Biosystems Investigated by Scanning Probe Microscopy (Berlin; Heidelberg, 2010). - ОГЛАВЛЕНИЕ / CONTENTS
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ОбложкаBiosystems Investigated by Scanning Probe Microscopy / ed. by H.Fuchs, B.Bhushan. - Berlin; Heidelberg: Springer, 2010. - xxxii, 834 p. - Incl. bibl. ref. - ISBN 978-3-642-02404-7
 

Оглавление / Contents
 
Part I
Instrumentation and Methods

Scanning Probe Microscopy in Biological Research ................ 3
Tatsuo Ushiki, Kazushige Kawabata
Introduction .................................................... 3
SPM for Visualization of the Surface of Biomaterials ............ 4
   Advantages of AFM in Biological Studies ...................... 4
   AFM of Biomolecules .......................................... 5
   AFM of Isolated Intracellular and Extracellular Structures ... 7
   AFM of Tissue Sections ...................................... 10
   AFM of Living Cells and Their Movement ...................... 10
   Combination of AFM with Scanning Near-Field Optical
   Microscopy for Imaging Biomaterials ......................... 12
SPM for Measuring Physical Properties of Biomaterials .......... 14
   Evaluation Methods of Viscoelasticity ....................... 14
   Examples for Viscoelasticity Mapping Measurements ........... 17
   Combination of Viscoelasticity Measurement with Other 
   Techniques .................................................. 20
SPM as a Manipulation Tool in Biology .......................... 22
Conclusion ..................................................... 24
References ..................................................... 24

Scanning Probes for the Life Sciences .......................... 27
Andrea M. Ho, Horacio D. Espinosa
Introduction ................................................... 27
Microarray Technology .......................................... 28
   Microcontact Printing ....................................... 29
   Optical Lithography ......................................... 30
   Protein Arrays .............................................. 32
Nanoarray Technology ........................................... 33
   The Push for Nanoscale Detection ............................ 33
   Probe-Based Patterning ...................................... 35
   Alternative Patterning Methods .............................. 46
Nanoscale Deposition Mechanisms ................................ 48
AFM Parallelization ............................................ 51
   One-Dimensional Arrays ...................................... 52
   Two-Dimensional Arrays ...................................... 53
Future Prospects for Nanoprobes ................................ 56
References ..................................................... 58

New AFM Developments to Study Elasticity and Adhesion
at the Nanoscale ............................................... 63
Robert Szoszkiewicz, Elisa Riedo
Introduction ................................................... 64
Contact Mechanics Theories and Their Limitations ............... 65
Modulated Nanoindentation ...................................... 67
   Force-Indentation Curves .................................... 67
   Elastic Moduli .............................................. 70
Ultrasonic Methods at Local Scales ............................. 72
   Brief Description of Ultrasonic Methods ..................... 72
   Applications of Ultrasonic Techniques in Elasticity
   Mapping ..................................................... 75
   UFM Measurements of Adhesion Hysteresis and Their 
   Relations to Friction at the Tip-Sample Contact ............. 76
References ..................................................... 78

Application of SPM and Related Techniques to the Mechanical
Properties of Biotool Materials ................................ 81
Thomas Schöberl, Ingomar L. Jäger, Helga С. Lichtenegger
Introduction ................................................... 82
Typical Biotool Materials ...................................... 84
   Chemistry ................................................... 84
   Structures .................................................. 86
   Mechanical Properties ....................................... 88
Experimental Methods and Setups ................................ 89
   SPM and Indentation ......................................... 89
   Scratch and Wear Tests ...................................... 91
   Dynamic Modes ............................................... 92
   Fracture Toughness Tests .................................... 93
Samples ........................................................ 94
   Choice ...................................................... 94
   Storage ..................................................... 95
   Preparation ................................................. 96
Experimental Conditions ........................................ 97
   Moisture .................................................... 97
   Temperature ................................................. 98
   Probe Tips .................................................. 98
   Test Velocity ............................................... 99
Results ........................................................ 99
   Sources of Error ............................................ 99
   Interpretation ............................................. 104
Examples from the Literature .................................. 105
References .................................................... 108

