| Preface |
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xxi | |
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Chapter 1 The Past, Present, and Future of Electron Microscopy |
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2 | (14) |
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7 | (2) |
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DEVELOPMENT OF THE ELECTRON MICROSCOPE |
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9 | (2) |
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HISTORICAL MILESTONES IN THE DEVELOPMENT OF THE ELECTRON MICROSCOPE |
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11 | (1) |
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DEVELOPMENT OF PREPARATIVE TECHNIQUES |
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11 | (1) |
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CONTRIBUTIONS TO BIOLOGY AND THE FUTURE OF ELECTRON MICROSCOPY |
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12 | (1) |
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JOURNALS DEVOTED PRIMARILY TO ELECTRON MICROSCOPY |
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13 | (1) |
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SELECTED JOURNALS PUBLISHING ELECTRON MICROGRAPHS |
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13 | (1) |
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14 | (2) |
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Chapter 2 Specimen Preparation for Transmission Electron Microscopy |
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16 | (32) |
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19 | (15) |
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The Mechanism of Chemical Fixation for Electron Microscopy |
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20 | (1) |
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20 | (1) |
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21 | (1) |
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Selection of a Fixative and a Buffer |
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22 | (1) |
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22 | (1) |
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22 | (2) |
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Obtaining and Preparing Buffered Glutaraldehyde Fixative |
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24 | (1) |
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Obtaining and Preparing Osmium Fixative |
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24 | (1) |
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Immersion and Perfusion Fixation |
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24 | (1) |
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25 | (1) |
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26 | (1) |
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26 | (1) |
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Microwave-Assisted Specimen Preparation |
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27 | (3) |
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Freezing Method for Specimen Preparation |
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30 | (1) |
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Popular Fixation Protocols Other than Glutaraldehyde-Osmium Tetroxide |
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31 | (1) |
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31 | (1) |
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Osmium-Reduced Ferrocyanide |
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31 | (2) |
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33 | (1) |
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33 | (1) |
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Fixation of Plant Tissues |
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33 | (1) |
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34 | (1) |
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34 | (1) |
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USE OF TRANSITIONAL SOLVENTS |
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35 | (1) |
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35 | (1) |
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36 | (3) |
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36 | (1) |
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Measuring Embedding Media |
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36 | (1) |
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36 | (1) |
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Other Embedments and Their Use |
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36 | (1) |
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37 | (1) |
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37 | (1) |
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37 | (1) |
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38 | (1) |
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Low Pressure Removal of Solvents and Air Bubbles |
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39 | (1) |
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39 | (2) |
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39 | (1) |
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39 | (1) |
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Removal of Tissue Supports |
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39 | (2) |
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41 | (2) |
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EMBEDDING TISSUE CULTURE CELLS |
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43 | (1) |
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SUITABLE CONTAINERS FOR TISSUE PROCESSING |
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44 | (1) |
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RAPID TISSUE PROCESSING PROTOCOLS |
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44 | (1) |
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AUTOMATIC TISSUE PROCESSORS |
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44 | (1) |
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TISSUE VOLUME CHANGES DURING SPECIMEN PREPARATION |
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45 | (1) |
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JUDGING ADEQUATE SPECIMEN PREPARATION |
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45 | (1) |
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46 | (2) |
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Chapter 3 Specimen Preparation for Scanning Electron Microscopy |
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48 | (24) |
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50 | (1) |
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50 | (3) |
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53 | (1) |
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SPECIMEN DRYING TECHNIQUES |
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54 | (4) |
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54 | (2) |
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56 | (2) |
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58 | (1) |
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SPECIMEN FRACTURING PROCEDURES |
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58 | (3) |
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58 | (2) |
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60 | (1) |
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61 | (2) |
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Negative Surface Replication Using Cellulose Acetate Film |
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61 | (1) |
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Negative/Positive Replication Using Silicone, Resin |
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61 | (1) |
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Corrosion Casting of Animal Vasculatures |
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62 | (1) |
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63 | (2) |
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SPECIMEN COATING FOR CONDUCTIVITY |
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65 | (4) |
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Sputter Coating Procedure |
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65 | (3) |
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68 | (1) |
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69 | (1) |
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70 | (2) |
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72 | (48) |
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SHAPING THE SPECIMEN BLOCK |
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76 | (6) |
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76 | (1) |
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Rough Trimming by Machine |
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77 | (2) |
