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