1. Introduction ................................................. 1
1.1. Imaging Capabilities .................................... 2
1.2. Structure Analysis ..................................... 10
1.3. Elemental Analysis ..................................... 10
1.4. Summary and Outline of This Book ....................... 17
Appendix A. Overview of Scanning Electron Microscopy ........ 18
Appendix B. Overview of Electron Probe X-Ray
Microanalysis ................................... 19
References .................................................. 20
2. The SEM and Its Modes of Operation .......................... 21
2.1. How the SEM Works ......................................... 21
2.1.1. Functions of the SEM Subsystems .................... 21
2.1.1.1. Electron Gun and Eenses Produce
a Small Electron Beam ..................... 22
2.1.1.2. Deflection System Controls
Magnification ............................. 22
2.1.1.3. Electron Detector Collects the
Signal .................................... 24
2.1.1.4. Camera or Computer Records the
Image ..................................... 25
2.1.1.5. Operator Controls ......................... 25
2.1.2. SEM Imaging Modes .................................. 25
2.1.2.1. Resolution Mode ........................... 27
2.1.2.2. High-Current Mode ......................... 27
2.1.2.3. Depth-of-Focus Mode ....................... 28
2.1.2.4. Low-Voltage Mode .......................... 29
2.1.3. Why Learn about Electron Optics? ................... 29
2.2. Electron Guns ............................................. 29
2.2.1. Tungsten Hairpin Electron Guns ..................... 30
2.2.1.1. Filament .................................. 30
2.2.1.2. Grid Cap .................................. 31
2.2.1.3. Anode ..................................... 31
2.2.1.4. Emission Current and Beam Current.......... 32
2.2.1.5. Operator Control of the Electron
Gun ....................................... 32
2.2.2. Electron Gun Characteristics ....................... 33
2.2.2.1. Electron Emission Current ................. 33
2.2.2.2. Brightness ................................ 33
2.2.2.3. Lifetime .................................. 34
2.2.2.4. Source Size, Energy Spread,
Beam Stability ............................ 34
2.2.2.5. Improved Electron Gun
Characteristics ........................... 34
2.2.3. Lanthanum Hexaboride (LaB6) Electron Guns .......... 35
2.2.3.1. Introduction .............................. 35
2.2.3.2. Operation of the LaB6 Source .............. 36
2.2.4. Field Emission Electron Guns ....................... 37
2.3. Electron Lenses ........................................... 40
2.3.1. Making the Beam Smaller ............................ 40
2.3.1.1. Electron Focusing ......................... 40
2.3.1.2. Demagnification of the Beam ............... 41
2.3.2. Lenses in SEMs ..................................... 42
2.3.2.1. Condenser Lenses .......................... 42
2.3.2.2. Objective Lenses .......................... 42
2.3.2.3. Real and Virtual Objective
Apertures ................................. 44
2.3.3. Operator Control of SEM Lenses ..................... 44
2.3.3.1. Effect of Aperture Size ................... 45
2.3.3.2. Effect of Working Distance ................ 45
2.3.3.3. Effect of Condenser Lens Strength ......... 46
2.3.4. Gaussian Probe Diameter ............................ 47
2.3.5. Lens Aberrations ................................... 48
2.3.5.1. Spherical Aberration ...................... 48
2.3.5.2. Aperture Diffraction ...................... 49
2.3.5.3. Chromatic Aberration ...................... 50
2.3.5.4. Astigmatism ............................... 51
2.3.5.5. Aberrations in the Objective Lens ......... 53
2.4. Electron Probe Diameter versus Electron Probe
Current ................................................... 54
2.4.1. Calculation of dmin and imax ........................ 54
2.4.1.1. Minimum Probe Size ........................ 54
2.4.1.2. Minimum Probe Size at 10-30 kV ............ 54
2.4.1.3. Maximum Probe Current at 10-30 kV ......... 55
2.4.1.4. Low-Voltage Operation ..................... 55
2.4.1.5. Graphical Summary ......................... 56
2.4.2. Performance in the SEM Modes ....................... 56
2.4.2.1. Resolution Mode ........................... 56
2.4.2.2. High-Current Mode ......................... 58