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79 | (1) |
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80 | (2) |
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TYPES OF ULTRAMICROTOME KNIVES |
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82 | (7) |
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82 | (2) |
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84 | (1) |
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Attachment of Water Trough or Boat |
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85 | (1) |
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86 | (3) |
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89 | (1) |
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89 | (1) |
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90 | (3) |
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93 | (4) |
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93 | (1) |
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93 | (1) |
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94 | (1) |
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Perforated or Holey Films |
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94 | (2) |
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Carbon-Coated Plastic Films |
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96 | (1) |
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96 | (1) |
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THE ULTRAMICROTOME AND THE SECTIONING PROCESS |
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97 | (12) |
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Development of the Ultramicrotome |
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97 | (1) |
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Basic Features of All Ultramicrotomes |
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97 | (1) |
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98 | (1) |
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99 | (1) |
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99 | (2) |
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101 | (1) |
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Clearance Angle Adjustment |
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101 | (1) |
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101 | (1) |
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101 | (1) |
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102 | (1) |
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102 | (1) |
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103 | (3) |
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Factors Affecting Sectioning |
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106 | (2) |
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108 | (1) |
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109 | (8) |
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117 | (3) |
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Chapter 5 Specimen Staining and Contrast Methods for Transmission Electron Microscopy |
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120 | (28) |
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122 | (8) |
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Preembedding, Positive Staining with Uranyl Salts |
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123 | (1) |
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Postembedding Staining with Uranyl Salts |
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124 | (1) |
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124 | (2) |
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126 | (1) |
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Postembedding Lead Staining |
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127 | (1) |
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127 | (2) |
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129 | (1) |
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129 | (1) |
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130 | (5) |
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Commonly Used Negative Stains |
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131 | (2) |
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Imaging Subcellular Components by Negative Staining |
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133 | (1) |
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Revealing Viruses Using Negative Staining |
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133 | (2) |
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METAL SHADOWING TECHNIQUES |
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135 | (11) |
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Metal Evaporation Procedures |
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136 | (1) |
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136 | (4) |
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Some Applications of Metal Shadowing and Negative Staining |
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140 | (1) |
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Making Height Measurements Using Metal Shadowing |
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140 | (1) |
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Replication of Biological Surfaces for Transmission Electron Microscopy |
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140 | (4) |
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Visualizing Macromolecules |
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144 | (2) |
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146 | (2) |
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Chapter 6 The Transmission Electron Microscope |
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148 | (54) |
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VISIBLE LIGHT, ELECTRONS, AND LENSES |
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151 | (7) |
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Electromagnetic Radiation and the Diffraction Phenomenon |
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151 | (2) |
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Effect of Diffraction on Resolution |
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153 | (2) |
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Electrons, Waves, and Resolution |
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155 | (1) |
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156 | (1) |
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Design of Electromagnetic Lenses |
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157 | (1) |
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158 | (4) |
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162 | (1) |
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DESIGN OF THE TRANSMISSION ELECTRON MICROSCOPE |
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163 | (25) |
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Comparison of Light Microscope to Transmission Electron Microscope |
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163 | (1) |
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Basic Systems Making Up a Transmission Electron Microscope |
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163 | (1) |
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164 | (9) |
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Specimen Manipulation System |
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173 | (2) |
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175 | (5) |
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180 | (8) |
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PREPARING THE TRANSMISSION ELECTRON MICROSCOPE FOR USE |
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188 | (13) |
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188 | (4) |
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Major Operational Modes of the Transmission Electron Microscope |
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192 | (2) |
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192 | (1) |
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193 | (1) |
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194 | (1) |
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195 | (1) |
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195 | (1) |
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195 | (1) |
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196 | (1) |
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196 | (1) |
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197 | (1) |
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197 | (1) |
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Magnification Calibration |
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197 | (1) |
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198 | (2) |
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Levels of Usage of the Transmission Electron Microscope |
