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VetBooks.ir  CONTENTS i FIFTEENTH EDITION VetBooks.ir Junqueira’s Basic Histology T E X T A N D AT L A S Anthony L. Mescher, PhD Professor of Anatomy and Cell Biology Indiana University School of Medicine Bloomington, Indiana New York Chicago San Francisco Athens London Madrid Mexico City Milan New Delhi Singapore Sydney Toronto 00_Mescher_FM_pi-x.indd 1 27/04/18 11:03 am  Copyright © 2018 by McGraw-Hill Education. All rights reserved. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written permission of the publisher. VetBooks.ir ISBN: 978-1-26-002618-4 MHID: 1-26-002618-3 The material in this eBook also appears in the print version of this title: ISBN: 978-1-26-002617-7, MHID: 1-26-002617-5. eBook conversion by codeMantra Version 1.0 All trademarks are trademarks of their respective owners. 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This limitation of liability shall apply to any claim or cause whatsoever whether such claim or cause arises in contract, tort or otherwise.  Contents VetBooks.ir PREFACE VII | ACKNOWLEDGMENTS IX 1 Histology & Its Methods 5 Connective Tissue 96 of Study 1 Cells of Connective Tissue 96 Preparation of Tissues for Study 1 Fibers 103 Light Microscopy 4 Ground Substance 111 Electron Microscopy 8 Types of Connective Tissue 114 Autoradiography 9 Summary of Key Points 119 Cell & Tissue Culture 10 Assess Your Knowledge 120 Enzyme Histochemistry 10 Visualizing Specific Molecules 10 6 Adipose Tissue 122 Interpretation of Structures in Tissue White Adipose Tissue 122 Sections 14 Brown Adipose Tissue 126 Summary of Key Points 15 Summary of Key Points 127 Assess Your Knowledge 16 Assess Your Knowledge 128 2 The Cytoplasm 17 7 Cartilage 129 Cell Differentiation 17 Hyaline Cartilage 129 The Plasma Membrane 17 Elastic Cartilage 133 Cytoplasmic Organelles 27 Fibrocartilage 134 The Cytoskeleton 42 Cartilage Formation, Growth, & Repair 134 Inclusions 47 Summary of Key Points 136 Summary of Key Points 51 Assess Your Knowledge 136 Assess Your Knowledge 52 8 Bone 138 3 The Nucleus 53 Bone Cells 138 Components of the Nucleus 53 Bone Matrix 143 The Cell Cycle 58 Periosteum & Endosteum 143 Mitosis 61 Types of Bone 143 Stem Cells & Tissue Renewal 65 Osteogenesis 148 Meiosis 65 Bone Remodeling & Repair 152 Apoptosis 67 Metabolic Role of Bone 153 Summary of Key Points 69 Joints 155 Assess Your Knowledge 70 Summary of Key Points 158 Assess Your Knowledge 159 4 Epithelial Tissue 71 Characteristic Features of Epithelial Cells 72 9 Nerve Tissue & the Nervous Specializations of the Apical Cell Surface 77 System 161 Types of Epithelia 80 Development of Nerve Tissue 161 Transport Across Epithelia 88 Neurons 163 Renewal of Epithelial Cells 88 Glial Cells & Neuronal Activity 168 Summary of Key Points 90 Central Nervous System 175 Assess Your Knowledge 93 Peripheral Nervous System 182 iii 00_Mescher_FM_pi-x.indd 3 27/04/18 11:03 am  iv CONTENTS Neural Plasticity & Regeneration 187 15 Digestive Tract 295 Summary of Key Points 190 VetBooks.ir General Structure of the Digestive Tract 295 Assess Your Knowledge 191 Oral Cavity 298 Esophagus 305 10 Muscle Tissue 193 Stomach 307 Skeletal Muscle 193 Small Intestine 314 Cardiac Muscle 207 Large Intestine 318 Smooth Muscle 208 Summary of Key Points 326 Regeneration of Muscle Tissue 213 Assess Your Knowledge 327 Summary of Key Points 213 Assess Your Knowledge 214 16 Organs Associated with the Digestive Tract 329 11 The Circulatory System 215 Salivary Glands 329 Heart 215 Pancreas 332 Tissues of the Vascular Wall 219 Liver 335 Vasculature 220 Biliary Tract & Gallbladder 345 Lymphatic Vascular System 231 Summary of Key Points 346 Summary of Key Points 235 Assess Your Knowledge 348 Assess Your Knowledge 235 17 The Respiratory System 349 12 Blood 237 Nasal Cavities 349 Composition of Plasma 237 Pharynx 352 Blood Cells 239 Larynx 352 Summary of Key Points 250 Trachea 354 Assess Your Knowledge 252 Bronchial Tree & Lung 354 Lung Vasculature & Nerves 366 13 Hemopoiesis 254 Pleural Membranes 368 Stem Cells, Growth Factors, & Differentiation 254 Respiratory Movements 368 Bone Marrow 255 Summary of Key Points 369 Maturation of Erythrocytes 258 Assess Your Knowledge 369 Maturation of Granulocytes 260 Maturation of Agranulocytes 263 18 Skin 371 Origin of Platelets 263 Epidermis 372 Summary of Key Points 265 Dermis 378 Assess Your Knowledge 265 Subcutaneous Tissue 381 Sensory Receptors 381 14 The Immune System & Lymphoid Hair 383 Organs 267 Nails 384 Innate & Adaptive Immunity 267 Skin Glands 385 Cytokines 