2.4.2.3. Depth-of-Focus Mode ....................... 59
2.4.2.4. Low-Voltage SEM ........................... 59
2.4.2.5. Environmental Barriers to
High-Resolution Imaging ................... 59
References ................................................ 60
3. Electron Beam-Specimen Interactions ......................... 61
3.1. The Story So Far .......................................... 61
3.2. The Beam Enters the Specimen .............................. 61
3.3. The Interaction Volume .................................... 65
3.3.1. Visualizing the Interaction Volume ................. 65
3.3.2. Simulating the Interaction Volume .................. 67
3.3.3. Influence of Beam and Specimen Parameters on
the Interaction Volume ............................. 68
3.3.3.1. Influence of Beam Energy on the
Interaction Volume ........................ 68
3.3.3.2. Influence of Atomic Number on the
Interaction Volume ........................ 69
3.3.3.3. Influence of Specimen Surface Tilt
on the Interaction Volume ................. 71
3.3.4. Electron Range: A Simple Measure of the
Interaction Volume ................................. 72
3.3.4.1. Introduction .............................. 72
3.3.4.2. The Electron Range at Low Beam
Energy .................................... 73
3.4. Imaging Signals from the Interaction Volume ............... 75
3.4.1. Backscattered Electrons ............................ 75
3.4.1.1. Atomic Number Dependence of BSE ........... 75
3.4.1.2. Beam Energy Dependence of BSE ............. 77
3.4.1.3. Tilt Dependence of BSE .................... 79
3.4.1.4. Angular Distribution of BSE ............... 80
3.4.1.5. Energy Distribution of BSE ................ 82
3.4.1.6. Lateral Spatial Distribution of BSE ....... 84
3.4.1.7. Sampling Depth of BSE ..................... 86
3.4.2. Secondary Electrons ................................ 88
3.4.2.1. Definition and Origin of SE ............... 88
3.4.2.2. SE Yield with Primary Beam Energy ......... 89
3.4.2.3. SE Energy Distribution .................... 91
3.4.2.4. Range and Escape Depth of SE .............. 91
3.4.2.5. Relative Contributions of SE1 and
SE2 ....................................... 93
3.4.2.6. Specimen Composition Dependence
of SE ..................................... 95
3.4.2.7. Specimen Tilt Dependence of SE ............ 96
3.4.2.8. Angular Distribution of SE ................ 97
References ................................................ 97
4. Image Formation and Interpretation .......................... 99
4.1. The Story So Far .......................................... 99
4.2. The Basic SEM Imaging Process ............................. 99
4.2.1. Scanning Action ................................... 101
4.2.2. Image Construction (Mapping) ...................... 103
4.2.2.1. Line Scans ............................... 103
4.2.2.2. Image (Area) Scanning .................... 104
4.2.2.3. Digital Imaging: Collection and
Display .................................. 107
4.2.3. Magnification ..................................... 108
4.2.4. Picture Element (Pixel) Size ...................... 110
4.2.5. Low-Magnification Operation ....................... 114
4.2.6. Depth of Field (Focus) ............................ 114
4.2.7. Image Distortion .................................. 118
4.2.7.1. Projection Distortion: Gnomonic
Projection ............................... 118
4.2.7.2. Projection Distortion: Image
Foreshortening ........................... 119
4.2.7.3. Scan Distortion: Pathological
Defects .................................. 123
4.2.7.4. Moire Effects ............................ 125
4.3. Detectors ................................................ 125
4.3.1. Introduction ...................................... 125
4.3.2. Electron Detectors ................................ 127
4.3.2.1. Everhart-Thornley Detector ............... 128
4.3.2.2. "Through-the-Lens" (TTL) Detector ........ 132
4.3.2.3. Dedicated Backscattered Electron
Detectors ................................ 133
4.4. The Roles of the Specimen and Detector in
Contrast Formation ....................................... 139
4.4.1. Contrast .......................................... 139
4.4.2. Compositional (Atomic Number) Contrast ............ 141
4.4.2.1. Introduction ............................. 141