Direct Force Measurements of Receptor-Ligand Interactions
on Living Cells ............................................... 115
Robert H. Eibl
Introduction .................................................. 116
Procedure ..................................................... 122
   Principle of AFM Force Spectroscopy ........................ 123
   Cell-Cell Interactions ..................................... 123
   Cell-Substrate Measurements ................................ 126
   Specificity and Blocking Antibodies ........................ 128
   Activation by SDF-1 ........................................ 131
Protocols ..................................................... 134
   Cantilever Functionalization ............................... 134
   AFM Measurement on Living Cells ............................ 136
   Inhibition with Blocking Antibodies, Peptidomimetic
   Inhibitors or EDTA ......................................... 139
   Activation with Mg2+, Mn2+ Ions, Activating Antibodies,
   Phorbolester or Chemokines ................................. 140
   AFM Measurement-Cell Free .................................. 140
Conclusion and Future Developments ............................ 141
References .................................................... 143

Self-Sensing Cantilever Sensor for Bioscience ................. 147
Hayato Sone, Sumio Hosaka
Introduction .................................................. 147
Basics of the Cantilever Mass Sensor .......................... 148
Finite Element Method Simulation of the Cantilever 
Vibration ..................................................... 151
Detection of Cantilever Deflection ............................ 154
   Using a Position Sensor .................................... 154
   Using a Piezoresistive Sensor .............................. 155
Self-Sensing Systems .......................................... 160
   Vibration Systems .......................................... 160
   Vibration-Frequency Detection Systems ...................... 160
Applications .................................................. 161
   Water Molecule Detection in Air ............................ 161
   Antigen and Antibody Detection in Water .................... 166
Prospective Applications ...................................... 172
References .................................................... 172

Microfabricated Cantilever Array Sensors for (Bio-)Chemical
Detection ..................................................... 175
Hans Peter Lang, Martin Hegner, Christoph Gerber
Introduction .................................................. 175
   Sensors .................................................... 175
   Cantilevers ................................................ 176
   Cantilever Operating Modes ................................. 178
   Cantilever Arrays .......................................... 184
Experimental Setup ............................................ 188
   Measurement Chamber ........................................ 188
   Cantilever Functionalization ............................... 190
Measurements .................................................. 195
   Artificial Nose for Detection of Perfume Essences .......... 196
   Label-Free DNA Hybridization Detection ..................... 198
Applications and Outlook ...................................... 201
References .................................................... 202

Nanomechanics and Microfluidics as a Tool for Unraveling
Blood Clotting Disease ........................................ 207
D.M. Steppich, S. Thalhammer, A. Wixforth, M.F. Schneider
Introduction .................................................. 207
Topography .................................................... 208
   Little Story of Blood Clotting ............................. 209
   High-Resolution Imaging .................................... 212
Lab-on-a-Chip ................................................. 219
   Nanomechanical Diagnostics ................................. 220
   Mimicking Blood Flow Conditions on a Surface Acoustic
   Wave-Driven Biochip ........................................ 222
The Lab on a Chip - AFM - Hybrid .............................. 224
   Experimental Setup ......................................... 224
   Bundle Relaxation .......................................... 226
   Stream Line Manipulation and Flow Sensoring ................ 230
Summary and Outlook ........................................... 234
References .................................................... 235

Quantitative Nanomechanical Measurements in Biology ........... 239
Malgorzata Lekka, Andrzej J. Kulik
Stiffness of Biological Samples ............................... 239
   Cell Structure ............................................. 239
   Determination of Young's Modulus ........................... 242
   Brief Overview of the Application of AFM to Studies of
   Living Cells ............................................... 251
   Summary .................................................... 256
Friction Force Microscopy ..................................... 258
   Friction and Chemical Force Microscopy ..................... 259
   Applications of FFM/CFM .................................... 263
   Summary .................................................... 270
References .................................................... 271

