B/C M D/CAL LECTRC N M/C RC C C py ILLUSTRATED METHODS AND INTERPRETATIONS Arvid B. Maunsbach Department of Cell Biology Institute of Anatomy University of Aarhus Aarhus, Denmark Bj0rn A. Afzelius Department of Ultrastructure Research The Arrhenius Laboratories Stockholm University Stockholm, Sweden ACADEMIC PRESSSan Diego London Boston New York Sydney Tokyo Toronto
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CONTENTSFOREWORD xi 5. Long Fixation Times 40PREFACE xiii 6. Formaldehyde-GlutaraldehydeACKNOWLEDGMENTS XV Combinations 42 7. Potassium Permanganate, Picric Acid, and Ruthenium Red 44 CHAPTER 1 8. Lead Salts and Tannic Acid 46 9. Uranyl Acetate Postfixation 48 MICROGRAPH INTERPRETATION 10. Tannic Acid-Uranyl Acetate Variations 50 11. Osmium Tetroxide-Potassium Ferrocyanide 52 1. Classical Preparation Method 2 12. Osmium Tetroxide Artifacts 54 2. Low Temperature Approach 4 13. Glutaraldehyde Artifacts 56 3. A Common Test Specimen 6 4. Detection of Objects 8 5. Identification of Artifacts 10 CHAPTER 3 6. Analysis of Geometry 12 7. Biological Identification 14 FIXATIVE VEHICLE 8. Biological Diversity 16 1. Absence and Presence of Buffer 60 9. Analysis of Dynamics: Endocytosis 18 2. Comparison of Buffers 6210. Analysis of Dynamics: Synthesis 2011. Comparison of Methods 22 3. Osmolality of Perfusion Fixatives 6412. Variations in Magnifications 24 4. Effects of Osmolality on Cell Shape 6613. Interpretation Difficulties 26 5. Effects of Osmolality on Cell Organelles 6814. Diagnostic Pathology 28 6. Adjustment of Osmolality with Sucrose 70 7. Colloid Osmotic Pressure: Low Magnification 72 CHAPTER 2 8. Colloid Osmotic Pressure: High Magnification 74 FIXATIVES 9. Phosphate Buffer Precipitate 76 1. Osmium Tetroxide and Glutaraldehyde at Low Magnification 32 CHAPTER 4 2. Osmium Tetroxide and Glutaraldehyde at High Magnification 34 FIXATIVE APPLICATION 3. Glutaraldehyde Concentration: Perfusion Fixation 36 1. Perfusion-Fixation versus 4. Glutaraldehyde Concentration: Immersion Immersion-Fixation 80 Fixation 38 2. Perfusion-Fixation with Pressure Control 82
vi CONTENTS 3. Fixation by Dripping in Vivo 84 3. Holey Films 146 4. Immersion-Fixation 86 4. Thick and Thin Support Films 148 5. Variability within the Tissue 88 5. Folds in Support Film 150 6. Unsuccessful Perfusion-Fixation 90 6. Defects in Formvar Films 152 7. Superficial Tissue Damage 92 7. Common Contaminants 154 8. Early Postmortal Changes 94 8. Volatile Contamination 156 9. Late Postmortal Changes 9610. Influence of Biopsy Method 9811. Microwave Treatment 100 CHAPTER 8 ULTRAMICROTOMY CHAPTER 5 1. Correlation of Light and Electron DEHYDRATION AND EMBEDDING Microscopy 160 2. Section Thickness: Low Magnification 162 1. Stepwise versus Direct Dehydration 104 3. Section Thickness: High Magnification 164 2. Prolonged Dehydration in Ethanol 106 4. Section Thickness: Half-Micron Section 166 3. Prolonged Dehydration in Acetone 108 5. Determination of Section Thickness 168 4. Inert Dehydration 110 6. Folds in the Section 170 5. Choice of Intermediate Solvent 112 7. Collection of Sections 172 6. Epon, Araldite, and Vestopal: Unstained 8. Surface Topography of Sections 174 Sections 114 9. Knife Scratches 176 7. Epon, Araldite, and Vestopal: Stained 10. Mottling and Flaking 178 Sections 116 11. Worn Glass Knives 180 8. Different Brands of Epoxy Resins 118 12. Transmitted Vibrations 182 9. Spurr and LR White 120 13. Vibrations and Knife Marks 18410. Embedding of Isolated Cells 122 14. Selective Chatter 186 15. Compression 188 CHAPTER 6 16. Holes and Deformations 190 17. Contamination during Microtomy 192 FREEZING A N D L O W - 18. Extraction during Sectioning 194 TEMPERATURE EMBEDDING 19. Cryoultramicrotomy: Survey Sections 196 20. Collection of Cryosections 1981. Plunge Freezing 126 21. Thickness of Cryosections 2002. Contact Freezing of Unfixed Tissue 128 22. Staining of Cryosections 2023. Contact Freezing of Fixed Tissue 130 23. Defects in Cryosections 2044. High-Pressure Freezing 1325. Freeze-Substitution in Methanol/Uranyl Acetate 134 CHAPTER 96. Freeze-Substitution in Osmium Tetroxide/ Acetone 136 SECTION-STAINING7. Progressive Lowering of Temperature Embedding in Lowicryl 138 1. Lead Citrate Staining 208 2. Uranyl Acetate Staining 210 3. Enhanced Section Staining 212 CHAPTER 7 4. Effects of Grid Storage 214 5. Section Exposed to Electron Beam 216 SUPPORT FILMS 6. Effect of Electron Beam 2181. Surface Topography 142 7. Lead-Staining Granularity 2202. Stability of Film or Section 144 8. Contamination 222