269 Skin Repair 388 Antigens & Antibodies 270 Summary of Key Points 391 Antigen Presentation 271 Assess Your Knowledge 391 Cells of Adaptive Immunity 273 Thymus 276 19 The Urinary System 393 Mucosa-Associated Lymphoid Tissue 281 Kidneys 393 Lymph Nodes 282 Blood Circulation 394 Spleen 286 Renal Function: Filtration, Secretion, & Summary of Key Points 293 Reabsorption 395 Assess Your Knowledge 294 Ureters, Bladder, & Urethra 406 00_Mescher_FM_pi-x.indd 4 27/04/18 11:03 am  CONTENTS v Summary of Key Points 411 22 The Female Reproductive System 460 Assess Your Knowledge 412 VetBooks.ir Ovaries 460 Uterine Tubes 470 20 Endocrine Glands 413 Major Events of Fertilization 471 Pituitary Gland (Hypophysis) 413 Uterus 471 Adrenal Glands 423 Embryonic Implantation, Decidua, & the Placenta 478 Pancreatic Islets 427 Cervix 482 Diffuse Neuroendocrine System 429 Vagina 483 Thyroid Gland 429 External Genitalia 483 Parathyroid Glands 432 Mammary Glands 483 Pineal Gland 434 Summary of Key Points 488 Summary of Key Points 437 Assess Your Knowledge 489 Assess Your Knowledge 437 23 The Eye & Ear: Special Sense Organs 490 21 The Male Reproductive Eyes: The Photoreceptor System 490 System 439 Ears: The Vestibuloauditory System 509 Testes 439 Summary of Key Points 522 Intratesticular Ducts 449 Assess Your Knowledge 522 Excretory Genital Ducts 450 Accessory Glands 451 APPENDIX 525 Penis 456 Summary of Key Points 457 FIGURE CREDITS 527 Assess Your Knowledge 459 INDEX 529 00_Mescher_FM_pi-x.indd 5 27/04/18 11:03 am VetBooks.ir This page intentionally left blank 00_Mescher_FM_pi-x.indd 6 27/04/18 11:03 am  Preface VetBooks.ir With this 15th edition, Junqueira’s Basic Histology continues as U.S. medical schools) can access a complete human histology the preeminent source of concise yet thorough information Laboratory Guide linked to the virtual microscope at the URL on human tissue structure and function. For over 45 years given below. This digital Laboratory Guide, which is new with this educational resource has met the needs of learners for a this edition of the text and unique among learning resources well-organized and concise presentation of cell biology and offered by any histology text and atlas, provides both links histology that integrates the material with that of biochemistry, to the appropriate microscope slides needed for each topic immunology, endocrinology, and physiology and provides and links to the correlated figures or tables in the text. Those an excellent foundation for subsequent studies in pathology. without AccessMedicine will lack the digital Laboratory Guide, The text is prepared specifically for students of medicine but may still study and utilize the 150 virtual microscope and other health-related professions, as well as for advanced slides of all human tissues and organs, which are available at: undergraduate courses in tissue biology. As a result of its value http://medsci.indiana.edu/junqueira/virtual/junqueira.htm. and appeal to students and instructors alike, Junqueira’s Basic As with the previous edition, the book facilitates learning Histology has been translated into a dozen different languages by its organization: and is used by medical students throughout the world. An opening chapter reviews the histological techniques Unlike other histology texts and atlases, the present that allow understanding of cell and tissue structure. work again includes with each chapter a set of multiple- Two chapters then summarize the structural and choice Self-Test Questions that allow readers to assess their functional organization of human cell biology, comprehension and knowledge of important points in that presenting the cytoplasm and nucleus separately. chapter. At least a few questions in each set utilize clinical The next seven chapters cover the four basic tissues that vignettes or cases to provide context for framing the medical make up our organs: epithelia, connective tissue (and its relevance of concepts in basic science, as recommended by the US major sub-types), nervous tissue, and muscle. National Board of Medical Examiners. As with the last edition, Remaining chapters explain the organization and each chapter also includes a Summary of Key Points designed to functional significance of these tissues in each of guide the students concerning what is clearly important and what the body’s organ systems, closing with up-to-date is less so. Summary Tables in each chapter organize and condense consideration of cells in the eye and ear. important information, further facilitating efficient