Applications of Scanning Near-Field Optical Microscopy
in Life Science ............................................... 275
Pietro Giuseppe Gucciardi
Introduction .................................................. 276
Experimental Techniques in Near-Field Optical Microscopy ...... 277
   Principles of Near-Field Optical Microscopy ................ 277
   Fluorescence Near-Field Optical Microscopy ................. 279
   Near-Field Optical Microscopy in Liquid .................... 281
   Tip-Enhanced Near-Field Optical Microscopy ................. 283
Applications of Near-Field Optical Microscopy in Life 
Science ....................................................... 284
   Infrared Imaging of Tobacco Mosaic Virus with Nanoscale
   Resolution ................................................. 284
   Co-Localization of Malarial and Host Skeletal Proteins in
   Infected Erythrocytes by Dual-Color Near-Field 
   Fluorescence Microscopy .................................... 285
   Co-Localization of a-Sarcoglycan and ВШ-Integrin in Human
   Muscle Cells by Near-Field Fluorescence Microscopy ......... 287
   Single Molecule Near-Field Fluorescence Microscopy of
   Dendritic Cells ............................................ 288
   Chemical Information of Bacterial Surfaces and Detection 
   of DNA Nucleobases by Tip-Enhanced Raman Spectroscopy ...... 290
Conclusions ................................................... 291
References .................................................... 292

Scanning Ion Conductance Microscopy ........................... 295
Tilman E. Schäffer, Boris Anczykowski, Harold Fuchs
Introduction .................................................. 296
Fundamental Principles ........................................ 296
   Basic Setup ................................................ 296
   Nanopipettes ............................................... 299
   Electrodes ................................................. 300
Ion Currents Through Nanopipettes ............................. 301
   Background Theory .......................................... 301
   Simple Analytical Model .................................... 301
   Finite Element Modeling .................................... 303
   Experimental Current-Distance Curves ....................... 305
   Imaging with Ion Current Feedback .......................... 306
Advanced Techniques ........................................... 307
   Modulation Methods ......................................... 308
   Applications in Bioscience ................................. 310
Combination with Other Scanning Techniques .................... 311
   Combination with Atomic Force Microscopy ................... 312
   Application in Material Science ............................ 312
   Combination with Shear Force Microscopy .................... 315
   Application in Bioscience .................................. 318
Outlook ....................................................... 319
References .................................................... 320

Scanning Probe Lithography for Chemical, Biological and
Engineering Applications ...................................... 325
Joseph M. Kinsella, Albena Ivanisevic
Introduction .................................................. 326
Modeling of the DPN Process ................................... 328
Patterning of Biological and Biologically Active Molecules .... 331
   DNA Patterning ............................................. 332
   Protein Patterning ......................................... 334
   Peptide Patterning ......................................... 337
   Patterning of Templates for Biological Bottom-Up
   Assembly ................................................... 339
Chemical Patterning ........................................... 341
   Thiols ..................................................... 341
   ω-Substituted Thiols ....................................... 342
   Silanes and Silazanes ...................................... 343
   Deposition of Solid Organic Inks ........................... 344
   Polymers ................................................... 345
   Polyelectrolytes ........................................... 347
   Dendrimers ................................................. 347
   Deposition of Supramolecular Materials ..................... 348
   Deposition of Metals ....................................... 349
   Deposition of Solid-State Materials ........................ 350
   Deposition of Magnetic Materials ........................... 351
Engineering Applications of DPN ............................... 352
Future Challenges and Applications ............................ 354
Conclusions ................................................... 355
References .................................................... 355

Scanning Probe Microscopy: From Living Cells to the
Subatomic Range ............................................... 359
Ille C. Gebeshuber, Manfred Drack, Friedrich Aumayr,
Hannspeter Winter, Friedrich Franek Introduction .............. 359
Cells In Vivo as Exemplified by Diatoms ....................... 360
   Introduction to Diatoms ....................................
Interaction of Large Organic Molecules ........................ 365
Nanodefects on Atomically Flat Surfaces ....................... 369
   Ion Bombardment of Highly Oriented Pyrolytic Graphite
   (HOPG) ..................................................... 370
   Bombardment of Single Crystal Insulators with
   Multicharged Ions .......................................... 374
Subatomic Features ............................................ 377
   Atom Orbitals .............................................. 377
   Single Electron Spin Detection with AFM and STM ............ 379
Conclusions and Outlook ....................................... 382
References .................................................... 383

Part II
AFM of Biomolecules

Atomic Force Microscopy of DNA Structure and Interactions ..... 389
Neil H. Thomson
Introduction: The Single-Molecule, Bottom-Up Approach ......... 389
DNA Structure and Function .................................... 391
The Atomic Force Microscope ................................... 393
Binding of DNA to Support Surfaces ............................ 399
   Properties of Support Surfaces for Biological AFM .......... 399
   DNA Binding to Surfaces .................................... 400
   DNA Transport to Surfaces .................................. 404
AFM of DNA Systems ............................................ 405
   Static Imaging versus Dynamic Studies ...................... 405
   The Race for Reproducible Imaging of Static DNA ............ 406
   Applications of Tapping-Mode AFM to DNA Systems ............ 408
Outlook ....................................................... 419
References .................................................... 421