CONTENTS vii 9. Block-Staining Precipitate 224 6. Damage to Negatives 30010. Removal of Contamination 226 7. Damage to Wet Negatives 302 8. Film/Imaging Plate/Charge-Coupled Device (CCD) Camera 304 CHAPTER 1 0 9. Enlarged Digital Recordings 306 10. Variation in Electron Dose 308 MICROSCOPY 11. Corrections of CCD Camera 310 1. Resolving Power 230 2. Through-Focus Series: Hole and Latex CHAPTER 1 2 Particle 232 3. Through-Focus Series: Myelin Sheath 234 PHOTOGRAPHIC AND 4. Through-Focus Series: Cells 236 DIGITAL PRINTING 5. Minimum Contrast Focusing 238 6. Wobbler Focusing 240 1. Photographic Paper of Different Grades 314 7. Accelerating Voltages 20-100 kV 242 2. Multigrade Paper 316 8. Accelerating Voltages 80-200 kV 244 3. Exposure and Development 318 9. Unsaturated Electron Beam 246 4. Enlargement of Micrograph Details 32010. Condenser Apertures 248 5. Objective Lens in Enlarger 32211. Objective Aperture 250 6. Focusing of Enlarger 32412. Through-Focus Series: Astigmatism 252 7. Intermediate Diapositive 32613. Image Distortion 254 8. Errors in Photographic Printing 32814. Chromatic Aberration 256 9. Retouch 33015. Mechanical Instability 258 10. Comparison of Printers: Low16. Specimen Drift versus Astigmatism 260 Magnification 33217. Focus Drift 262 11. Comparison of Printers: Enlarged Prints 33418. Electrical Instabilities 264 12. Pixel Size at Printing 33619. Contamination in the Electron Beam 26620. Radiation Damage 268 CHAPTER 1321. Radiation Damage and Contamination 27022. Low-Dose Exposure 272 NEGATIVE STAINING23. Spectroscopic Imaging: Thin Film 27424. Spectroscopic Imaging: Thick Section 276 1. Negative Staining Methods 34025. Spectroscopic Imaging: Carbon 278 2. Properties of Support Film 34226. Spectroscopic Imaging: Calcium 280 3. Comparison of Stains 34427. Spectroscopic Imaging: Contrast Changes 282 4. Thickness of Stain 34628. Cryoelectron Microscopy: Na, K-ATPase 5. Concentration of Specimen 348 Crystals 284 6. Deformation of Specimen 35029. Defects in Cryoelectron Micrographs 286 7. Radiation Damage 352 CHAPTER 1 1 CHAPTER 14 IMAGE RECORDING AUTORADIOGRAPHY 1. Exposure Time 290 1. Undeveloped Emulsion 356 2. Over/Underexposure 292 2. Developed Emulsion 358 3. Effects of Development 294 3. Resolution 360 4. Exposure Dose Adjustment 296 4. Quantitation 362 5. Primary Magnification 298 5. Preparatory Defects 364
viii CONTENTS CHAPTER 15 7. Complementary Replicas and Stereo Images 440 CYTOCHEMISTRY 8. Ice Crystals and Etching 442 9. Quick-Freeze Deep Etching 4441. Influence of Fixation 368 10. Identification of Transport Molecules 4462. Preincubation Treatment 370 11. Contamination 4483. Appearance of Reaction Product 372 12. Plastic Distortion 4504. Composition of Incubation Medium 374 13. Replica Defects 4525. Cytochemical Resolution 3766. Unspecific Staining 3787. Extraction of Reaction Product 380 CHAPTER 18 SAMPLING AND QUANTITATION CHAPTER 16 1. Calibration of Magnification 456 IMMUNOCYTOCHEMISTRY 2. Sampling and Object Variability 458 3. Sampling of Pellets: Differential 1. Fixation of Sensitive Antigens 384 Centrifugation 460 2. Fixation of Insensitive Antigens 386 4. Sampling of Pellets: Gradient 3. Comparison of Embedding Media 388 Centrifugation 462 4. Influence of Preincubation Solutions 390 5. Micrograph Montages 464 5. Comparison of Primary Antibodies 392 6. Automated Digital Montages 466 6. Dilution of Primary Antibody 394 7. Resolution of Digital Montages 468 7. Quantitation of Gold Particles 396 8. Measurements on Digital Images 