4.4.2.2. Compositional Contrast with
Backscattered Electrons .................. 141
4.4.3. Topographic Contrast .............................. 145
4.4.3.1. Origins of Topographic Contrast .......... 146
4.4.3.2. Topographic Contrast with the
Everhart-Thornley Detector ............... 147
4.4.3.3. Light-Optical Analogy .................... 151
4.4.3.4. Interpreting Topographic Contrast
with Other Detectors ..................... 158
4.5. Image Quality ............................................ 173
4.6. Image Processing for the Display of Contrast
Information .............................................. 178
4.6.1. The Signal Chain .................................. 178
4.6.2. The Visibility Problem ............................ 180
4.6.3. Analog and Digital Image Processing ............... 182
4.6.4. Basic Digital Image Processing .................... 184
4.6.4.1. Digital Image Enhancement ................ 187
4.6.4.2. Digital Image Measurements ............... 192
References ............................................... 192
5. Special Topics in Scanning Electron Microscopy ............. 195
5.1. High-Resolution Imaging .................................. 195
5.1.1. The Resolution Problem ............................ 195
5.1.2. Achieving High Resolution at High Beam
Energy ............................................ 197
5.1.3. High-Resolution Imaging at Low Voltage ............ 201
5.2. STEM-in-SEM: High Resolution for the Special Case
of Thin Specimens ........................................ 203
5.3. Surface Imaging at Low Voltage ........................... 207
5.4. Making Dimensional Measurements in the SEM ............... 209
5.5. Recovering the Third Dimension: Stereomicroscopy ......... 212
5.5.1. Qualitative Stereo Imaging and Presentation ....... 212
5.5.2. Quantitative Stereo Microscopy .................... 217
5.6. Variable-Pressure and Environmental SEM .................. 220
5.6.1. Current Instruments ............................... 221
5.6.2. Gas in the Specimen Chamber ....................... 222
5.6.2.1. Units of Gas Pressure .................... 222
5.6.2.2. The Vacuum System ........................ 222
5.6.3. Electron Interactions with Gases .................. 225
5.6.4. The Effect of the Gas on Charging ................. 231
5.6.5. Imaging in the ESEM and the VPSEM ................. 236
5.6.6. X-Ray Microanalysis in the Presence of
a Gas ............................................. 241
5.7. Special Contrast Mechanisms .............................. 242
5.7.1. Electric Fields ................................... 243
5.7.2. Magnetic Fields ................................... 245
5.7.1. Type 1 Magnetic Contrast .......................... 245
5.7.2.2. Type 2 Magnetic Contrast ................. 247
5.7.3. Crystallographic Contrast ......................... 247
5.8. Electron Backscatter Patterns ............................ 256
5.8.1. Origin of EBSD Patterns ........................... 260
5.8.2. Hardware for EBSD ................................. 262
5.8.3. Resolution of EBSD ................................ 264
5.8.3.1. Lateral Spatial Resolution ............... 264
5.8.3.2. Depth Resolution ......................... 266
5.8.4. Applications ...................................... 267
5.8.4.1. Orientation Mapping ...................... 267
5.8.4.2. Phase Identification ..................... 267
References ............................................... 269
6. Generation of X-Rays in the SEM Specimen ................... 271
6.1. Continuum X-Ray Production (Bremsstrahlung)............... 271
6.2. Characteristic X-Ray Production .......................... 274
6.2.1. Origin ............................................ 274
6.2.2. Fluorescence Yield ................................ 275
6.2.3. Electron Shells ................................... 276
6.2.4. Energy-Level Diagram .............................. 277
6.2.5. Electron Transitions .............................. 277
6.2.6. Critical Ionization Energy ........................ 278
6.2.7. Moseley's Law ..................................... 279
6.2.8. Families of Characteristic Lines .................. 279
6.2.9. Natural Width of Characteristic X-Ray Lines ....... 281
6.2.10. Weights of Lines .......................... 282
6.2.11. Cross Section for Inner Shell
Ionization ................................ 283