Nanostructuration and Nanoimaging of Biomolecules for
Biosensors .................................................... 427
Claude Martelet, Nicole Jaffrezic-Renault, Yanxia Hou,
Abdelhamid Errachid, Frangois Bessueille
Introduction and Definition of Biosensors ..................... 427
   Definition ................................................. 427
   Biosensor Components ....................................... 427
   Immobilization of the Bioreceptor .......................... 428
Langmuir-Blodgett and Self-Assembled Monolayers as
Immobilization Techniques ..................................... 429
   Langmuir-Blodgett Technique ................................ 429
   Self-Assembled Monolayers .................................. 438
   Characterization of SAMs and LB Films ...................... 450
Prospects and Conclusion ...................................... 455
References .................................................... 457

Part III
AFM of Biological Membranes, Cells and Tissue

Imaging Chemical Groups and Molecular Recognition Sites
on Live Cells Using AFM ....................................... 463
David Alsteens, Vincent Dupres, Etienne Dague, Claire
Verbelen, Guillaume André, Grégory Francius, Yves 
F. Dufrêne
Introduction .................................................. 463
Chemical Force Microscopy ..................................... 464
   Methods .................................................... 464
   Probing Hydrophobic Forces ................................. 465
   Chemical Force Microscopy of Live Cells .................... 468
Molecular Recognition Imaging ................................. 472
   Spatially Resolved Force Spectroscopy ...................... 473
   Immunogold Imaging ......................................... 475
Conclusions ................................................... 477
References .................................................... 477

Single-Molecule Studies on Cells and Membranes Using the
Atomic Force Microscope ....................................... 479
Ferry Kienberger, Lilia A. Chtcheglova, Andreas Ebner, 
Theeraporn Puntheeranurak, Hermann J. Gruber, Peter 
Hinterdorfer
Introduction .................................................. 480
Principles of Atomic Force Microscopy ......................... 481
Imaging of Membrane-Protein Complexes ......................... 482
   Membranes of Photosynthetic Bacteria and Bacterial
   S-Layers ................................................... 482
   Nuclear Pore Complexes ..................................... 484
   Cell Membranes with Attached Viral Particles ............... 484
Single-Molecule Recognition on Cells and Membranes ............ 488
   Principles of Recognition Force Measurements ............... 488
   Force-Spectroscopy Measurements on Living Cells ............ 491
Unfolding and Refolding of Single-Membrane Proteins ........... 495
Simultaneous Topography and Recognition Imaging on Cells
(TREC) ........................................................ 497
Concluding Remarks ............................................ 500
References .................................................... 501

Atomic Force Microscopy: Interaction Forces Measured in
Phospholipid Monolayers, Bilayers, and Cell Membranes ......... 505
Zoya Leonenko, David Cramb, Matthias Amrein, Eric Finot
Introduction .................................................. 505
Phase Transitions of Lipid Bilayers in Water .................. 507
   Morphology Change During Lamellar Phase Transition ......... 508
   Change in Forces During Phase Transition ................... 510
Force Measurements on Pulmonary Surfactant Monolayers in
Air ........................................................... 517
   Adhesion Measurements: Monolayer Stiffness and Function .... 519
   Repulsive Forces: The Interaction of Charged Airborne 
   Particles with Surfactant .................................. 520
Interaction Forces Measured on Lung Epithelial Cells in
Buffer ........................................................ 522
   Cell Culture/Force Measurement Setup ....................... 523
   Mechanical Properties ...................................... 525
Conclusions ................................................... 528
References .................................................... 529

Atomic Force Microscopy Studies of the Mechanical Properties
of Living Cells ............................................... 533
Félix Rico, Ewa P. Wojcikiewicz, Vincent T. Moy
Introduction .................................................. 533
Principle of Operation ........................................ 534
   AFM Imaging ................................................ 536
   Force Measurements ......................................... 536
Cell Viscoelasticity .......................................... 537
   AFM Tip Geometries ......................................... 538
   Elasticity: Young's Modulus ................................ 538
   Viscoelasticity: Complex Shear Modulus ..................... 540
   Cell Adhesion .............................................. 542
Concluding Remarks and Future Directions ...................... 548
References .................................................... 549