470 8. Controls 398 9. Stereological Grids 472 9. Comparison of Gold Probes 400 10. Cycloid Test System 47410. Amplification of Gold Particles 40211. Section Staining 404 CHAPTER 1912. Resolution 40613. Background Labeling 408 IMAGE PROCESSING14. Antigen Retrieval by Etching 41015. Antigen Retrieval with Sodium Dodecyl 1. Digital Contrast Changes 478 Sulfate 412 2. Processing of Scanned Image 48016. Double Labeling 414 3. Translational Image Enforcement 48217. Immunonegative Staining 416 4. Averaging of Macromolecular Assemblies 48418. Freeze-Fracture Replica Labeling 418 5. Rotational Image Enforcement 48619. Preembedding Labeling 420 6. Photographic versus Computer Averaging 48820. Semithin Light Microscopic Sections 422 7. Fourier Correction of Section Chatter 490 8. Removal of Image Defects 492 9. Scientific Fraud: Removal of Objects 494 CHAPTER 17 496 10. Scientific Fraud: Manipulation of Labeling FREEZE FRACTURING AND SHADOWING CHAPTER 2 0 1. Shadowing of DNA Molecules 428 THREE-DIMENSIONAL 2. Shadowing of Protein Molecules 430 RECONSTRUCTIONS 3. Freeze-Fractured Membrane Faces 432 4. Thickness of Replica: Low Magnification 434 1. Comparison between Transmission and Scanning 5. Thickness of Replica: High Magnification 436 Electron Microscopy 500 6. Rotary Shadowing 438 2. Serial Sectioning 502
CONTENTS ix3. Large Three-Dimensional Objects 504 4. Support Films 5264. Tilting of Section Cell Nucleus 506 5. Ultramicrotomy 5285. Tilting of Section" Nuclear Envelope 508 6. Section Staining 5306. Helical Structures 510 7. Microscopy and Image Recording 5327. Computer-Analyzed Helices 512 8. Photographic Work 5338. A Three-Dimensional Model of 9. Negative Staining 534 Na, K-ATPase 514 10. Autoradiography 535 11. immunolabeling 536 APPENDIX 12. Freeze Fracture 538 PRACTICAL M E T H O D S AUTHOR BIOGRAPHIES 539 1. Fixation 517 ACKNOWLEDGMENTS FOR REPRODUCTION 2. Dehydration and Embedding 522 OF FIGURES 541 3. Low Temperature Embedding 524 INDEX 545
FOREWORD Professors Arvid Maunsbach and Bj6rn Afzelius are publish early, and often, to compete for dwindling grantuniquely qualified to author a book on methods of speci- funds have led to a decline in the quality of publishedmen preparation for biomedical electron microscopy. electron micrographs. Meticulous preparation of tissuesThey are not newcomers to this field. They were among is essential to avoid artifacts that may lead to interpreta-the pioneers in the application of the electron microscope tions of questionable validity. This well-written, abun-to the study of cells and tissues. In distinguished research dantly illustrated, book describes in detail the procedurescareers spanning some 40 years, they have witnessed the required to obtain images of cells and cell organelles freedevelopment of a broad range of methods of specimen of artifact, and provides guidance for image processingpreparation for transmission electron microscopy, and and interpretation. It will be a rich source of information,have made important contributions to the improvement and inspiration, for younger investigators struggling toof some of those methods. The electron micrographs in attain optimal results with traditional methods and fortheir classic papers on the kidney, liver, and spermatozoa older investigators desirous of applying some of the newerhave set a high standard of quality that others have sought techniques to their own research.to attain. There is a real need for this book. Regrettably, the Don W. Fawcett, M.D.current preoccupation of biomedical scientists with the Hersey Professor of Anatomy and Cell Biology, Emeritusnewer field of molecular biology, and the pressures to Harvard Medical School xi
