Table of Contents

Acknowledgements xiii

Chapter 1 An Introduction 1

Chapter 2 Apparatus 5

2.1 The atomic force microscope 5

2.2 Piezoelectric scanners 7

2.3 Probes and cantilevers 10

2.3.1 Cantilever geometry 10

2.3.2 Tip shape 12

2.3.3 Tip functionality 14

2.4 Sample holders 14

2.4.1 Liquid cells 15

2.5 Detection methods 16

2.5.1 Optical detectors: laser beam deflection 16

2.5.2 Optical detectors: interferometry 18

2.5.3 Electrical detectors: electron tunnelling 19

2.5.4 Electrical detectors: capacitance 20

2.5.5 Electrical detectors: piezoelectric cantilevers 21

2.6 Control systems 21

2.6.1 AFM electronics 21

2.6.2 Operation of the electronics 24

2.6.3 Feedback control loops 25

2.6.4 Design limitations 27

2.6.5 Enhancing the performance of large scanners 28

2.7 Vibration isolation: thermal and mechanical 28

2.8 Calibration 30

2.8.1 Piezoelectric scanner non-linearity 30

2.8.2 Tip related factors: convolution 31

2.8.3 Calibration standards 32

2.8.4 Tips for scanning a calibration specimen 33

2.9 Integrated AFMs 34

2.9.1 Combined AFM-light microscope (AFM-LM) 34

2.9.2 'Submarine' AFM - the combined AFM-Langmuir Trough 35

2.9.3 Combined AFM-surface plasmon resonance (AFM-SPR) 36

2.9.4 Cryo-AFM 36

Chapter 3 Basic Principles 41

3.1 Forces 41

3.1.1 The Van der Waals force and force-distance curves 41

3.1.2 The electrostatic force 44

3.1.3 Capillary and adhesive forces 44

3.1.4 Double layer forces 46

3.2 Imaging modes 47

3.2.1 Contact dc mode 47

3.2.2 Ac modes: Tapping and non-contact 47

3.2.3 Deflection mode 54

3.3 Image types 55

3.3.1 Topography 55

3.3.2 Frictional force 56

3.3.3Phase 56

3.4 Substrates 58

3.4.1 Mica 58

3.4.2 Glass 58

3.4.3 Graphite 58

3.5 Common problems 59

3.5.1 Thermal drift 59

3.5.2 Multiple tip effects 59

3.5.3 The 'pool' artifact 61

3.5.4 Optical interference on highly reflective samples 61

3.5.5 Sample roughness 62

3.5.6 Sample mobility 63

3.5.7 Imaging under liquid 64

3.6 Getting started 65

3.6.1 DNA 65

3.6.2 Troublesome large samples 68

3.7 Image optimisation 70

3.7.1 Grey levels and colour tables 70

3.7.2 Brightness and contrast 71

3.7.3 High and low pass filtering 71

3.7.4 Normalisation and plane fitting 71

3.7.5 Despike 71

3.7.6 Fourier filtering 72

3.7.7 Correlation averaging 73

3.7.8 Stereographs and anaglyphs 73

3.7.9 Do your homework! 74

Chapter 4 Macromolecules 76

4.1 Imaging methods 76

4.1.1 Tip adhesion, molecular damage and displacement 76

4.1.2 Depositing macromolecules onto substrates 77

4.1.3 Metal coated samples 78

4.1.4 Imaging in air 79

4.1.5 Imaging under non-aqueous liquids 80

4.1.6 Binding molecules to the substrate 81

4.1.7 Imaging under water or buffers 85

4.2 Nucleic acids: DNA 86

4.2.1 Imaging DNA 87

4.2.2 DNA conformation, size and shape 88

4.2.3 DNA-protein interactions 94

4.2.4 Location and mapping of specific sites 99

4.2.5 Chromosomes 102

4.3 Nucleic acids: RNA 105

4.4 Polysaccharides 106

4.4.1 Imaging polysaccharides 107

4.4.2 Size, shape, structure and conformation 108

4.4.3 Aggregates, networks and gels 117

4.4.4 Cellulose, plant cell walls and starch 122

4.4.5 Proteoglycans and mucins 128

4.5 Proteins 130

4.5.1 Globular proteins 131

4.5.2 Antibodies 136

4.5.3 Fibrous proteins 139

Chapter 5 Interfacial Systems 181

5.1 Introduction to interfaces 181

5.1.1 Surface activity 181

5.1.2 AFM of interracial systems 184

5.1.3 The Langmuir trough 185

5.1.4 Langmuir-Blodgett film transfer 186

5.2 Sample preparation 188

5.2.1 Cleaning protocols: glassware and trough 188

5.2.2 Substrates 189

5.2.3 Performing the dip 191

5.3 Phospholipids 192

5.3.1 Early AFM studies of phospholipid films 193

