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

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Springer Science & Business Media, Dec 31, 2003 - Science - 534 pages
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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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Contents

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
72
Preparation of Virus Samples for Transmission Electron Microscopy
281
Ultracentrifugation Technique for Viral Sample Preparation
282
Immune Electron Microscopy for Concentrating Viruses
283
Viral Concentration with the Beckman Airfuge
284
Transmission Electron Microscopy
287
II THEORY OF ELECTRON OPTICS
288
III FOUR ASPECTS OF IMAGE FORMATION MEEK 1976
300
IV GENERAL TEM FEATURES
301

CHAPTER 1 TECHNIQUES
74
Preparation of Primary Aldehyde Fixatives
80
Preparation of Osmium Osmium Tetroxide Osmium Tetraoxide
81
Buffering Systems
85
Cacodylate Buffers
86
Phosphate Buffers
87
TrisHCI Buffer Chayen et al 1969
89
Sodium Acetate Buffer Chayen et al 1969
90
Dulbeccos PhosphateBuffered Saline
91
Resin Formulations
92
Spurr Resin
93
PolyBed 812 Resin
96
SPIPon 812
97
Araldite 6005 Resin
99
Mollenhauers EponAraldite Resin Adapted for Use with Epon Substitutes
100
London Resin Co LR White Resin
101
Lowicryl and LR Gold Resins
103
PEG Method for TEM Sections
104
JB4 Glycol Methacrylate Techniques for HighResolution Light Microscopy
105
Agar Embedment of Cell Suspensions or Subcellular Particulates for TEM
107
Preparing Adherent Tissue Culture Monolayers in Situ for TEM
109
Flat Embedding of Cell Cultures Grown on Permanox Tissue Culture Dishes for TEM
111
Sperm Fixation
114
Central Nervous System Fixation Brain Spinal Cord
116
Using Vacuum to Help Wet Fungal Plant or Insect Samples during Primary Fixation
117
Simultaneous GlutaraldehydeOsmium Fixation for Protozoan Samples or Samples with a Large Amount of Lipid
118
Killing Cells Prior to Chemical Fixation
119
Flat Embedding on Microscope Slides
120
Procedure for Deparaffining Samples
122
Cryotechniques
125
I HISTORY
126
II PURPOSE
127
III CRYOGENS
128
IV SAFETY PRECAUTIONS
129
VI USES OF FROZEN SPECIMENS
135
VII ARTIFACTS AND THEIR CORRECTION
146
REFERENCES
149
Cryoultramicrotomy for Structural Examinations or Consequent Immunolabeling
150
Ultramicrotomy
153
II KNIVES
155
III BLOCK TRIMMING
157
IV ULTRAMICROTOMY WORKING AREA
158
CHAPTER 3 TECHNIQUES
159
Making a Section Manipulation Tool
160
Making a Locking Ring for Forceps
161
Making Glass Knife Boats
164
Block Trimming
165
Sectioning Procedures
167
Semithin Sections
170
Grid Selection
171
Ultrathin Sections
172
Staining Methods for Semithins and Ultrathins
175
II ULTRATHIN SECTION STAINING
179
REFERENCES
189
CHAPTER 4 TECHNIQUES
190
Polychrome Stain for Semithin Sections
191
Staining Ultrathin Sections
192
Subbing Slides
196
Cytochemistry
197
II SPECIFIC REACTION PRODUCTS
199
III EXAMPLES OF ENZYME CYTOCHEMISTRY
200
IV EXAMPLES OF NONENZYMATIC CYTOCHEMISTRY
203
REFERENCES
207
CHAPTER 5 TECHNIQUES
209
Silver Methenamine Staining for Polysaccharides
210
Calcium Staining
213
Postfixation Calcium Staining with Pyroantimonate
215
Immunocytochemistry
219
II PREPARATIVE TECHNIQUES
220
III IMMUNOGLOBULINS
224
