Contentsodin.ces.edu.co/Contenidos_Web/41028572.pdf · Development of Specimen Preparation Methods...
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Contents Preface. ................................................................... xii Introduction .............................................................. xiv About the Authors ........................................................ xvi Chapter 1 The History of Microscopy 1 Development of the Light Microscope. .............................. 1 Development of the Electron Microscope. ........................... 4 Development of Other Imaging Technologies. ....................... 6 Development of Specimen Preparation Methods for Light and Electron Microscopy ................................... 7 Conclusion 9 References and Suggested Reading ................................ 12 History of Microscopic Image Reproduction 13 Photographic lIIustration 19 Photographic Darkroom Procedures 20 Loading and Exposing the Film Cassette. ............................ 20 Processingthe Film 20 Enlargingand Printingthe Negative 21 Digital Photography ............................................... 23 Low-Volume Printing of Electronic Image Files 24 High-Speed Commercial Printing 24 Color Reproduction 27 References and Suggested Reading ................................ 28 Chapter 2 Chapter 3 Preparation of Specimens for Lightand Electron Microscopy .. ............................................ 29 Fixation 29 Aldehydes 30 Osmium Tetroxide 34 ColdMethanol/Acetone/Ethanol 34 Bouin'sFluid 35 En BlocStaining 35 Dehydration 35 Infiltration and Embedding 36 Paraff1n ............................................................. 37 Epoxy Resin ......................................................... 37 Water-Compatible Resins 40 Microtomy-Cutting Sections. . . .. .. . . . .. . . . . . .. .. . . . . . . . .. .. . . . .. .40 Staining ...........................................................47 Staining of Paraff1nSections for LightMicroscopy 47 Staining of Epoxy Resin Sections with Heavy Metals for Electron Microscopy ............................................. 51 Methods That Facilitate the Overall Tissue Processing ............... 52 v
Transcript of Contentsodin.ces.edu.co/Contenidos_Web/41028572.pdf · Development of Specimen Preparation Methods...
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
About the Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi
Chapter 1 The History of Microscopy 1Development of the Light Microscope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1
Development of the Electron Microscope. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Development of Other Imaging Technologies. . . . . . . . . . . . . . . . . . . . . . . . 6
Development of Specimen Preparation Methodsfor Light and Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Conclusion 9
References and Suggested Reading . . . .. . . .. . . . .. . . . .. . . .. . . . .. . . .. 12
History of Microscopic Image Reproduction 13Photographic lIIustration 19Photographic Darkroom Procedures 20
Loading and Exposing the Film Cassette. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Processingthe Film 20Enlargingand Printingthe Negative 21
Digital Photography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Low-Volume Printing of Electronic Image Files 24High-Speed Commercial Printing 24Color Reproduction 27References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Chapter 2
Chapter 3 Preparation of Specimens for Lightand ElectronMicroscopy .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Fixation 29
Aldehydes 30Osmium Tetroxide 34ColdMethanol/Acetone/Ethanol 34Bouin'sFluid 35
En BlocStaining 35Dehydration 35Infiltration and Embedding 36
Paraff1n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Epoxy Resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Water-CompatibleResins 40Microtomy-Cutting Sections. . . .. .. . . . .. . . . . . .. .. . . . . . . . .. .. . . . .. .40Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Stainingof Paraff1nSections for LightMicroscopy 47Staining of Epoxy Resin Sections with Heavy Metalsfor Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Methods That Facilitate the Overall Tissue Processing . . . . . . . . . . . . . . . 52
v
vi (ontents
Chapter 4
Chapter 5
Nonstandard Methods forTissue Processing 52Artifacts ofTissue Processing 53Interpretation ofThree-Dimensional Tissue Structure UsingTwo-Dimensional Sections 53
Storage and Documentation of Microscopic Specimens 56References and Suggested Reading . . . .. . . . .. . . .. . . . .. . . .. . . .. . . . .. 57
A Brief Introduction to Cel! Structure . . . . . . . . . . . . . . . . . . . . . . 59
TheAnimal Cell 59The Cell Surface and Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
The PlasmaMembrane 59The Extracellular Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Membrane Junctions 62The Cytoskeleton 62CellShape and Motility 65The Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
CellOrganelles 69The Nucleus 69The Mitochondrion 69Rough Endoplasmic Reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
The Golgi Apparatus .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Smooth EndoplasmicReticulum 73
