Biological Electron Microscopy: Theory, Techniques, and Troubleshooting, Teil 997

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Springer Science & Business Media, 31.12.2003 - 534 Seiten
Electron microscopy is frequently portrayed as a discipline that stands alone, separated from molecular biology, light microscopy, physiology, and biochemistry, among other disciplines. It is also presented as a technically demanding discipline operating largely in the sphere of "black boxes" and governed by many absolute laws of procedure. At the introductory level, this portrayal does the discipline and the student a disservice. The instrumentation we use is complex, but ultimately understandable and, more importantly, repairable. The procedures we employ for preparing tissues and cells are not totally understood, but enough information is available to allow investigators to make reasonable choices concerning the best techniques to apply to their parti cular problems. There are countless specialized techniques in the field of electron and light microscopy that require the acquisition of specialized knowledge, particularly for interpretation of results (electron tomography and energy dispersive spectroscopy immediately come to mind), but most laboratories possessing the equipment to effect these approaches have specialists to help the casual user. The advent of computer operated electron microscopes has also broadened access to these instruments, allowing users with little technical knowledge about electron microscope design to quickly become operators. This has been a welcome advance, because earlier instru ments required a level of knowledge about electron optics and vacuum systems to produce optimal photographs and to avoid "crashing" the instruments that typically made it difficult for beginners.
 

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Inhalt

Specimen Preparation for Electron Microscopy
1
II TRADITIONAL CHEMICAL FIXATION
2
III BUFFERS
24
IV DEHYDRATION
29
V EMBEDDING MEDIA
31
VI EXAMINATION OF TISSUES PREPARED WITH A VARIETY OF FIXATIVES AND BUFFERS
36
VII A QUASIUNIVERSAL FIXATION DEHYDRATION AND EMBEDMENT SCHEDULE SUCCESSFULLY USED FOR ORGANISMS FROM TH...
40
REFERENCES
74
Negative Staining with Ammonium Molybdate
282
Preparation of Virus Samples for Transmission Electron Microscopy
283
Ultracentrifugation Technique for Viral Sample Preparation
284
Immune Electron Microscopy for Concentrating Viruses
285
Transmission Electron Microscopy
289
II THEORY OF ELECTRON OPTICS
290
III FOUR ASPECTS OF IMAGE FORMATION MEEK 1976
302
IV GENERAL TEM FEATURES
303