IV COMMON IMMUNOLABELING TECHNIQUES FOR ELECTRON MICROSCOPY
226
REFERENCES
230
CHAPTER 6 TECHNIQUES
232
Preparation of 13nm Colloidal Gold
233
Conjugation of Gold to Proteins
235
Indirect Immunolabeling Procedure for Sections of Materials Embedded in LR White or Lowicryl K4M Resin Acrylic Resins
237
Procedure for Immunolabeling Intact Cells Preembedment Labeling of Cell Surfaces
238
Support Films
241
III METHODS
242
REFERENCES
244
CHAPTER 7 TECHNIQUES
245
FormvarCoated Aluminum Bridges for Slot Grids
250
Coating Grids with Butvar B98
252
Coating Grids with Collodion Films
253
Making Carbon Support Films
256
Replicas Shadowing and Negative Staining
259
II NEGATIVE STAINING
266
REFERENCES
269
CHAPTER 8 TECHNIQUES
271
DNA Plasmid Preparation for TEM
274
Negative Staining
275
Negative Staining with Phosphotungstic Acid
276
Negative Staining with Uranyl Acetate
279
Negative Staining with Ammonium Molybdate
280
FUNCTIONAL ASPECTS
302
DECISION MAKING
313
REFERENCES
321
Vacuum Systems
323
I TYPES OF GAUGES
324
II VACUUM PUMPS
326
III SEQUENTIAL OPERATION OF A COMPLETE VACUUM SYSTEM TO ACHIEVE HIGH VACUUM
334
IV LUBRICATION OF VACUUM SEALS AND LEAK DETECTION
336
REFERENCES
337
HighVoltage Transmission Electron Microscopes HVEM
339
III FUNCTIONAL ASPECTS OF HVEMs A Resolution
340
IV MICROSCOPE CONSTRUCTION
342
V SAMPLE PREPARATION
344
REFERENCES
345
Intermediate Voltage Electron Microscopes IVEM Electron Tomography and SingleParticle Electron Microscopy
347
II ELECTRON TOMOGRAPHY AND SINGLEPARTICLE ELECTRON MICROSCOPY
349
REFERENCES
355
Scanning Electron Microscopy
357
II THE USE OF SEM IN BIOLOGICAL RESEARCH AND MEDICINE
358
III PRINCIPLES OF THE SEM
360
IV OPERATION OF THE SEM
362
V INTERACTION OF THE ELECTRON BEAM AND SPECIMEN
363
VI SPECIMEN PREPARATION
371
VII ARTIFACTS AND THEIR CORRECTION
376
FEG LV AND ESEM INSTRUMENTS
377
REFERENCES
382
CHAPTER 13 TECHNIQUES
384
Organosilane CoverglassSlide Treatment
385
Critical Point Drying for SEM
386
Drying Samples with Hexamethyldisilazane
388
Sputter Coating
389
Vascular Casting with Mercox CL2B Resin
391
Microanalysis
395
II ELECTRON ENERGY LOSS SPECTROSCOPY EELS
401
REFERENCES
402
Photography
405
I EMULSION COMPOSITION
406
II FILM TYPES
407
III PRODUCING A LATENT IMAGE
409
V DEVELOPMENT CONTROLS
412
VI PAPER TYPES
413
VII KEEPING PROPERTIES OF CHEMICALS AND PRECAUTIONS
414
VIII SHARPNESS
415
X COPY WORK
417
XI TYPES OF ENLARGERS
422
XII VIEWING A PRINT IN PERSPECTIVE
424
REFERENCES
425
CHAPTER 15 TECHNIQUES
426
Films in 35mm Format
427
Ilford PanF and FP4
428
Kodak TMax 100 Film
429
Kodak Kodalith Film
430
Kodak LPD4Precision Line Film
431
TungstenBalanced Kodak Ektachrome
432
Polaroid Copy Negatives Using Type 55 PN Film
433
Making Photographic Prints
434
Poster Preparation
437
Digital Imaging and Telemedicine
439
II TELEMEDICINETELEPATHOLOGY CONSIDERATIONS
446
REFERENCES
449
Morphometry and Stereology
451
II RESOLUTION AND DISCRIMINATION
452
IV STEREOLOGY
455
V COMPUTERASSISTED ANALYSIS OF MOVEMENT
457
REFERENCES
458
Photomicroscopy
459
II LIGHTMICROSCOPE OCULARS
465
III LIGHTMICROSCOPE CONDENSER ASSEMBLIES
466
IV SLIDE THICKNESS
467
VI TYPES OF OPTICAL SYSTEMS
468
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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