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200 | (1) |
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201 | (1) |
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201 | (1) |
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Chapter 7 The Scanning Electron Microscope |
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202 | (38) |
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208 | (11) |
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Electron Optical and Beam Control Systems |
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208 | (1) |
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208 | (1) |
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208 | (5) |
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Interaction of Electron Beam with Specimen |
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213 | (1) |
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214 | (1) |
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Electron Detector, Signal Processing, and Recording Systems |
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214 | (1) |
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214 | (1) |
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Secondary Electron Detector |
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214 | (1) |
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214 | (3) |
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217 | (2) |
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CONTRAST AND THREE-DIMENSIONALITY OF THE SEM IMAGE |
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219 | (2) |
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STEREO IMAGING WITH THE SEM |
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221 | (3) |
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Generating Two Micrographs with Separate Views |
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221 | (3) |
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MAJOR OPERATIONAL MODES OF THE SEM |
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224 | (1) |
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224 | (1) |
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225 | (1) |
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IMAGING OTHER TYPES OF SPECIMEN SIGNALS |
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225 | (6) |
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225 | (1) |
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Backscattered Electron Detection |
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226 | (1) |
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Important Considerations when Using Backscattered Imaging with Biological Specimens |
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227 | (1) |
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228 | (3) |
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SPECIALIZED INSTRUMENTATION FOR OBSERVING UNFIXED TISSUES |
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231 | (3) |
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Observation of Frozen Specimens |
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231 | (1) |
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Observation of Fresh Specimens |
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231 | (3) |
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ALIGNMENT AND OPERATION OF THE SCANNING ELECTRON MICROSCOPE |
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234 | (4) |
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DIGITAL SCANNING ELECTRON MICROSCOPES |
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238 | (1) |
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238 | (2) |
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Chapter 8 Production of the Electron Micrograph |
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240 | (22) |
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242 | (8) |
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Negative Recording Medium |
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242 | (1) |
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Exposure Process in the SEM |
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243 | (1) |
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Exposure Process in the TEM |
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243 | (1) |
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244 | (1) |
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Improving Resolution and Contrast in TEM Negatives |
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245 | (1) |
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Commercial Films, Handling, Developing, and Troubleshooting |
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245 | (1) |
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Negative Recording Media for TEM |
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245 | (1) |
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Handling of Negative Materials and Conventional Processing |
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246 | (1) |
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Standardization of Procedures |
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247 | (1) |
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248 | (1) |
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Automatic Processing of Negative Materials |
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249 | (1) |
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250 | (6) |
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Work Prints and Final Prints |
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250 | (1) |
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The Enlarger and Accessories |
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251 | (1) |
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252 | (2) |
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254 | (1) |
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255 | (1) |
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255 | (1) |
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Techniques to Enhance Contrast |
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256 | (1) |
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Matte and Glossy Electron Micrographs |
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256 | (1) |
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PREPARING MICROGRAPHS FOR PUBLICATION |
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256 | (5) |
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256 | (1) |
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Choosing a Representative Print |
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256 | (1) |
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Matching Several Prints Placed Together |
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256 | (1) |
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Selecting the Dimensions of a Final Print |
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257 | (1) |
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257 | (1) |
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257 | (1) |
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257 | (1) |
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258 | (1) |
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259 | (1) |
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259 | (1) |
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259 | (1) |
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260 | (1) |
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Determining Print Magnification from a Negative |
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260 | (1) |
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261 | (1) |
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Chapter 9 Immunocytochemistry |
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262 | (20) |
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THE ANTIGEN-ANTIBODY REACTION |
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264 | (2) |
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266 | (2) |
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EPITOPE TAGGING OF PROTEINS |
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268 | (1) |
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268 | (6) |
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GENERAL CONSIDERATIONS IN PERFORMING AN IMMUNOCYTOCHEMICAL EXPERIMENT |
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274 | (3) |
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Immunohistochemistry/Immunofluoresence |
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274 | (1) |
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Obtaining and Applying the Primary Antibody |
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274 | (1) |
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Obtaining the Secondary Antibody |