PREFACE Electron microscopy has fundamentally changed the procedures in biological electron microscopy, rangingknowledge about cell structure and function. It is now from fixation through microtomy and microscopy toan indispensable tool in many fields of cell biology and photographic procedures. The second part exemplifiesmedicine and includes a large variety of preparatory more special preparation techniques, including autoradi-methods. Each of these has its own sets of information ography, cytochemistry, immunoelectron microscopy,possibilities along with interpretative difficulties and pit- and computer-assisted image analysis of electron micro-falls. A profound understanding of the methods is there- graphs. Each chapter begins with an introduction, whichfore required for an in-depth analysis and evaluation of we have kept quite short, but which includes a note onstructure-function relationships. In this volume we illus- the early development in the field. The presentations oftrate the basic preparatory methods for transmission elec- the micrographs start with a short section on the prepara-tron microscopy in biology and medicine, and we hope tion method. Next to this is a description of what can bethat this monograph will be a guide through the broad observed and finally we have some comments on thespectrum of methods. results. Details of the preparatory procedures used for From discussions with our research students we have most of the illustrations are included in the Appendixrealized that the interpretation or "reading" of electron (Practical Methods) or can be found in the literaturemicrographs often presents more of a problem than the listed at the end of each chapter. These reference listsactual preparation and microscopy of the biological speci- include both classical studies and more recent key publi-mens. We have found that the best way to help students cations.in critically evaluating their micrographs is to show exam- Most illustrations come from preparations that weples of t h e i r ~ a n d of o u r ~ g o o d and failed preparations. have performed specifically for this monograph. SomeThese comparisons have then formed the basis of discus- originate from our own published papers (see Acknowl-sions of the biological significance of various structures edgments for Reproduction of Figures). As experimen-in electron micrographs. tal material we have mainly used tissues and cell types This monograph should be useful for investigators that have been extensively studied in our laboratories,working with transmission electron microscopy in biology particularly kidney tubule cells, hepatocytes, and sper-and medicine~beginners as well as more experienced matozoa. To a large extent, the content of this volumeresearchers. Investigators in other fields of biology may therefore reflects our own hands-on experiences. Thisuse it as a guide in evaluating and interpreting electron focus on a few types of cells and tissues may appear tomicrographs. Research technicians may find it useful for be a limitation, but will facilitate comparisons of dif-choosing optimal preparation methods and identifying ferent procedures on the same tissue. Moreover, manyartifacts. methods apply almost equally well to different cell The first part of this monograph presents the basic types. XlII
ACKNOWLEDGMENTS Much of the inspiration for the present work can be This monograph would not have been completed with-traced back to our early years of training in biological out the technical help of several persons, particularlyelectron microscopy under the critical scientific guidance Karen Thomsen and Else-Merete LOcke in Aarhus andof Professor Fritiof S. SjOstrand. Ulla Afzelius in Stockholm, who participated in this ven- Plans for this monograph were already laid in the 1970s ture with great skill and enthusiasm. We also acknowl-in connection with a series of postgraduate courses in edge the outstanding photographic work of Albert Meierbiological electron microscopy organized by Jos6 David- and thank him for many valuable suggestions. At previousFerreira, then director of the Laboratorio Biologia Celu- stages of this project we also received important help fromlar at the Gulbenkian Institute, Oeiras, Portugal. Over Margrethe Aarup, Gunilla Almesj/5, Christina Bohman,the following years, we continued to assemble