Atomic force microscopy for biologists

Atomic force microscopy (AFM) is part of a range of emerging microscopic methods for biologists which offer the magnification range of both the light and electron microscope, but allow imaging under the 'natural' conditions usually associated with the light microscope. To biologists, AFM offers the prospect of high resolution images of biological material, images of molecules and their interactions even under physiological conditions, and the study of molecular processes in living systems. This book provides a realistic appreciation of the advantages and limitations of the technique and the present and future potential for improving the understanding of biological systems.The second edition of this bestseller has been updated to describe the latest developments in this exciting field, including a brand new chapter on force spectroscopy. The dramatic developments of AFM over the past ten years from a simple imaging tool to the multi-faceted, nano-manipulating technique that it is today are conveyed in a lively and informative narrative, which provides essential reading for students and experienced researchers alike.

Acknowledgements	p. xiii
Chapter 1 An Introduction	p. 1
Chapter 2 Apparatus	p. 5
2.1 The atomic force microscope	p. 5
2.2 Piezoelectric scanners	p. 7
2.3 Probes and cantilevers	p. 10
2.3.1 Cantilever geometry	p. 10
2.3.2 Tip shape	p. 12
2.3.3 Tip functionality	p. 14
2.4 Sample holders	p. 14
2.4.1 Liquid cells	p. 15
2.5 Detection methods	p. 16
2.5.1 Optical detectors: laser beam deflection	p. 16
2.5.2 Optical detectors: interferometry	p. 18
2.5.3 Electrical detectors: electron tunnelling	p. 19
2.5.4 Electrical detectors: capacitance	p. 20
2.5.5 Electrical detectors: piezoelectric cantilevers	p. 21
2.6 Control systems	p. 21
2.6.1 AFM electronics	p. 21
2.6.2 Operation of the electronics	p. 24
2.6.3 Feedback control loops	p. 25
2.6.4 Design limitations	p. 27
2.6.5 Enhancing the performance of large scanners	p. 28
2.7 Vibration isolation: thermal and mechanical	p. 28
2.8 Calibration	p. 30
2.8.1 Piezoelectric scanner non-linearity	p. 30
2.8.2 Tip related factors: convolution	p. 31
2.8.3 Calibration standards	p. 32
2.8.4 Tips for scanning a calibration specimen	p. 33
2.9 Integrated AFMs	p. 34
2.9.1 Combined AFM-light microscope (AFM-LM)	p. 34
2.9.2 'Submarine' AFM - the combined AFM-Langmuir Trough	p. 35
2.9.3 Combined AFM-surface plasmon resonance (AFM-SPR)	p. 36
2.9.4 Cryo-AFM	p. 36
Chapter 3 Basic Principles	p. 41
3.1 Forces	p. 41
3.1.1 The Van der Waals force and force-distance curves	p. 41
3.1.2 The electrostatic force	p. 44
3.1.3 Capillary and adhesive forces	p. 44
3.1.4 Double layer forces	p. 46
3.2 Imaging modes	p. 47
3.2.1 Contact dc mode	p. 47
3.2.2 Ac modes: Tapping and non-contact	p. 47
3.2.3 Deflection mode	p. 54
3.3 Image types	p. 55
3.3.1 Topography	p. 55
3.3.2 Frictional force	p. 56
3.3.3 Phase	p. 56
3.4 Substrates	p. 58
3.4.1 Mica	p. 58
3.4.2 Glass	p. 58
3.4.3 Graphite	p. 58
3.5 Common problems	p. 59