learning. Each chapter has been revised and shortened, while For additional review of what’s been learned or to coverage of specific topics has been expanded and updated as assist rapid assimilation of the material in Junqueira’s Basic needed. Study is facilitated by modern page design. Inserted Histology, McGraw-Hill has published a set of 200 full-color throughout each chapter are more numerous, short paragraphs Basic Histology Flash Cards, also authored by Anthony that indicate how the information presented can be used Mescher. Each card includes images of key structures to medically and which emphasize the foundational relevance of identify, a summary of important facts about those structures, the material learned. and a clinical comment. This valuable learning aid is available The art and other figures are presented in each chapter, as a set of actual cards from Amazon.com, or as an app for with the goal to simplify learning and integration with smartphones or tablets from the online App Store. related material. The McGraw-Hill medical illustrations, With its proven strengths and the addition of new features, now used throughout the text, are the most useful, thorough, I am confident that Junqueira’s Basic Histology will continue and attractive of any similar medical textbook. Electron and as one of the most valuable and most widely read educational light micrographs have been replaced throughout the book resources in histology. Users are invited to provide feedback as needed, and they again make up a complete atlas of cell, to the author with regard to any aspect of the book’s features. tissue, and organ structures fully compatible with the students’ own collection of glass or digital slides. Health science Anthony L. Mescher students whose medical library offers AccessMedicine among Indiana University School of Medicine its electronic resources (which includes more than 95% of [email protected] vii 00_Mescher_FM_pi-x.indd 7 27/04/18 11:03 am VetBooks.ir This page intentionally left blank 00_Mescher_FM_pi-x.indd 8 27/04/18 11:03 am  Acknowledgments VetBooks.ir I wish to thank the students at Indiana University School of Review, by Robert Klein and George Enders. The use here Medicine and the undergraduates at Indiana University with of questions from these valuable resources is gratefully whom I have studied histology and cell biology for over 35 years acknowledged. Students are referred to those review books for and from whom I have learned much about presenting basic hundreds of additional self-assessment questions. concepts most effectively. Their input has greatly helped in the I am also grateful to my colleagues and reviewers from task of maintaining and updating the presentations in this classic throughout the world who provided specialized expertise or textbook. As with the last edition, the help of Sue Childress and original photographs, which are also acknowledged in figure Dr. Mark Braun was invaluable in slide preparation and the captions. I thank those professors and students in the United virtual microscope for human histology respectively. States and countries throughout the world who provided useful As with the last edition, the present text includes ten suggestions that have improved the new edition of Junqueira’s multiple-choice questions at the end of each chapter, aimed Basic Histology. Finally, I am pleased to acknowledge the help to test the learner’s retention and understanding of important and collegiality provided by the staff of McGraw-Hill, especially points in that body of material. Many of these questions editors Michael Weitz and Brian Kearns, whose work made were used in my courses, but others are taken or modified possible publication of this 15th edition of Junqueira’s Basic from a few of the many excellent review books published by Histology. McGraw-Hill/Lange for students preparing to take the U.S. Medical Licensing Examination. These include Histology and Anthony L. Mescher Cell Biology: Examination and Board Review, by Douglas Indiana University School of Medicine Paulsen; USMLE Road Map: Histology, by Harold Sheedlo; and Anatomy, Histology, & Cell Biology: PreTest Self-Assessment & [email protected] ix 00_Mescher_FM_pi-x.indd 9 27/04/18 11:03 am VetBooks.ir This page intentionally left blank 00_Mescher_FM_pi-x.indd 10 27/04/18 11:03 am  1 Histology & Its VetBooks.ir C H A P T E R Methods of Study PREPARATION OF TISSUES FOR STUDY 1 AUTORADIOGRAPHY 9 Fixation 1 CELL & TISSUE CULTURE 10 Embedding & Sectioning 3 ENZYME HISTOCHEMISTRY 10 Staining 