Secretory Granulesand Exocytosis 74Endocytosisand the Lysosomal System 74
Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
The Cell Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Chloroplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Vacuoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Storage Organelles 78Plasmodesmata 81
Fungal Cells ..............................81Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Prokaryotic Cells 83Bacteria 83Viruses 85
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Electromagnetic Radiation and Its Interactionwith Matter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Interaction of Electromagnetic Radiationwith Specimens 92The Speed of Light 94Refraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Reflection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Diffraction 99Interference 102Polarization ... . . .. . . . .. . . .. . . . .. . . . .. . . .. . . . .. .. 104
Absorption and Emission of Radiation 106
Chapter 6
Chapter 7
Chapter 8
(ontents vii
Qualities of Electromagnetic Radiation Summarized 109References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
The Light Microscope and Image Formation . . . . . . . . . . . . . . 111
Image Formation Requires Diffraction and Interference 113Lens Aberrations .. . . .. . . .. .. 120
Objective Lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Immersion Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 123
FluorescenceObjectives 123Phase ContrastObjectives 124DIC Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 124
Correction-Collar Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 124
Plan Apochromatic Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 124
Oculars 124
Light Sources and Lamp Houses . . . . . . . . . . . . . . . 125The Specimen Stage 130Design Plan of the Compound Light Microscope 130Light Paths in the Compound Microscope 132K6hler lIIumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Infinity-Corrected Optics 136Prisms and Beamsplitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 139
Phase, Interference, and Polarization Methodsfor Optical Contrast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Vital Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Phase-Contrast Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Darkfield Microscopy 145PolarizationMicroscopy . . .. . . .. . . . .. . . .. . . . .. . . .. . . .. . 147Differentiallnterference Contrast Microscopy 151Hoffman Interference Contrast 154
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
The Transmission Electron Microscope .. . . . . . . . . . . . . . . . . . 156lIIuminationSystem . . .. . . .. . . .. . . 156
TheElectronGun 156Condenser Lens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 162
Deflector Coi/s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 164
Specimen Chamber and Control System 164Imaging System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Objective Lens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 166
Intermediate and ProjectorLenses 168ViewingSystem 168Camera Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 169
Vacuum System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
RotaryandDiffusionPumps 169Other VacuumSystems 171Vacuum Meters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 171
viii (ontents
Chapter 9
Chapter 10
Practical Guide far Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 171
The Stand-By Positions 171
Steps to Using the TEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 172
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
The Scanning Electron Microscope 177The Systems and Principies af the SEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
/1/uminationSystem 178The Specimen Stage and Manipulation 182Imaging System 183
Image Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
PrimaryBeam Spot Size 191FinalAperture Size 191WorkingDistance 192Accelerating Voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 193
Sample Preparatian 194BasicProtocol 194Fixation 794Dehydration and Critical-PointDrying 796Mounting the Specimen 797Specimen Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 198
Analytical Microscopy .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
X-RayMicroanalysis 799Additianal Mades af SEMAnalysisand Operatians 201
Environmental andVariable PressureSEM 207Crya-SEM 202Practical Guide far Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Specimen Change (Based on a Drawer Design) . . . . . . . . . . . . . . . . . . . . .202
Optimizing the Image 203Making a Micrograph 203General Rules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Cryogenic Techniques in Electron Microscopy . . . . . . . . . . . . 205
Freezing Methads Using Cryaprotectants 205Ultrarapid Freezing by Immersian 207Propane Jet Freezing 209Metal Mirrar Freezing 210High-Pressure Freezing . .. .. . . . . . . . . . .. .. . . . . .. . . . . . . . . . . . " . . . . ..212Cryafixatian Avaids Artifacts af Chemical Fixatian But CanCause Freezing Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Freeze Substitutian 215Freeze Fracture and Replica Productian 218Imaging and Presenting Freeze Fracture Replicas 225LiveCellsCan Be Rapidly Frazen and Freeze Fractured . . .. . . . .. . . .. 228