material Poul Boldsen, Marianne Ellegaard, Maj Hasselgren, andduring our postgraduate courses in Denmark and Sweden Inger Kristoffersen. Stig Sundelin in Stockholm and Arneuntil we decided to finalize the work. Christensen, Ole Moeskja~r, and the late Ejnar Hansen We are indebted to the late Sven-Olof Bohman, to in Aarhus carefully maintained microscopes and com-Hans Hebert and BjCrn Johansen for very useful advice puter equipment.on various chapters of this book; to Jos6 and Karin David- The task of bringing this volume into readable shapeFerreira for allowing us to include some micrographs; was performed by Dorrit Ipsen, Jytte Kragelund, andand to our co-authors over the years for inspiring discus- Bente Kragh. We thank them for very competent secre-sions. We also thank several colleagues and friends who tarial help and editing and for being enormously patientgenerously shared their specimens or antibodies with us, with our constant rewriting of the text.helped to prepare specimens or micrographs, and/or gave Last but not least we thank the staff at Academicus their constructive comments: Ulla Afzelius, Peter Press in San Diego for very constructive and stimulatingAgre, Pier Luigi Bellon, Emile L. Boulpaep, Erik Ilsr cooperation: Craig Panner for advice and planning, LoriChristensen, Gunna Christiansen, Romano Dallai, Carl Asbury for production, Michael Remener for design, andChristian Danielsen, Jens DCrup, Hans JCrgen Gund- Debby Bicher for artwork.ersen, Anders H/56g, Kaj Josephsen, Peter Leth This project was financially supported in part by theJ0rgensen, Lars Kihlborg, Salvatore Lanzavecchia, Else- Danish Medical Research Council, the Swedish NaturalMerete LOcke, S0ren Mogensen, Jesper Vuust M011er, Science Research Council, the Danish Biomembrane Re-S0ren Nielsen, T. Steen Olsen, Peter Ottosen, Kaarina search Center, the Karen Elise Jensen Foundation, thePihakaski-Maunsbach, Finn Reinholt, Elisabeth Skriver, Novo Nordisk Foundation, and the Aarhus UniversityMargaret SOderholm, Karen Thomsen, Pierre Verroust, Research Foundation.Hans Orskov, and several others, who may not always A.B.M. and B.A.A.have been aware of how valuable their comments were Aarhus and Stockholmto us. February 1998 XV
MICROGRAPH INTERPRETATION 1. Classical Preparation Method 8. Biological Diversity 2. Low Temperature Approach 9 Analysis of Dynamics: Endocytosis 3. A Common Test Specimen 10 Analysis of Dynamics: Synthesis 4. Detection of Objects 11 Comparison of Methods 5. Identification of Artifacts 12 Variations in Magnifications 6. Analysis of Geometry 13 Interpretation Difficulties 7. Biological Identification 14 Diagnostic Pathology Present-day ultrastructure research aims at the under- significance of which may not be readily apparent. Inter-standing of biological structure-function relationships at pretation requires an understanding of the principles ofcellular and molecular levels using a wide variety of pre- the techniques as well as experience in detecting method-paratory procedures, such as cytochemistry, immunocyto- ological errors. The electron microscope image is charac-chemistry, negative staining, and image analysis. These terized by its wealth of information. However, only partmethods all depend on a series of basic procedures that of the information is directly related to the biologicalare crucial for the outcome of the analyses, e.g., fixation object itself; other parts depend on various preparatoryprocedures, ultramicrotomy, and, not the least, mi- steps and to instrument characteristics. As a consequence,croscopy. electron micrographs are far less accessible to interpreta- For optimal results a number of methodological as- tion than they appear to be.pects have to be considered: What preparation method The process of electron micrograph interpretation canshould be used? Should different methods be applied in intentionally or unintentionally be divided into a seriesparallel? In what ways will the preparation procedure of consecutive steps or levels of analysis, for example:influence the object? Is the object frequent or rare? 9 detection of objectsWhich magnification should be used for optimal results? 