6.2.12. X-Ray Production in Thin Foils ............ 284
6.2.13. X-Ray Production in Thick Targets ......... 284
6.2.14. X-Ray Peak-to-Background Ratio ............ 285
6.3. Depth ofX-Ray Production (X-Ray Range) ................... 286
6.3.1. Anderson-Hasler X-Ray Range ....................... 286
6.3.2. X-Ray Spatial Resolution .......................... 286
6.3.3. Sampling Volume and Specimen Homogeneity .......... 288
6.3.4. Depth Distribution of X-Ray Production,
φ(pz) ............................................. 288
6.4. X-Ray Absorption ......................................... 289
6.4.1. Mass Absorption Coefficient for an Element ........ 290
6.4.2. Effect of Absorption Edge on Spectrum ............. 291
6.4.3. Absorption Coefficient for Mixed-Element
Absorbers ......................................... 291
6.5. X-Ray Fluorescence ....................................... 292
6.5.1. Characteristic Fluorescence ....................... 293
6.5.2. Continuum Fluorescence ............................ 294
6.5.3. Range of Fluorescence Radiation ................... 295
References ............................................... 295
7. X-Ray Spectral Measurement: EDS and WDS .................... 297
7.1. Introduction ............................................. 297
7.2. Energy-Dispersive X-Ray Spectrometer ..................... 297
7.2.1. Operating Principles .............................. 297
9.8.4.5. Trace Element Analysis ................... 446
9.8.4.6. Trace Element Analysis
Geochronologic Applications .............. 448
9.8.4.7. Biological and Organic Specimens ......... 449
References ............................................... 449
10.Special Topics in Electron Beam X-Ray Microanalysis ........ 453
10.1.Introduction ............................................. 453
10.2.Thin Film on a Substrate ................................. 454
10.3.Particle Analysis ........................................ 462
10.3.1.Particle Mass Effect .............................. 463
10.3.2.Particle Absorption Effect ........................ 463
10.3.3.Particle Fluorescence Effect ...................... 464
10.3.4.Particle Geometric Effects ........................ 465
10.3.5.Corrections for Particle Geometric Effects ........ 466
10.3.5.1.The Consequences of Ignoring
Particle Effects ......................... 466
10.3.5.2.Normalization ............................ 466
10.3.5.3.Critical Measurement Issues for
Particles ................................ 468
10.3.5.4.Advanced Quantitative Methods for
Particles ................................ 470
10.4.Rough Surfaces ........................................... 476
10.4.1.Introduction ...................................... 476
10.4.2.Rough Specimen Analysis Strategy .................. 479
10.4.2.1.Reorientation ............................ 479
10.4.2.2.Normalization ............................ 479
10.4.2.3.Peak-to-Background Method ................ 479
10.5.Beam-Sensitive Specimens (Biological, Polymeric) ......... 480
10.5.1.Thin-Section Analysis ............................. 480
10.5.2.Bulk Biological and Organic Specimens ............. 483
10.6.X-Ray Mapping ............................................ 485
10.6.1.Relative Merits of WDS and EDS for Mapping ........ 486
10.6.2.Digital Dot Mapping ............................... 487
10.6.3.Gray-Scale Mapping ................................ 488
10.6.3.1.The Need for Scaling in Gray-Scale
Mapping .................................. 489
10.6.3.2.Artifacts in X-Ray Mapping ............... 491
10.6.4.Compositional Mapping ............................. 492
10.6.4.1.Principles of Compositional
Mapping .................................. 492
10.6.4.2.Advanced Spectrum Collection
Strategies for Compositional
Mapping .................................. 494
10.6.5.The Use of Color in Analyzing and
Presenting X-Ray Maps ............................. 497
10.6.5.1.Primary Color Superposition .............. 497
10.6.5.2.Pseudocolor Scales ....................... 497
10.7.Light Element Analysis ................................... 499
10.7.1.Optimization of Light Element X-Ray
Generation ........................................ 499
10.7.2.X-Ray Spectrometry of the Light Elements .......... 503
10.7.2.1.Si EDS ................................... 503