3 LIGHT MICROSCOPY 4 VISUALIZING SPECIFIC MOLECULES 10 Bright-Field Microscopy 4 Immunohistochemistry 11 Fluorescence Microscopy 5 Hybridization Techniques 12 Phase-Contrast Microscopy 5 INTERPRETATION OF STRUCTURES IN TISSUE Confocal Microscopy 5 SECTIONS 14 Polarizing Microscopy 7 SUMMARY OF KEY POINTS 15 ELECTRON MICROSCOPY 8 ASSESS YOUR KNOWLEDGE 16 Transmission Electron Microscopy 8 Scanning Electron Microscopy 9 H istology is the study of the tissues of the body and how these tissues are arranged to constitute organs. This subject involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ. a better knowledge of tissue biology. Familiarity with the tools and methods of any branch of science is essential for a proper understanding of the subject. This chapter reviews common methods used to study cells and tissues, focusing on micro- scopic approaches. Tissues have two interacting components: cells and extra- cellular matrix (ECM). The ECM consists of many kinds of macromolecules, most of which form complex structures, such as collagen fibrils. The ECM supports the cells and con- ››PREPARATION OF TISSUES tains the fluid transporting nutrients to the cells, and carry- FOR STUDY ing away their wastes and secretory products. Cells produce The most common procedure used in histologic research is the ECM locally and are in turn strongly influenced by matrix the preparation of tissue slices or “sections” that can be exam- molecules. Many matrix components bind to specific cell ined visually with transmitted light. Because most tissues and surface receptors that span the cell membranes and connect organs are too thick for light to pass through, thin translu- to structural components inside the cells, forming a contin- cent sections are cut from them and placed on glass slides for uum in which cells and the ECM function together in a well- microscopic examination of the internal structures. coordinated manner. The ideal microscopic preparation is preserved so that the During development, cells and their associated matrix tissue on the slide has the same structural features it had in the become functionally specialized and give rise to fundamen- body. However, this is often not feasible because the prepara- tal types of tissues with characteristic structural features. tion process can remove cellular lipid, with slight distortions Organs are formed by an orderly combination of these tissues, of cell structure. The basic steps used in tissue preparation for and their precise arrangement allows the functioning of each light microscopy are shown in Figure 1–1. organ and of the organism as a whole. The small size of cells and matrix components makes his- tology dependent on the use of microscopes and molecular Fixation methods of study. Advances in biochemistry, molecular biol- To preserve tissue structure and prevent degradation by ogy, physiology, immunology, and pathology are essential for enzymes released from the cells or microorganisms, pieces of 1 01_Mescher_ch01_p001-016.indd 1 27/04/18 6:44 pm  2 CHAPTER 1 Histology & Its Methods of Study FIGURE 1–1 Sectioning fixed and embedded tissue. VetBooks.ir 52°- 60°C (a) Fixation Dehydration Clearing Infiltration Embedding Drive wheel Block holder Paraffin block Tissue Steel knife b Most tissues studied histologically are prepared as shown, with Similar steps are used in preparing tissue for transmission elec- this sequence of steps (a): tron microscopy (TEM), except special fixatives and dehydrating solutions are used with smaller tissue samples and embedding Fixation: Small pieces of tissue are placed in solutions of involves epoxy resins which become harder than paraffin to allow chemicals that cross-link proteins and inactivate degradative very thin sectioning. enzymes, which preserve cell and tissue structure. Dehydration: The tissue is transferred through a series of (b) A microtome is used for sectioning paraffin-embedded tissues increasingly concentrated alcohol solutions, ending in 100%, for light microscopy. The trimmed tissue specimen is mounted which removes all water. in the paraffin block holder, and each turn of the drive wheel by Clearing: Alcohol is removed in organic solvents in which the histologist advances the holder a controlled distance, gener- both alcohol and paraffin are miscible. ally from 1 to 10 μm. After each forward move, the tissue block Infiltration: The tissue is then placed in melted paraffin until it passes over the steel knife edge and a section is cut at a thickness becomes completely infiltrated with this