Application of Atomic Force Microscopy to the Study of
Expressed Molecules in or on a Single Living Cell ............. 555
Hyonchol Kim, Hironori Uehara, Rehana Afrin, Hiroshi 
Sekiguchi, Hideo Arakawa, Toshiya Osada, Atsushi Ikai
Introduction .................................................. 556
Methods of Manipulation To Study Molecules in or on a Living 
Cell Using an AFM ............................................. 557
   AFM Tip Preparation To Manipulate Receptors on a Cell 
   Surface .................................................... 557
   Analysis of Molecular Interactions Where Multiple Bonds
   Formed ..................................................... 559
   Measurement of Single-Molecule Interaction Strength on
   Soft Materials ............................................. 561
Observation of the Distribution of Specific Receptors on 
a Living Cell Surface ......................................... 562
   Distribution of Fibronectin Receptors on a Living
   Fibroblast Cell ............................................ 562
   Distribution of Vitronectin Receptors on a Living
   Osteoblast Cell ............................................ 565
   Quantification of the Number of Prostaglandin Receptors
   on a Chinese Hamster Ovary Cell Surface .................... 567
Further Application of the AFM to the Study of Single-Cell
Biology ....................................................... 570
   Manipulation of Expressed mRNAs in a Living Cell Using an
   AFM ........................................................ 570
   Manipulation of Membrane Receptors on a Living Cell 
   Surface Using an AFM ....................................... 576
References .................................................... 579

Towards a Nanoscale View of Microbial Surfaces Using the
Atomic Force Microscope ....................................... 583
Claire Verbelen, Guillaume Andre, Xavier Haulot, Yann 
Gilbert, David Alsteens, Etienne Dague, Yves F. Dufrêne
Introduction .................................................. 583
Imaging ....................................................... 584
   Sample Preparation ......................................... 584
   Visualizing Membrane Proteins at Subnanometer Resolution ... 584
   Live-Cell Imaging .......................................... 585
Force Spectroscopy ............................................ 588
   Customized Tips ............................................ 588
   Probing Nanoscale Elasticity and Surface Properties ........ 589
   Stretching Cell Surface Polysaccharides and Proteins ....... 591
   Nanoscale Mapping and Functional Analysis of Molecular
   Recognition Sites .......................................... 592
Conclusions ................................................... 595
References .................................................... 596

Cellular Physiology of Epithelium and Endothelium ............. 599
Christoph Riethmüller, Hans Oberleithner
Introduction .................................................. 599
Epithelium .................................................... 600
   Transport Through a Septum ................................. 600
   In the Kidney .............................................. 602
Endothelium ................................................... 608
   Paracellular Gaps .......................................... 609
   Cellular Drinking .......................................... 611
   Wound Healing .............................................. 614
   Transmigration of Leukocytes ............................... 615
Technical Remarks ............................................. 616
Summary ....................................................... 617
References .................................................... 617

Nanotribological Characterization of Human Hair and Skin
Using Atomic Force Microscopy (AFM) ........................... 621
Bharat Bhushan, Carmen LaTorre
Introduction................................................... 621
Human Hair, Skin, and Hair Care Products ...................... 625
   Human Hair and Skin ........................................ 625
   Hair Care: Cleaning and Conditioning Treatments, and
   Damaging Processes ......................................... 632
Experimental Techniques ....................................... 637
   Experimental Procedure ..................................... 639
   Hair and Skin Samples ...................................... 643
Results and Discussion ........................................ 645
   Surface Roughness, Friction, and Adhesion for Various
   Ethnicities of Hair ........................................ 645
   Surface Roughness, Friction, and Adhesion for Virgin and
   Chemically Damaged Caucasian Hair (with and without
   Commercial Conditioner Treatment ........................... 656
   Surface Roughness, Friction, and Adhesion for Hair
   Treated with Various Combinations of Conditioner
   Ingredients ................................................ 664
   Investigation of Directionality Dependence and Scale
   Effects on Friction and Adhesion of Hair ................... 671
   Surface Roughness and Friction of Skin ..................... 684
Closure ....................................................... 684
References .................................................... 688
Appendix ...................................................... 689