Quick Freezing,Deep Etching,and Ratary Shadawing 229References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Chapter 11
Chapter 12
Chapter 13
Contents ix
Video Microscopy and Electronic Imaging . . . . . . . . . . . . . . . . 234
The Video Signal 237Video Cameras: The Old Tube Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Solid-State Cameras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Solid-State Color Cameras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
Digitallmage Processing 253Signal-to-Noise Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
Storage of Electronic Signals 255Video Cassette Recorders 259
Playback ofVideo and Electronic Images 262Digital Video Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
Compression 267GenerationalLoss 267
Image Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
The MolecularBasisof Fluorescence 270OpticalFilters 273Fluorescent Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Quantum Dots 279Using Multiple Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
Autofluorescence and Background Fluorescence . .. . . .. . . .. . . . .. . .284Laser Scanning Confocal Microscopy 285Lasers 288
Delivery of Laser Ught to the Scanning Head . . . . . . . . . . . . . . . . . . . . . . 290
Scanning the Beam Onto the Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Ught Collection and Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Une Scanning and Spinning Disc Confocal Microscopy . . . . . . . . . . . . 294
Multiphoton Confocal Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
Formats for Presentation ofThree-DimensionalData Sets 296
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Microscopic Localization and Dynamicsof Biological Molecules 301Cytochemistry 301Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Use of Fluorescent Probes 305
Immunocytochemistry 308Fluorescent Proteins (Green and Otherwise) 317In Situ Hybridization 319Multispectrallmaging Microscopy and ChromosomePainting 320Localization of Upids, Carbohydrates, and OtherMolecules Based on Chemical Affinity 323
Probes of Membrane Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
x Contents
Chapter 14
Chapter 15
Chapter 16
Carbohydrate Polymers and Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . 324
Organelle-Specific Probes 327Single Molecule Fluorescence 327Detection of Molecular Motions and Interactions: FluorescenceResonance EnergyTransfer, Fluorescence Recovery afterPhotobleaching, Fluorescence Correlation Spectroscopy,Fluorescence Polarization, and Speckle Microscopy 331
Fluorescence Polarization . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . 331
Fluorescence Correlation Spectroscopy (FCS) .... ..... .... ..... .... .332
Fluorescence Recovery after Photobleaching (FRAP) . . . . . . . . . . . . . . . . 333
Fluorescence Resonance Energy Transfer (FRET) .. . . . . . . . . . . . . . . . . . . 335
Speckle Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Single Particle Tracking Using Video Microscopy 338Optical Tweezers- The LaserTrapping of Small Organelles,Particles, and Motile Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
Fluorescence-Activated Flow Cytometry 340References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Imaging lons and Intracellular Messengers . . . . . . . . . . . . . . . 345
Precipitation Techniques 345Rapid-Freezing Techniques 346
Calcium Detection in Live Cells Using Fluorescentand Luminescent Probes 346Calcium Homeostasis 348Fluorescent Calcium Chelators: A New Generationof Probes 349
Fura-2 and Indo-1:Second-Generation Calcium Probes 351
Requirements for Live Celllmaging 352Quantitation of Fluorescence Signals 356Calcium Signals Visualized by Fluorescent Probes 359Fluorescent Probes for Other Ions 361Potential-Sensitive Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Cyclic Nucleotide and Protein Kinase Probes 364
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Imaging Macromolecules and SupermolecularComplexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Rotary Platinum Shadowing 367Negative Staining 373Single Particle Analysis of Frozen Macromoleculesby Cryoelectron Microscopy 374
Electron Microscope Tomography 378X-Rayand Electron Diffraction Methods 381
Scanning Probe Microscopy 385Near-Field Microscopy 389
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Image Processing and Presentation . . . . . . . . . . . . . . . . . . . . . . 393
Start with the Right Specimen and MicroscopyTechnique 393
Index
Figure Credits
Contents xi
Getting the Most Out ofYour Microscopy 394Postproduction Analysis and Optimization of Images 395When Is Image Processing Reasonable? . . . .. .. . . . .. .. .. . . . . . .. .. . .404ObtainingQuantitativeDatafrom Images 405Motion Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Three-Dimensional Reconstructions 409
Methods Used for Presentation of Images . . . . . . . . . . . . . . . . . . . . . . . . . 410
Developing Figure Layouts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
Journal Figures .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
Poster Presentations 411Seminars/Slide Shows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .412
Referencesand Suggested Reading 414
.415
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