10.7.2.2.WDS ...................................... 507
10.7.3.Special Measurement Problems for the Light
Elements .......................................... 511
10.7.3.1.Contamination ............................ 511
10.7.3.2.Overvoltage Effects ...................... 512
10.7.3.3.Absorption Effects .............. 514
10.7.4.Light Element Quantification ...................... 515
10.8.Low-Voltage Microanalysis ................................ 518
10.8.1."Low-Voltage" versus "Conventional"
Microanalysis ..................................... 518
10.8.2.X-Ray Production Range ............................ 519
10.8.2.1.Contribution of the Beam Size to
the X-Ray Analytical Resolution .......... 520
10.8.2.2.A Consequence of the X-Ray Range
under Low-Voltage Conditions ............. 523
10.8.3.X-Ray Spectrometry in Low-Voltage
Microanalysis ..................................... 525
10.8.3.1.The Oxygen and Carbon Problem ............ 526
10.8.3.2.Quantitative X-Ray Microanalysis
at Low Voltage ........................... 528
10.9.Report of Analysis ....................................... 531
References .................................................... 535
11.Specimen Preparation of Hard Materials: Metals,
Ceramics, Rocks, Minerals, Microelectronic and
Packaged Devices, Particles, and Fibers .................... 537
11.1.Metals ................................................... 537
11.1.1.Specimen Preparation for Surface Topography ....... 537
11.1.2.Specimen Preparation for Microstructural and
Microchemical Analysis ............................ 538
11.1.2.1.Initial Sample Selection and
Specimen Preparation Steps ............... 538
11.1.2.2.Final Polishing Steps .................... 539
11.1.2.3.Preparation for Microanalysis ............ 540
11.2.Ceramics and Geological Samples .......................... 541
11.2.1.Initial Specimen Preparation: Topography
and Microstructure ................................ 542
11.2.2.Mounting and Polishing for Microstructural
and Microchemical Analysis ........................ 542
11.2.3.Final Specimen Preparation for
Microstructural and Microchemical Analysis ........ 542
11.3.Microelectronics and Packages ............................ 543
11.3.1.Initial Specimen Preparation ...................... 543
11.3.2.Polishing ......................................... 544
11.3.3.Final Preparation ................................. 545
11.4.Imaging of Semiconductors ................................ 545
11.4.1.Voltage Contrast .................................. 546
11.4.2.Charge Collection ................................. 546
11.5.Preparation for Electron Diffraction in the SEM .......... 547
11.5.1.Channeling Patterns and Channeling Contrast ....... 547
11.5.2.Electron Backscatter Diffraction .................. 547
11.6.Special Techniques ....................................... 551
11.6.1.Plasma Cleaning ................................... 551
11.6.2.Focused-Ion-Beam Sample Preparation for SEM ....... 553
11.6.2.1.Application of FIB for
Semiconductors ........................... 554
11.6.2.2.Applications of FIB in Materials
Scie 557
11.7.1.Particle Substnce ................................. 555
11.7.Particles and Fibers rates and Supports .................. 559
11.7.1.1.Bulk Particle Substrates ................. 559
11.7.1.2.Thin Particle Supports ................... 560
11.7.2.Particle Mounting Techniques ...................... 560
11.7.3.Particles Collected on Filters .................... 562
11.7.4.Particles in a Solid Matrix ....................... 563
11.7.5.Transfer of Individual Particles .................. 563
References .................................................... 564
12.Specimen Preparation of Polymer Materials .................. 565
12.1.Introduction ............................................. 565
12.2.Microscopy of Polymers ................................... 565
12.2.1.Radiation Effects ................................. 566
12.2.2.Imaging Compromises ............................... 567
12.2.3.Metal Coating Polymers for Imaging ................ 567
12.2.4.X-Ray Microanalysis of Polymers ................... 570
12.3.Specimen Preparation Methods for Polymers ................ 570
12.3.1.Simple Preparation Methods ........................ 571
12.3.2.Polishing of Polymers ............................. 571