3.5.1 Thermal drift	p. 59
3.5.2 Multiple tip effects	p. 59
3.5.3 The 'pool' artifact	p. 61
3.5.4 Optical interference on highly reflective samples	p. 61
3.5.5 Sample roughness	p. 62
3.5.6 Sample mobility	p. 63
3.5.7 Imaging under liquid	p. 64
3.6 Getting started	p. 65
3.6.1 DNA	p. 65
3.6.2 Troublesome large samples	p. 68
3.7 Image optimisation	p. 70
3.7.1 Grey levels and colour tables	p. 70
3.7.2 Brightness and contrast	p. 71
3.7.3 High and low pass filtering	p. 71
3.7.4 Normalisation and plane fitting	p. 71
3.7.5 Despike	p. 71
3.7.6 Fourier filtering	p. 72
3.7.7 Correlation averaging	p. 73
3.7.8 Stereographs and anaglyphs	p. 73
3.7.9 Do your homework!	p. 74
Chapter 4 Macromolecules	p. 76
4.1 Imaging methods	p. 76
4.1.1 Tip adhesion, molecular damage and displacement	p. 76
4.1.2 Depositing macromolecules onto substrates	p. 77
4.1.3 Metal coated samples	p. 78
4.1.4 Imaging in air	p. 79
4.1.5 Imaging under non-aqueous liquids	p. 80
4.1.6 Binding molecules to the substrate	p. 81
4.1.7 Imaging under water or buffers	p. 85
4.2 Nucleic acids: DNA	p. 86
4.2.1 Imaging DNA	p. 87
4.2.2 DNA conformation, size and shape	p. 88
4.2.3 DNA-protein interactions	p. 94
4.2.4 Location and mapping of specific sites	p. 99
4.2.5 Chromosomes	p. 102
4.3 Nucleic acids: RNA	p. 105
4.4 Polysaccharides	p. 106
4.4.1 Imaging polysaccharides	p. 107
4.4.2 Size, shape, structure and conformation	p. 108
4.4.3 Aggregates, networks and gels	p. 117
4.4.4 Cellulose, plant cell walls and starch	p. 122
4.4.5 Proteoglycans and mucins	p. 128
4.5 Proteins	p. 130
4.5.1 Globular proteins	p. 131
4.5.2 Antibodies	p. 136
4.5.3 Fibrous proteins	p. 139
Chapter 5 Interfacial Systems	p. 181
5.1 Introduction to interfaces	p. 181
5.1.1 Surface activity	p. 181
5.1.2 AFM of interracial systems	p. 184
5.1.3 The Langmuir trough	p. 185
5.1.4 Langmuir-Blodgett film transfer	p. 186
5.2 Sample preparation	p. 188
5.2.1 Cleaning protocols: glassware and trough	p. 188
5.2.2 Substrates	p. 189
5.2.3 Performing the dip	p. 191
5.3 Phospholipids	p. 192
5.3.1 Early AFM studies of phospholipid films	p. 193
5.3.2 Modification of phospholipid bilayers with the AFM	p. 194
5.3.3 Studying intrinsic bilayer properties by AFM	p. 196
5.3.4 Ripple phases in phospholipid bilayers	p. 199
5.3.5 Mixed phospholipid films	p. 202
5.3.6 Effect of supporting layers	p. 205
5.3.7 Dynamic processes of phopholipid layers	p. 208
5.4 Liposomes and intact vesicles	p. 211
5.5 Lipid-protein mixed films	p. 213
5.5.1 High resolution studies of phospholipid bilayers	p. 217
5.6 Miscellaneous lipid films/surfactant films	p. 219
5.7 Interfacial protein films	p. 219
5.7.1 Specific precautions	p. 220
5.7.2 AFM studies of interfacial protein films	p. 222
Chapter 6	p. 231
6.1 Three-dimensional crystals	p. 231
6.1.1 Crystalline cellulose	p. 231
6.1.2 Protein crystals	p. 232
6.1.3 Nucleic acid crystals	p. 235
6.1.4 Viruses and virus crystals	p. 236
6.2 Two dimensional protein crystals: an introduction	p. 240
6.2.1 What does AFM have to offer?	p. 241