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274 | (1) |
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275 | (1) |
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Preembedding or Postembedding Labeling |
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275 | (1) |
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276 | (1) |
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Blocking Nonspecific Labeling |
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276 | (1) |
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276 | (1) |
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276 | (1) |
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276 | (1) |
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Use of Tag or Unlabeled Antibody |
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276 | (1) |
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Omission of Primary or Secondary Antibodies |
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276 | (1) |
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277 | (1) |
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DEALING WITH SOLUBLE ANTIGENS |
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277 | (1) |
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IMMUNOLOCALIZATION USING CRYOULTRAMICROTOMY |
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277 | (1) |
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LR WHITE EMBEDDING MEDIUM |
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277 | (1) |
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277 | (1) |
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INTERPRETATION OF MICROGRAPHS |
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278 | (1) |
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278 | (4) |
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Chapter 10 Enzyme Cytochemistry |
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282 | (10) |
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BASIS OF ENZYME CYTOCHEMISTRY |
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284 | (1) |
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REQUIREMENTS FOR PERFORMING ENZYME CYTOCHEMISTRY |
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284 | (1) |
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Preservation of Tissue Structure and Enzymatic Activity |
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285 | (1) |
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Maximization of Reaction Conditions |
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285 | (1) |
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Facilitation of Substrate Penetration |
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285 | (1) |
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Use of Appropriate Controls |
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285 | (1) |
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Visualization of the Reaction Product |
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285 | (1) |
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285 | (1) |
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286 | (1) |
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287 | (1) |
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EXAMPLES OF CYTOCHEMISTRY FOR SELECTED ENZYMES |
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287 | (3) |
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290 | (2) |
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Chapter 11 Autoradiography/Radioautography |
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292 | (18) |
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295 | (1) |
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EMULSION USED IN AUTORADIOGRAPHY |
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296 | (2) |
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HOW TO PERFORM AUTORADIOGRAPHY |
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298 | (4) |
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299 | (1) |
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Administration of the Radioactive Substance and Tissue Preparation |
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299 | (1) |
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Light Microscope Autoradiography |
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299 | (1) |
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299 | (2) |
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Exposure of the Autoradiograph |
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301 | (1) |
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Development of the Autoradiograph |
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301 | (1) |
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301 | (1) |
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Placement of Exposed Tissue on Grids |
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302 | (1) |
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INTERPRETATION OF AUTORADIOGRAPHS |
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302 | (3) |
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IN SITU HYBRIDIZATION USING EM AUTORADIOGRAPHY |
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305 | (2) |
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307 | (3) |
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Chapter 12 Miscellaneous Localization and Enhancement Techniques |
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310 | (10) |
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312 | (1) |
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CARBOHYDRATES/OLIGOSACCHARIDES |
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313 | (2) |
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313 | (1) |
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Using Tannic Acid and Metals |
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313 | (1) |
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Using a Modified PA-Schiff Reaction |
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313 | (1) |
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Using Colloidal Iron and Colloidal Thorium |
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313 | (2) |
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GOLGI COMPLEX/MULTIVESICULAR BODY |
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315 | (1) |
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315 | (1) |
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315 | (2) |
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317 | (1) |
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317 | (1) |
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317 | (1) |
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317 | (3) |
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Chapter 13 Quantitative Electron Microscopy |
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320 | (22) |
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323 | (1) |
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GENERAL SCHEME OF STEREOLOGY |
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324 | (1) |
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PARAMETERS MEASURED AND SYMBOLS USED IN STEREOLOGY |
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325 | (1) |
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TISSUE COMPARTMENTS OR SPACES |
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325 | (2) |
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327 | (1) |
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BASIC TYPES OF DETERMINATIONS AND ASSOCIATED FORMULAS |
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328 | (8) |
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328 | (2) |
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Volume Determination Methods for Spherical Objects |
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330 | (2) |
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Volume Determinations for Nonspherical Objects |
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332 | (1) |
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332 | (2) |
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Surface Density and Surface-to-Volume Determinations |
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334 | (1) |
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Numerical Density Determinations |
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335 | (1) |
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ASSUMPTIONS AND CONDITIONS |
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336 | (1) |
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336 | (1) |
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HOW MUCH DATA FROM TEST SYSTEMS IS NEEDED? |
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337 | (1) |
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COMPUTER-ASSISTED STEREOLOGY |