Evaluating Tribological Properties of Materials for
Total Joint Replacements Using Scanning Probe Microscopy ...... 691
Sriram Sundararajan, Kanaga Karuppiah Kanaga Subramanian
Introduction .................................................. 691
   Total Joint Replacements ................................... 691
   Social and Economic Significance ........................... 692
Problems Associated with Total Joint Replacements ............. 692
   Tribology .................................................. 694
   Materials .................................................. 694
   Lubrication in Joints-the Synovial Fluid ................... 695
Conventional Tribological Testing of Material Pairs for
Total Joint Replacements ...................................... 696
   Wear Tests ................................................. 696
   Friction Tests ............................................. 696
Scanning Probe Microscopy as a Tool to Study Tribology of
Total Joint Replacements ...................................... 696
   Nanotribology of Ultrahigh Molecular Weight Polyethylene ... 697
   Fretting Wear of Cobalt-Chromium Alloy ..................... 705
Summary and Future Outlook .................................... 709
References .................................................... 710

Atomic Force Microscopy in Nanomedicine ....................... 713
Dessy Nikova, Tobias Lange, Hans Oberleithner, Hermann 
Schillers, Andreas Ebner, Peter Hinterdorfer
AFM in Biological Sciences .................................... 713
Plasma Membrane Preparation for AFM Imaging ................... 716
   Introduction ............................................... 716
   Plasma Membrane Preparation ................................ 717
   Atomic Force Microscopy .................................... 719

Part IV
Functional Bio(-inspired) Surfaces

Lotus Effect: Roughness-Induced Superhydrophobicity ........... 741
Michael Nosonovsky, Bharat Bhushan
Introduction .................................................. 741
Contact Angle Analysis ........................................ 744
   Homogeneous Solid-Liquid Interface ......................... 745
   Composite Solid-Liquid-Air Interface ....................... 748
   Stability of the Composite Interface ....................... 751
Calculation of the Contact Angle for Selected Rough Surfaces
and Surface Optimization ...................................... 759
   Two-Dimensional Periodic Profiles .......................... 760
   Three-Dimensional Surfaces ................................. 763
   Surface Optimization for Maximum Contact Angle ............. 769
Meniscus Force ................................................ 771
   Sphere in Contact with a Smooth Surface .................... 771
   Multiple-Asperity Contact .................................. 773
Experimental Data ............................................. 774
Closure ....................................................... 777
References .................................................... 778

Gecko Feet: Natural Attachment Systems for Smart Adhesion-
Mechanism, Modeling, and Development of Bio-Inspired 
Materials ..................................................... 781
Bharat Bhushan, Robert A. Sayer
Introduction .................................................. 781
Tokay Gecko ................................................... 782
   Construction of Tokay Gecko ................................ 782
   Other Attachment Systems ................................... 785
   Adaptation to Surface Roughness ............................ 787
   Peeling .................................................... 788
   Self-Cleaning .............................................. 790
Attachment Mechanisms ......................................... 792
   Van der Waals Forces ....................................... 792
   Capillary Forces ........................................... 793
Experimental Adhesion Test Techniques and Data ................ 794
   Adhesion Under Ambient Conditions .......................... 795
   Effects of Temperature ..................................... 797
   Effects of Humidity ........................................ 798
   Effects of Hydrophobicity .................................. 798
Adhesion Modeling ............................................. 799
   Spring Model ............................................... 801
   Single Spring Contact Analysis ............................. 801
   The Multilevel Hierarchical Spring Analysis ................ 803
   Adhesion Results for the Gecko Attachment System 
   Contacting a Rough Surface ................................. 806
   Capillarity Effects ........................................ 810
   Adhesion Results that Account for Capillarity Effects ...... 811
Modeling of Biomimetic Fibrillar Structures ................... 814
   Fiber Model ................................................ 814
   Single Fiber Contact Analysis .............................. 814
   Constraints ................................................ 815
   Numerical Simulation ....................................... 819
   Results and Discussion ..................................... 821
Fabrication of Biomimetric Gecko Skin ......................... 828
   Single-Level Hierarchical Structures ....................... 829
   Multilevel Hierarchical Structures ......................... 833
Closure ....................................................... 835
Appendix ...................................................... 836
References .................................................... 839


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