12.3.3.Microtomy of Polymers ............................. 572
12.3.4.Fracture of Polymer Materials ..................... 573
12.3.5.Staining of Polymers .............................. 576
12.3.5.1.Osmium Tetroxide and Ruthenium
Tetroxide ................................ 578
12.3.5.2.Ebonite .................................. 578
12.3.5.3.Chlorosulfonic Acid and
Phosphotungstic Acid ..................... 578
12.3.6.Etching of Polymers ............................... 579
12.3.7.Replication of Polymers ........................... 580
12.3.8.Rapid Cooling and Drying Methods for
Polymers .......................................... 580
12.3.8.1.Simple Cooling Methods ................... 580
12.3.8.2.Freeze-Drying ............................ 581
12.3.8.3.Critical-Point Drying .................... 581
12.4.Choosing Specimen Preparation Methods .................... 581
12.4.1.Fibers ............................................ 582
12.4.2.Films and Membranes ............................... 582
12.4.3.Engineering Resins and Plastics ................... 583
12.4.4.Emulsions and Adhesives ........................... 587
12.5.Problem-Solving Protocol ................................. 588
12.6.Image Interpretation and Artifacts ....................... 589
References ............................................... 590
13.Ambient-Temperature Specimen Preparation
of Biological Material ..................................... 591
13.1.Introduction ............................................. 591
13.2.Preparative Procedures for the Structural
SEM of Single Celluar Biological Particles,
and Fibers ............................................... 592
13.2.1.Particulate, Cellular, and Fibrous Organic
Material .......................................... 592
13.2.2.Dry Organic Particles and Fibers .................. 593
13.2.2.1.Organic Particles and Fibers on
a Filter ................................. 594
13.2.2.2.Organic Particles and Fibers
Entrained within a Filter ................ 594
13.2.2.3.Organic Particulate Matter
Suspended in a Liquid .................... 594
13.2.2.4.Manipulating Individual Organic
Particles ................................ 595
13.3.Preparative Procedures for the Structural
Observation of Large Soft Biological Specimens ........... 596
13.3.1.Introduction ...................................... 596
13.3.2.Sample Handling before Fixation ................... 596
13.3.3.Fixation .......................................... 596
13.3.4.Microwave Fixation ................................ 597
13.3.5.Conductive Infiltration ........................... 597
13.3.6.Dehydration ....................................... 597
13.3.7.Embedding ......................................... 602
13.3.8.Exposing the Internal Contents of Bulk
Specimens ......................................... 602
13.3.8.1.Mechanical Dissection .................... 602
13.3.8.2.High-Energy-Beam Surface Erosion ......... 602
13.3.8.3.Chemical Dissection ...................... 603
13.3.8.4.Surface Replicas and Corrosion
Casts .................................... 604
13.3.9.Specimen Supports and Methods of Sample
Attachment ........................................ 605
13.3.10.Artifacts ........................................ 607
13.4.Preparative Procedures for the in Situ Chemical
Analysis of Biological Specimens in the SEM .............. 607
13.4.1.Introduction ...................................... 607
13.4.2.Preparative Procedures for Elemental
Analysis Using X-Ray Microanalysis ................ 608
13.4.2.1.The Nature and Extent of the
Problem .................................. 608
13.4.2.2.Types of Sample That May be
Analyzed ................................. 609
13.4.2.3.The General Strategy for Sample
Preparation .............................. 609
13.4.2.4.Criteria for Judging Satisfactory
Sample Preparation ....................... 610
13.4.2.5.Fixation and Stabilization ............... 610
13.4.2.6.Precipitation Techniques ................. 611
13.4.2.7.Procedures for Sample Dehydration,
Embedding, and Staining .................. 611
13.4.2.8.Specimen Supports ........................ 611
13.4.3.Preparative Procedures for Localizing
Molecules Using Histochemistry .................... 612
13.4.3.1.Staining and Histochemical Methods ....... 612
13.4.3.2.Atomic Number Contrast with
Backscattered Electrons .................. 613