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337 | (2) |
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339 | (3) |
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Chapter 14 Freeze Fracture Replication |
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342 | (26) |
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347 | (5) |
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INTERPRETATION OF FREEZE FRACTURE REPLICAS |
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352 | (2) |
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352 | (1) |
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Orienting the Micrograph so that the Shading of IMPs is from Below |
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353 | (1) |
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Determining Which Membrane Is Being Viewed |
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353 | (1) |
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Determining Whether the Membrane Face Being Viewed Is a P-Face or an E-Face |
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353 | (1) |
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354 | (2) |
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356 | (1) |
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QUICK FREEZE, DEEP ETCH, ROTARY SHADOW TECHNIQUES |
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356 | (1) |
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FREEZE FRACTURE CYTOCHEMISTRY |
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357 | (1) |
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357 | (8) |
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365 | (3) |
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Chapter 15 Analytical Electron Microscopy |
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368 | (28) |
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INTERACTION OF AN ELECTRON BEAM WITH A SPECIMEN |
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370 | (1) |
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MICROSCOPES USED FOR DETECTING ANALYTICAL SIGNALS |
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371 | (2) |
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373 | (5) |
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Continuum (Bremsstrahlung) X Rays |
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373 | (1) |
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374 | (1) |
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X-Ray Microanalysis May Be Conducted to Achieve Several Goals |
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375 | (3) |
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INFORMATION OBTAINABLE USING X-RAY ANALYSIS |
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378 | (2) |
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SPECIMEN PREPARATION FOR X-RAY MICROANALYSIS |
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380 | (2) |
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380 | (1) |
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Single Cells, Isolated Organelles, Liquid Secretions, or Extracts |
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381 | (1) |
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381 | (1) |
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ELECTRON ENERGY LOSS SPECTROSCOPY (EELS) |
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382 | (6) |
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386 | (8) |
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Formation of Diffraction Patterns |
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386 | (3) |
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Single Crystal Versus Polycrystalline Specimens |
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389 | (3) |
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Types of Diffraction Modes |
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392 | (1) |
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Selected Area Diffraction (SAD) |
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392 | (1) |
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393 | (1) |
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Other Types of Diffraction |
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394 | (1) |
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394 | (2) |
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Chapter 16 Intermediate and High Voltage Microscopy |
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396 | (10) |
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398 | (1) |
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ADVANTAGES OF HIGH VOLTAGE MICROSCOPY |
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398 | (3) |
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CONTRIBUTIONS OF HIGH VOLTAGE MICROSCOPY |
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401 | (4) |
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405 | (1) |
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406 | (8) |
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SOME SPECIFIC TRACERS IN USE |
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409 | (3) |
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Cationic and Native Ferritin |
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409 | (1) |
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409 | (1) |
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409 | (2) |
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411 | (1) |
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411 | (1) |
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412 | (2) |
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Chapter 18 Image Processing and Image Analysis by Computer |
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414 | (28) |
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CAPTURING THE IMAGE: CONVENTIONAL VERSUS DIGITAL METHODS |
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416 | (7) |
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423 | (13) |
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Controlling Contrast, Brightness, and Gamma |
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423 | (1) |
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424 | (1) |
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424 | (5) |
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429 | (1) |
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429 | (1) |
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430 | (2) |
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Look-Up Tables, Thresholding, Binary Images, and Pseudocoloring |
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432 | (4) |
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Image Averaging and Computer Enhancement |
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436 | (1) |
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FINAL DISPLAY OF DIGITAL IMAGES FOR PUBLICATION AND PRESENTATION |
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436 | (1) |
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ELECTRONIC IMAGE PROCESSING AND IMAGE ANALYSIS FOR ELECTRON MICROSCOPY |
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437 | (3) |
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437 | (1) |
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438 | (1) |
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438 | (1) |
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Grid Overlays for Surface Density Measurements |
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438 | (1) |
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439 | (1) |
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440 | (1) |
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Center of a Mass (Centroid) |
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440 | (1) |
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440 | (1) |
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THREE-DIMENSIONAL IMAGING |
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440 | (1) |
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PRACTICALITY AND UTILITY OF COMPUTER IMAGE ANALYSIS: A CAVEAT |
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440 | (1) |
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441 | (1) |
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441 | (1) |
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Chapter 19 Interpretation of Micrographs |
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442 | (34) |
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INTRODUCTION TO VIEWING BIOLOGICAL ELECTRON MICROGRAPHS |
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444 | (2) |
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INTERPRETATION OF NORMAL TISSUE STRUCTURE |
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446 | (28) |
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Magnification and Resolution |
