectric Scanners 5

to the DSP. The HV electronics must be able to amplify small signals from thecomputer (of some volts) to hundreds of volts needed to move the piezoelectrictube over micrometer distances. It is therefore essential that this amplificationdoes not introduce electrical noises that may affect the resolution of the AFM.(iii) The DSP, the computer, and the software: The DSP performs all the signalprocessing and calculations involved in the real-time operation of the AFM. TheDSP is mainly located in a board plugged in the computer. It contains the chips toperform the translation from digital to analog signals (digital to analog converters(DACs)), which are further managed by the HV electronics. Analog signals fromthe HV amplifier are converted to digital signals also at the DSP board usinganalog to digital converters (ADCs). Finally, all computer-based systems needsoftware to run the setup. Nearly all AFMs in the market come with purpose-madeacquisition software. Raw images can later be processed with any of the manyimaging-processing freeware available in the Internet.

Operating the AFM in liquid conditions requires modifications of some partsto prevent wetting of electrical components such as piezoelectric ceramics. Forinstance, the sample holder must be large enough to accommodate the sample andthe buffer in which it is immersed. Some authors simply use a small droplet of sometens of microliters, which covers the sample; others use a small container filledwith several milliliters of buffer. The first approach has the advantage of a smallermass (droplet) added to the piezo, but experiments suffer from evaporation, whichresults in a change in the concentration of solvents. On the other hand, using thecontainer approach, concentrations are kept roughly constant, but a considerablemass must be moved by the piezo scanner, which reduces its resonance frequencyand therefore the range of imaging speeds. The tip holder, also known as liquidcell (Section 1.4.2.1), must be designed to prevent contact between the liquid andthe small piezo that drives movement of the cantilever. Finally, in some AFMs,the piezo tube is protected and covered to prevent wetting in case of liquid spill(Section 1.2.1).

1.2Piezoelectric Scanners

Piezoelectric ceramic transducers are used to accurately position the tip and samplein AFMs. The direct piezoelectric effect consists of the generation of a potentialdifference across the opposite faces of certain nonconductive crystals as a result ofthe application of stress. The reverse piezoelectric effect is also possible becauseof a change in dimensions of the crystal as a consequence of the application of apotential difference between two faces of the piezoelectric material. This method isused to position the tip and sample with respect to each other with subnanometerprecision since piezoelectric ceramic transducers are highly sensitive, stable, andreliable. Regardless, if the tip is moved over the sample or vice versa, the scan inAFM is performed using piezoelectric transducers.

6 1 AFM: Basic Concepts

z

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DNA DNA

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Figure 1.2 Piezoelectric scanners. (a) Piezo-tube architecture based on four sectors.Voltages are applied between opposite sides,and as a consequence, movement of thepiezo is generated. (b) Sequence of volt-ages applied to X (fast scan) and Y (slowscan) to generate an image scan. Each step

in y-axis is associated with the change of animaging line. (c,d)Two typical problems ofpiezotube scanners. (c) The plane where thesample is situated describes an arc ratherthan a straight line. (d) Clear effect of piezohysteresis when imaging DNA molecules.

Many AFMs use piezoelectric tube scanners such as the one shown in Figure 1.2a.They consist of a thin-walled hard piezoelectric ceramic, which is polarized radially[6]. The external face of the tube is divided into four longitudinal segments ofequal size and electrodes are welded to the internal and external faces of the tube.To achieve extension or contraction, a bias voltage is applied between the innerand all the outer electrodes. The scan movement is performed by applying a biasvoltage to one of the segments of the outer wall. To amplify this bending effectby a factor of two, a voltage with opposite sign is applied to the opposite segment.With a correct synchronization of applied voltages (±x and ± y), a sequential scancan be generated (Figure 1.2b). Typically, tube scanners of 10 × 10 μm rangehave sensitivities of ∼40 nm/V. This means that voltages up to ±125 V must begenerated with submilivolts precision to achieve a basal noise of ∼0.01 nm. Thisoperation is performed by the HV electronics.

1.2 Piezoelectric Scanners 7

Tube scanners have some drawbacks. For instance, the plane in which thesample is located describes an arc rather than a straight line. This effect is morepronounced when large areas are scanned and the height of the objects to be imagedis small compared to the scan area. For instance, Figure 1.2c shows a profile of aflat surface with adsorbed DNA molecules. The effect of the arc trajectory describedby the piezo is clearly visible, and the detection of small molecules such as DNA(height < 1 nm) is challenging. This effect can easily be corrected by subtractinga polynomial function to each scan line or to the overall surface. Piezo tubes haverelatively low resonance frequencies, of the order of kilohertz, which limits the scanspeed. Recently, some manufacturers have employed small stacks of piezoelectricceramics to increase the resonance frequency of these devices and therefore theimaging speed. However, stacked piezos have a quite limited scan range.