CHAPTER 1 TECHNIQUES
76
Preparation of Primary Aldehyde Fixatives
82
Preparation of Osmium Osmium Tetroxide Osmium Tetraoxide
83
Buffering Systems
87
Cacodylate Buffers
88
Phosphate Buffers
89
TrisHCI Buffer Chayen et al 1969
91
Sodium Acetate Buffer Chayen et al 1969
92
Dulbeccos PhosphateBuffered Saline
93
Resin Formulations
94
Spurr Resin
95
PolyBed 812 Resin
98
SPIPon 812
99
Araldite 6005 Resin
101
Mollenhauers EponAraldite Resin Adapted for Use with Epon Substitutes
102
London Resin Co LR White Resin
103
Lowicryl and LR Gold Resins
105
PEG Method for TEM Sections
106
JB4 Glycol Methacrylate Techniques for HighResolution Light Microscopy
107
Agar Embedment of Cell Suspensions or Subcellular Particulates for TEM
109
Preparing Adherent Tissue Culture Monolayers in Situ for TEM
111
Flat Embedding of Cell Cultures Grown on Permanox Tissue Culture Dishes for TEM
113
Sperm Fixation
116
Central Nervous System Fixation Brain Spinal Cord
118
Using Vacuum to Help Wet Fungal Plant or Insect Samples during Primary Fixation
119
Simultaneous GlutaraldehydeOsmium Fixation for Protozoan Samples or Samples with a Large Amount of Lipid
120
Killing Cells Prior to Chemical Fixation
121
Flat Embedding on Microscope Slides
122
Cryotechniques
127
I HISTORY
128
II PURPOSE
129
III CRYOGENS
130
IV SAFETY PRECAUTIONS
131
VI USES OF FROZEN SPECIMENS
137
VII ARTIFACTS AND THEIR CORRECTION
148
REFERENCES
151
Cryoultramicrotomy for Structural Examinations or Consequent Immunolabeling
152
Ultramicrotomy
155
II KNIVES
157
III BLOCK TRIMMING
159
IV ULTRAMICROTOMY WORKING AREA
160
CHAPTER 3 TECHNIQUES
161
Making a Section Manipulation Tool
162
Making a Locking Ring for Forceps
163
Making Glass Knife Boats
166
Block Trimming
167
Sectioning Procedures
169
Semithin Sections
172
Grid Selection
173
Ultrathin Sections
174
Staining Methods for Semithins and Ultrathins
177
II ULTRATHIN SECTION STAINING
181
REFERENCES
191
CHAPTER 4 TECHNIQUES
192
Polychrome Stain for Semithin Sections
193
Staining Ultrathin Sections
194
Subbing Slides
198
Cytochemistry
199
II SPECIFIC REACTION PRODUCTS
201
III EXAMPLES OF ENZYME CYTOCHEMISTRY
202
IV EXAMPLES OF NONENZYMATIC CYTOCHEMISTRY
205
REFERENCES
209
CHAPTER 5 TECHNIQUES
211
Silver Methenamine Staining for Polysaccharides
212
Calcium Staining
215
Postfixation Calcium Staining with Pyroantimonate
217
Immunocytochemistry
221
II PREPARATIVE TECHNIQUES
222
III IMMUNOGLOBULINS
226
IV COMMON IMMUNOLABELING TECHNIQUES FOR ELECTRON MICROSCOPY
228
REFERENCES
232
CHAPTER 6 TECHNIQUES
234
Preparation of 13nm Colloidal Gold
235
Conjugation of Gold to Proteins
237
Indirect Immunolabeling Procedure for Sections of Materials Embedded in LR White or Lowicryl K4M Resin Acrylic Resins
239
Support Films
243
III METHODS
244
REFERENCES
246
CHAPTER 7 TECHNIQUES
247
FormvarCoated Aluminum Bridges for Slot Grids
252
Coating Grids with Butvar B98
254
Coating Grids with Collodion Films
255
Making Carbon Support Films
258
Replicas Shadowing and Negative Staining
261
II NEGATIVE STAINING
268
REFERENCES
271
CHAPTER 8 TECHNIQUES
273
DNA Plasmid Preparation for TEM
276
Negative Staining
277
Negative Staining with Phosphotungstic Acid
278
Negative Staining with Uranyl Acetate
281
FUNCTIONAL ASPECTS
304
DECISION MAKING
315
REFERENCES
323
Vacuum Systems
325
I TYPES OF GAUGES
326
II VACUUM PUMPS
328
III SEQUENTIAL OPERATION OF A COMPLETE VACUUM SYSTEM TO ACHIEVE HIGH VACUUM
336
IV LUBRICATION OF VACUUM SEALS AND LEAK DETECTION
338
REFERENCES
339
HighVoltage Transmission Electron Microscopes HVEM
341
III FUNCTIONAL ASPECTS OF HVEMs A Resolution
342
IV MICROSCOPE CONSTRUCTION
344
REFERENCES
347
Intermediate Voltage Electron Microscopes IVEM Electron Tomography and SingleParticle Electron Microscopy
349
II ELECTRON TOMOGRAPHY AND SINGLEPARTICLE ELECTRON MICROSCOPY
351
REFERENCES
357
Scanning Electron Microscopy
359
II THE USE OF SEM IN BIOLOGICAL RESEARCH AND MEDICINE
360
III PRINCIPLES OF THE SEM
362
IV OPERATION OF THE SEM
364
V INTERACTION OF THE ELECTRON BEAM AND SPECIMEN
365
VI SPECIMEN PREPARATION
373
VII ARTIFACTS AND THEIR CORRECTION
378
FEG LV AND ESEM INSTRUMENTS
379
REFERENCES
384
CHAPTER 13 TECHNIQUES
386
Organosilane CoverglassSlide Treatment
387
Critical Point Drying for SEM
388
Drying Samples with Hexamethyldisilazane
390
Sputter Coating
391
Vascular Casting with Mercox CL2B Resin
393
Microanalysis
397
II ELECTRON ENERGY LOSS SPECTROSCOPY EELS
403
Photography
407
I EMULSION COMPOSITION
408
II FILM TYPES
409
III PRODUCING A LATENT IMAGE
411
V DEVELOPMENT CONTROLS
414
VI PAPER TYPES
415
VII KEEPING PROPERTIES OF CHEMICALS AND PRECAUTIONS
416
VIII SHARPNESS
417
X COPY WORK
419
XI TYPES OF ENLARGERS
424
XII VIEWING A PRINT IN PERSPECTIVE
426
REFERENCES
427
CHAPTER 15 TECHNIQUES
428
Films in 35mm Format
429
Ilford PanF and FP4
430
Kodak TMax 100 Film
431
Kodak Kodalith Film
432
Kodak LPD4Precision Line Film
433
TungstenBalanced Kodak Ektachrome
434
Polaroid Copy Negatives Using Type 55 PN Film
435
Making Photographic Prints
436
Poster Preparation
439
Digital Imaging and Telemedicine
441
II TELEMEDICINETELEPATHOLOGY CONSIDERATIONS
448
REFERENCES
451
Morphometry and Stereology
453
II RESOLUTION AND DISCRIMINATION
454
IV STEREOLOGY
457
V COMPUTERASSISTED ANALYSIS OF MOVEMENT
459
REFERENCES
460
Photomicroscopy
461
II LIGHTMICROSCOPE OCULARS
467
III LIGHTMICROSCOPE CONDENSER ASSEMBLIES
468
IV SLIDE THICKNESS
469
VI TYPES OF OPTICAL SYSTEMS
470
REFERENCES
479
CHAPTER 18 TECHNIQUES
480
Focusing Using a Focusing Telescope Bertrand Lens
481
Reading an Objective Lens
482
Use of Focusing Collars on High Dry 40X Objective Lenses
483
Using Oil or WaterImmersion Objectives
484
Use of Filters with BlackandWhite Films
485
Use of Filters with Color Films
486
Laboratory Safety
489
General Sources for Information Concerning Microscopy
491
III SOCIETIES
492
Electron Microscopy Equipment and Supplies
493
II LIGHT MICROSCOPES
494
III ELECTRON MICROSCOPES
495
V HIGHVACUUM PUMPS
496
VII EQUIPMENT FOR CRYOTECHNIQUES
497
VIII SPUTTER COATERS AND VACUUM EVAPORATORS
498
Computing Micrometer Bar Sizes
499
Calibrating the TEM and the SEM
501
Materials and Methods WriteUp Suggestions for Standard TEM and SEM Preparations
503
II MATERIALS AND METHODS FOR ROUTINE SEM PREPARATION
504
Index
505
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