13.4.4.Preparative Procedures for Localizing
Macromolecues Using Immunocytochemistry ........... 614
13.4.4.1.Introduction ............................. 614
13.4.4.2.The Antibody-Antigen Reaction ............ 614
13.4.4.3.General Features of Specimen
Preparation for
Immunocytochemistry ...................... 615
13.4.4.4.Imaging Procedures in the SEM ............ 616
References ............................................... 618
14.Low-Temperature Specimen Preparation ....................... 621
14.1.Introduction ............................................. 621
14.2.The Properties of Liquid Water and Ice ................... 622
14.3.Conversion of Liquid Water to Ice ........................ 623
14.4.Specimen Pretreatment before Rapid (Quench)
Cooling .................................................. 624
14.4.1.Minimizing Sample Size and Specimen Holders ....... 624
14.4.2.Maximizing Undercooling ........................... 626
14.4.3.Altering the Nucleation Process ................... 626
14.4.4.Artificially Depressing the Sample
Freezing Point .................................... 626
14.4.5.Chemical Fixation ................................. 626
14.5.Quench Cooling ........................................... 627
14.5.1.Liquid Cryogens ................................... 627
14.5.2.Solid Cryogens .................................... 628
14.5.3.Methods for Quench Cooling ........................ 629
14.5.4.Comparison of Quench Cooling Rates ................ 630
14.6.Low-Temperature Storage and Sample Transfer .............. 631
14.7.Manipulation of Frozen Specimens:
Cryosectioning, Cryofracturing, and Cryoplaning .......... 631
14.7.1.Cryosectioning .................................... 631
14.7.2.Cryofracturing .................................... 633
14.7.3.Cryopolishing or Cryoplaning ...................... 634
14.8.Ways to Handle Frozen Liquids within the Specimen ........ 635
14.8.1.Frozen-Hydrated and Frozen Samples ................ 636
14.8.2.Freeze-Drying ..................................... 637
14.8.2.1.Physical Principles Involved in
Freeze-Drying ............................ 637
14.8.2.2.Equipment Needed for Freeze-Drying ....... 638
14.8.2.3.Artifacts Associated with
Freeze-Drying ............................ 639
14.8.3.Freeze Substitution and Low-Temperature
Embedding ......................................... 639
14.8.3.1.Physical Principles Involved
in Freeze Substitution and
Low-Temperature Embedding ................ 639
14.8.3.2.Equipment Needed for Freeze
Substitution and Low-Temperature
Embedding ................................ 640
14.9.Procedures for Hydrated Organic Systems .................. 640
14.10.Procedures for Hydrated Inorganic Systems ............... 641
14.11.Procedures for Nonaqueous Liquids ....................... 642
14.12.Imaging and Analyzing Samples at Low
Temperatures ............................................ 643
References .............................................. 644
15.Procedures for Elimination of Charging
in Nonconducting Specimens ................................. 647
15.1.Introduction ............................................. 647
15.2.Recognizing Charging Phenomena ........................... 650
15.3.Procedures for Overcoming the Problems of Charging ....... 656
15.4.Vacuum Evaporation Coating ............................... 657
15.4.1.High-Vacuum Evaporation Methods ................... 658
15.4.2.Low-Vacuum Evaporation Methods .................... 661
15.5.Sputter Coating .......................................... 661
15.5.1.Plasma Magnetron Sputter Coating .................. 662
15.5.2.Ion Beam and Penning Sputtering ................... 664
15.6.High-Resolution Coating Methods .......................... 667
15.7.Coating for Analytical Studies ........................... 669
15.8.Coating Procedures for Samples Maintained
at Low Temperatures ...................................... 669
15.9.Coating Thickness ........................................ 670
15.10.Damage and Artifacts on Coated Samples .................. 672
15.11.Summary of Coating Guidelines ........................... 673
References .................................................... 673
Index ......................................................... 675
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