5.3.2 Modification of phospholipid bilayers with the AFM 194

5.3.3 Studying intrinsic bilayer properties by AFM 196

5.3.4 Ripple phases in phospholipid bilayers 199

5.3.5 Mixed phospholipid films 202

5.3.6 Effect of supporting layers 205

5.3.7 Dynamic processes of phopholipid layers 208

5.4 Liposomes and intact vesicles 211

5.5 Lipid-protein mixed films 213

5.5.1 High resolution studies of phospholipid bilayers 217

5.6 Miscellaneous lipid films/surfactant films 219

5.7 Interfacial protein films 219

5.7.1 Specific precautions 220

5.7.2 AFM studies of interfacial protein films 222

Chapter 6 Ordered Macromolecules 231

6.1 Three-dimensional crystals 231

6.1.1 Crystalline cellulose 231

6.1.2 Protein crystals 232

6.1.3 Nucleic acid crystals 235

6.1.4 Viruses and virus crystals 236

6.2 Two dimensional protein crystals: an introduction 240

6.2.1 What does AFM have to offer? 241

6.2.2 Sample preparation: membrane proteins 243

6.2.3 Sample preparation: soluble proteins 244

6.3 AFM studies of 2D membrane protein crystals 246

6.3.1 Purple membrane (bacteriorhodopsin) 246

6.3.2 Gap junctions 249

6.3.3 Photosynthelic protein membranes 252

6.3.4 ATPase in kidney membranes 252

6.3.5 OmpF porin 253

6.3.6 Bacterial Slayers 254

6.3.7 Bacteriophage Ø29 head-tail connector 257

6.3.8 AFM imaging of membrane dynamics 259

6.3.9 Force spectroscopy of membrane proteins 261

6.3.10 Gas vesicle protein 261

6.4 AFM studies of 2D crystals of soluble proteins 262

6.4.1 Imaging conditions 264

6.4.2 Electrostatic considerations 266

Chapter 7 Cells, Tissue and Biominerals 276

7.1 Imaging methods 276

7.1.1 Sample preparation 277

7.1.2 Force mapping and mechanical measurements 278

7.2 Microbial cells: bacteria, spores and yeasts 290

7.2.1 Bacteria 290

7.2.2 Yeasts 300

7.3 Blood cells 302

7.3.1 Erythrocytes 302

7.3.2 Leukocytes and lymphocytes 304

7.3.3 Platelets 304

7.4 Neurons and Glial cells 306

7.5 Epithelial cells 307

7.6 Non-confluent renal cells 309

7.7 Endothelial cells 311

7.8 Cardiocytes 313

7.9 Other mammalian cells 314

7.10 Plant cells 317

7.11 Tissue 321

7.11.1 Embedded sections 321

7.11.2 Embedment-free sections 322

7.11.3 Hydrated sections 323

7.11.4 Freeze-fracture replicas 324

7.11.5 Immunolabelling 324

7.12 Biominerals 325

7.12.1 Bone, tendon and cartilage 325

7.12.2 Teeth 327

7.12.3 Shells 328

Chapter 8 Other Probe Microscopes 342

8.1 Overview 342

8.2 Scanning tunnelling microscope (STM) 342

8.3 Scanning near-field optical microscope (SNOM) 345

8.4 Scanning ion conductance microscope (SICM) 347

8.5 Scanning thermal microscope (SThM) 349

8.6 Optical tweezers and the photonic force microscope (PFM) 351

Chapter 9 Force Spectroscopy 356

9.1 Force measurement with the AFM 356

9.2 First steps in force spectroscopy: from raw data to force-distance curves 357

9.2.1 Quantifying cantilever displacement 357

9.2.2 Determining cantilever spring constants 359

9.2.3 Anatomy of a force-distance curve 362

9.3 Pulling methods 364

9.3.1 Intrinsic elastic properties of molecules 364

9.3.2 Molecular recognition force spectroscopy 369

9.3.3 Chemical force microscopy (CFM) 373

9.4 Pushing methods 374

9.4.1 Colloidal probe microscopy (CPM) 374

9.4.2 How to make a colloid probe cantilever assembly 377

9.4.3 Deformation and indentation methods 380

9.5 Analysis of force-distance curves 381

9.5.1 Worm-like chain and freely jointed chain models 382

9.5.2 Molecular interactions 384

9.5.3 Deformation analysis 387

9.5.4 Adhesive force at pull-off 388

9.5.5 Elastic indentation depth, δ, and contact radius, a, during deformation 388

9.5.6 Contact radius at zero load 389

9.5.7 Colloidal forces 389

SPM Books 397

Index 399