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446 | (1) |
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447 | (1) |
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Shape, Kinds, and Number of Structures |
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448 | (5) |
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453 | (2) |
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Dehydration, Infiltration, and Embedding Artifacts |
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455 | (1) |
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456 | (8) |
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464 | (2) |
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466 | (7) |
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473 | (1) |
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INTERPRETING DYNAMIC PROCESSES FROM STATIC IMAGES |
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474 | (1) |
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ESTIMATION OF MICROGRAPH MAGNIFICATION |
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474 | (1) |
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475 | (1) |
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Chapter 20 Survey of Biological Ultrastructure |
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476 | (140) |
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480 | (26) |
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The Lipid Bilayer of the Plasmalemma |
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480 | (2) |
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482 | (1) |
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483 | (5) |
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Cell Surface Specializations |
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488 | (18) |
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506 | (7) |
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506 | (3) |
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509 | (1) |
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510 | (3) |
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Regionalization of Cytoskeletal Elements |
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513 | (1) |
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513 | (15) |
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The Nuclear Envelope and Nuclear Lamina |
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514 | (1) |
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515 | (3) |
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518 | (1) |
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518 | (3) |
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521 | (1) |
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521 | (7) |
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528 | (7) |
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PROTEIN SYNTHETIC AND SECRETORY STRUCTURES |
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535 | (14) |
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535 | (1) |
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535 | (1) |
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Rough Endoplasmic Reticulum |
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535 | (2) |
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Smooth Endoplasmic Reticulum |
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537 | (1) |
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538 | (4) |
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542 | (7) |
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549 | (2) |
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551 | (5) |
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551 | (1) |
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552 | (4) |
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556 | (7) |
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556 | (2) |
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558 | (5) |
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PEROXISOMES OR MICROBODIES |
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563 | (1) |
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564 | (1) |
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564 | (4) |
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568 | (3) |
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568 | (1) |
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568 | (3) |
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571 | (8) |
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571 | (4) |
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575 | (1) |
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Matrix of Bone and Cartilage |
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576 | (3) |
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SPECIAL FEATURES OF PLANT TISSUES |
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579 | (6) |
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579 | (1) |
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579 | (4) |
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583 | (2) |
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585 | (3) |
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ALGAE, FUNGI, YEAST, AND PROTOZOA |
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588 | (4) |
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592 | (16) |
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608 | (8) |
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Chapter 21 Safety in the Electron Microscope Laboratory |
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616 | (25) |
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PERSONAL SAFETY IN THE LABORATORY |
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618 | (4) |
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Safety Apparatus and Safe Practices |
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618 | (3) |
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Pathogens and Radioisotopes |
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|
621 | (1) |
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622 | (6) |
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Handling Chemicals in a Safe Manner |
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622 | (1) |
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623 | (2) |
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Some Chemicals Commonly Used in Electron Microscopy |
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|
625 | (1) |
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Disposal of Spent Chemicals |
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|
625 | (1) |
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626 | (1) |
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Cleaning Up Hazardous Spills |
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626 | (1) |
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627 | (1) |
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628 | (1) |
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628 | (2) |
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628 | (1) |
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629 | (1) |
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630 | (1) |
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630 | (1) |
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Vacuum Evaporators and Sputter Coaters |
|
|
630 | (1) |
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Proper Grounding of Equipment |
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|
630 | (1) |
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Servicing of Electron Microscopes and Small Equipment |
|
|
631 | (1) |
|
First Aid for Shock Victims |
|
|
631 | (1) |
|
PHYSICAL AND MECHANICAL HAZARDS |
|
|
631 | (6) |
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|
631 | (1) |
|
Critical Point Dryers (CPDs) |
|
|
631 | (1) |
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|
632 | (1) |
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|
632 | (1) |
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632 | (1) |
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632 | (1) |
|
Cryogenic Gases and Vacuum Dewars |
|
|
633 | (1) |
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|
633 | (2) |
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|
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635 | (1) |
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635 | (1) |
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636 | (1) |
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|
|
636 | (1) |
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|
|
637 | (1) |
|
TRAINING AND ORIENTATION PROGRAMS |
|
|
637 | (2) |
|
HOTLINES AND OTHER RESOURCES |
|
|
639 | (1) |
|
|
|
639 | (2) |
| Appendix A Review Questions and Problems |
|
641 | (12) |
| Index |
|
653 | |
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