9 identification of artifactsWhat quantitative aspects should be considered? What 9 analysis of geometryis the appearance of a "golden standard" preparation? 9 biological identification The first part of this chapter serves to illustrate briefly 9 analysis of dynamicssome of these questions. It starts with illustrations ofkidney and liver cells, which we regard to be almost opti- Examples of these five steps of analysis are given inmally prepared by present-day standards. Comparisons the following. The late steps of interpretation usuallyare then made between entirely different methods. The depend on the early ones. An ability to master the initialnecessity to use the right magnifications for the object sequence of interpretation is required for the analysis ofunder study is emphasized and we raise the important the late sequence to be meaningful.question of whether an abnormal pattern is due to a The validity of the final biological conclusions is invari-preparatory artifact or represents a pathological change ably correlated to the quality of the information obtained.of the biological object. Thus, emphasis on high technical standards and method- The last part of this chapter deals with the fact that ological insight is not lart pour lart in electron micros-electron micrographs represent scientific raw data, the copy but a requirement for the analysis.
2 1, MICROGRAPH INTERPRETATION1, Classical Preparation Method FIGURE 1.1 A transmission electron micrographshowing part of the cytoplasm in a rat liver cell. Theanesthetized animal was perfusion fixed through the ab-dominal aorta with 1% glutaraldehyde in 0.1 M cacodylatebuffer. Excised blocks of liver tissue were postfixed, firstin the same glutaraldehyde fixative and then in 1% os-mium tetroxide, dehydrated in ethanol, and embeddedin Epon 812. Ultrathin sections were cut on a diamondknife and stained with uranyl acetate and lead citrate.x 36,000. This micrograph is representative of a hepatocyte prepared by a conventional chemi-cal fixation and embedding procedure. This micrograph is considered as approachingthe "state-of-the-art" preparation. It is free of obvious artifacts, such as disruptionof membranes, extracted regions of ground substance, or organelles, and shows noinstrumental errors, such as astigmatism or specimen drift, yet there is no way to knowthe "true" structural appearance of a liver cell or any other cell in a transmissionelectron micrograph. For example, one may question whether the membranes of theR E R in reality have a slightly wavy conformation, as seen here, or are more planar.Have the mitochondria retained the same diameters and positions as they had in theliving cell? Are the separate mitochondrial profiles seen in this figure in reality incontinuity outside the plane of the section? The electron micrograph is to be regarded as representing a snapshot of a momentin the life of the cell. When interpreting the micrograph the investigator must also takethe dynamics and the movements of the organelles in consideration. Mitochondria maydivide or fuse with other mitochondria, various organelles may be transported alongthe microtubules, which themselves may grow or shorten, and secretory vesicles openat the cell surface. The ultrastructure of cells and tissues as they appear in conventionally fixed andresin-embedded specimens has not changed appreciably since the early 1960s, despitethe dramatic improvements of the transmission electron microscope itself, which nowfeatures increased flexibility in handling and extended automation due to computeriza-tion. Indeed, the major improvement in transmission electron microscopy of noncryo-preparations has been the result of an expanded repertoire of preparatory methodssuch as cytochemistry, autoradiography, and, not the least, immunoelectron microscopy.
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