A different approach used to move the sample is based on stick-slip motion.These positioners rely on the controllable use of the inertia of a sliding block.In brief, a sliding block slips along a guided rod, which is otherwise clampedin frictional engagement. A net step is obtained by first accelerating very rapidlythe guiding rod over a short period (typically microseconds) so that the inertia ofthe sliding block overcomes the friction. The sliding block disengages from theaccelerated rod and remains nearly nondisplaced. Then, the guiding rod movesback to its initial position slowly enough so that the sliding block sticks to it andthus makes a net step. Periodic repetition of this sequence leads to a step-by-stepmotion of the sliding block in one direction. The movement of the guiding rodis performed by a piezoelectric ceramic, which can pull or push as required.Stick-slip positioners have long travel ranges of several millimeters, but theirperformance is dependent on the mass to be moved, which can be significant inliquid imaging. These devices also have the limitation of a relatively large step(few nanometers) and a low resonance frequency (much lower than that of stackedpiezos). Hence the main use of these devices is as nanopositioners rather than as fastscanners.

Piezoelectric scanners are inherently nonlinear, and this nonlinearity becomesquite significant at large scans. Typical piezos suffer from hysteresis in the forwardand backward traces. This effect can be clearly seen in the forward and backwardprofiles shown in Figure 1.2d. Piezo scanners are also subjected to creep afterchanging the polarity (direction) of the scan or just after setting the voltage tozero. This is due to some sort of relaxation, which occurs under constant stress.It has an effect on the images distorting the dimensions of the objects to beimaged. In general, this problem can be solved by repeating the scan, allowing thepiezo to relax. To minimize unwanted motions in the piezoactuator, some AFMsincorporate a combination of piezoactuators and metal springs. These devices haveflexure-guided stages, acting as springs and restricted to move only in one direction.A piezoactuator pulls against the spring, and therefore a forward and backwardmovement of the flexure guide can be achieved by changing the voltage in thepiezoactuator. This combination effectively decouples the unwanted motions in thepiezoactuator and produces a pure linear translation while keeping high resonancefrequencies at relatively high loads (∼2 kHz for a 100 g load). Finally, many AFMs

8 1 AFM: Basic Concepts

have capacitive sensors incorporated in their piezos that allow for measuring theposition independent of the applied voltage. With this feature, a closed-loop circuitcan be designed, being able to cancel any hysteresis, creep, or nonlinearity byapplying additional correction voltages.

1.2.1Piezoelectric Scanners for Imaging in Liquids

In many AFMs, the piezoelectric used to image in air is the same as that usedto image in liquids, but some precautions must be taken. The main concern isrelated to the electrical isolation of the piezo to avoid any shortcut due to wetting.HVs (hundreds of volts) are applied to the piezo, and if any water gets into it, theexpensive piezo tube will almost certainly be destroyed. Therefore, some cautionmust be taken when imaging in liquids to avoid any spill of water into the piezo.In most AFMs, some silicone or rubber is added to prevent any liquid from gettinginto the piezo.

For imaging in liquids, it is often recommended to move the tip relative tothe sample instead of keeping the tip fixed and move the sample. In the latterscenario, volumes of milliliters should be moved at kilohertz frequencies, affectingthe mechanical stability of the piezo. This is equivalent to considering an effectivemass in Eq. (1.3). That will lower the piezo resonant frequency and will slow downthe imaging speed of the AFM. Instead, when moving the tip relative to the samplein liquids, the added effective mass is small because only the tip and parts of the tipholder are immersed in the buffer container. When imaging in environments withlarge viscosity such as liquids, it is important to keep the mass of moving objectsas low as possible. This is also important for oscillating the tip; a need in dynamicmodes. Some users oscillate the complete tip holder, exciting many mechanicalvibrations in the buffer container, which hide the genuine mechanical resonanceof the cantilever (Section 1.5.3.1).

1.3Tips and Cantilevers

In contact mode, to be able to feel the surface with atom resolution, the stiffness ofan AFM cantilever should be much smaller than the spring constant that maintainsthe atoms confined together on the surface. This bonding force constant in acrystalline lattice is of the order of 1 N m−1 [7], meaning that to use the AFM incontact mode (Section 1.5.1), the spring constant of the cantilevers (k) should bemuch smaller than 1 N m−1. To achieve this value of k, a beamlike cantilever madeof silicon or silicon nitride should have micrometer dimensions if one considersthe formula for the spring constant of a cantilever

k = Et3w

4l3(1.1)