6.2.2 Sample preparation: membrane proteins	p. 243
6.2.3 Sample preparation: soluble proteins	p. 244
6.3 AFM studies of 2D membrane protein crystals	p. 246
6.3.1 Purple membrane (bacteriorhodopsin)	p. 246
6.3.2 Gap junctions	p. 249
6.3.3 Photosynthelic protein membranes	p. 252
6.3.4 ATPase in kidney membranes	p. 252
6.3.5 OmpF porin	p. 253
6.3.6 Bacterial Slayers	p. 254
6.3.7 Bacteriophage Ø29 head-tail connector	p. 257
6.3.8 AFM imaging of membrane dynamics	p. 259
6.3.9 Force spectroscopy of membrane proteins	p. 261
6.3.10 Gas vesicle protein	p. 261
6.4 AFM studies of 2D crystals of soluble proteins	p. 262
6.4.1 Imaging conditions	p. 264
6.4.2 Electrostatic considerations	p. 266
Chapter 7 Cells, Tissue and Biominerals	p. 276
7.1 Imaging methods	p. 276
7.1.1 Sample preparation	p. 277
7.1.2 Force mapping and mechanical measurements	p. 278
7.2 Microbial cells: bacteria, spores and yeasts	p. 290
7.2.1 Bacteria	p. 290
7.2.2 Yeasts	p. 300
7.3 Blood cells	p. 302
7.3.1 Erythrocytes	p. 302
7.3.2 Leukocytes and lymphocytes	p. 304
7.3.3 Platelets	p. 304
7.4 Neurons and Glial cells	p. 306
7.5 Epithelial cells	p. 307
7.6 Non-confluent renal cells	p. 309
7.7 Endothelial cells	p. 311
7.8 Cardiocytes	p. 313
7.9 Other mammalian cells	p. 314
7.10 Plant cells	p. 317
7.11 Tissue	p. 321
7.11.1 Embedded sections	p. 321
7.11.2 Embedment-free sections	p. 322
7.11.3 Hydrated sections	p. 323
7.11.4 Freeze-fracture replicas	p. 324
7.11.5 Immunolabelling	p. 324
7.12 Biominerals	p. 325
7.12.1 Bone, tendon and cartilage	p. 325
7.12.2 Teeth	p. 327
7.12.3 Shells	p. 328
Chapter 8 Other Probe Microscopes	p. 342
8.1 Overview	p. 342
8.2 Scanning tunnelling microscope (STM)	p. 342
8.3 Scanning near-field optical microscope (SNOM)	p. 345
8.4 Scanning ion conductance microscope (SICM)	p. 347
8.5 Scanning thermal microscope (SThM)	p. 349
8.6 Optical tweezers and the photonic force microscope (PFM)	p. 351
Chapter 9 Force Spectroscopy	p. 356
9.1 Force measurement with the AFM	p. 356
9.2 First steps in force spectroscopy: from raw data to force-distance curves	p. 357
9.2.1 Quantifying cantilever displacement	p. 357
9.2.2 Determining cantilever spring constants	p. 359
9.2.3 Anatomy of a force-distance curve	p. 362
9.3 Pulling methods	p. 364
9.3.1 Intrinsic elastic properties of molecules	p. 364
9.3.2 Molecular recognition force spectroscopy	p. 369
9.3.3 Chemical force microscopy (CFM)	p. 373
9.4	p. 374
9.4.1 Colloidal probe microscopy (CPM)	p. 374
9.4.2 How to make a colloid probe cantilever assembly	p. 377
9.4.3 Deformation and indentation methods	p. 380
9.5 Analysis of force-distance curves	p. 381
9.5.1 Worm-like chain and freely jointed chain models	p. 382
9.5.2 Molecular interactions	p. 384
9.5.3 Deformation analysis	p. 387
9.5.4 Adhesive force at pull-off	p. 388
9.5.5 Elastic indentation depth, ¿, and contact radius, a, during deformation	p. 388
9.5.6 Contact radius at zero load	p. 389
9.5.7 Colloidal forces	p. 389
SPM Books	p. 397
Index	p. 399

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