substance. equal to the distance the block advanced. The paraffin sections Embedding: The paraffin-infiltrated tissue is placed in a small are placed on glass slides and allowed to adhere, deparaffinized, mold with melted paraffin and allowed to harden. and stained for light microscope study. For TEM, sections less than Trimming: The resulting paraffin block is trimmed to expose 1 μm thick are prepared from resin-embedded cells using an ultra- the tissue for sectioning (slicing) on a microtome. microtome with a glass or diamond knife. organs are placed as soon as possible after removal from the microscopy, react with the amine groups (NH2) of proteins, body in solutions of stabilizing or cross-linking compounds preventing their degradation by common proteases. Glutaral- called fixatives. Because a fixative must fully diffuse through dehyde also cross-links adjacent proteins, reinforcing cell and the tissues to preserve all cells, tissues are usually cut into ECM structures. small fragments before fixation to facilitate penetration. To Electron microscopy provides much greater magni- improve cell preservation in large organs, fixatives are often fication and resolution of very small cellular structures, introduced via blood vessels, with vascular perfusion allowing and fixation must be done very carefully to preserve addi- fixation rapidly throughout the tissues. tional “ultrastructural” detail. Typically in such studies, One widely used fixative for light microscopy is forma- glutaraldehyde-treated tissue is then immersed in buffered lin, a buffered isotonic solution of 37% formaldehyde. Both osmium tetroxide, which preserves (and stains) cellular lip- this compound and glutaraldehyde, a fixative used for electron ids as well as proteins. 01_Mescher_ch01_p001-016.indd 2 26/04/18 11:10 am  Preparation of Tissues for Study 3 Embedding & Sectioning Staining C H A P T E R VetBooks.ir To permit thin sectioning, fixed tissues are infiltrated and Most cells and extracellular material are completely color- embedded in a material that imparts a firm consistency. less, and to be studied microscopically tissue sections must Embedding materials include paraffin, used routinely for light be stained (dyed). Methods of staining have been devised that microscopy, and plastic resins, which are adapted for both make various tissue components not only conspicuous but also light and electron microscopy. distinguishable from one another. Dyes stain material more or Before infiltration with such media, the fixed tissue must less selectively, often behaving like acidic or basic compounds undergo dehydration by having its water extracted gradually and forming electrostatic (salt) linkages with ionizable radicals 1 by transfers through a series of increasing ethanol solutions, of macromolecules in tissues. Cell components, such as nucleic Histology & Its Methods of Study Preparation of Tissues for Study ending in 100% ethanol. The ethanol is then replaced by an acids with a net negative charge (anionic), have an affinity for organic solvent miscible with both alcohol and the embedding basic dyes and are termed basophilic; cationic components, medium, a step referred to as clearing because infiltration with such as proteins with many ionized amino groups, stain more the reagents used here gives the tissue a translucent appearance. readily with acidic dyes and are termed acidophilic. The fully cleared tissue is then placed in melted paraffin Examples of basic dyes include toluidine blue, alcian blue, in an oven at 52°-60°C, which evaporates the clearing solvent and methylene blue. Hematoxylin behaves like a basic dye, and promotes infiltration of the tissue with paraffin, and then staining basophilic tissue components. The main tissue com- embedded by allowing it to harden in a small container of ponents that ionize and react with basic dyes do so because of paraffin at room temperature. Tissues to be embedded with acids in their composition (DNA, RNA, and glycosaminogly- plastic resin are also dehydrated in ethanol and then infiltrated cans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain with plastic solvents that harden when cross-linking polymer- the acidophilic components of tissues such as mitochondria, izers are added. Plastic embedding avoids the higher tempera- secretory granules, and collagen. tures needed with paraffin, which helps avoid tissue distortion. Of all staining methods, the simple combination of The hardened block with tissue and surrounding embed- hematoxylin and eosin (H&E) is used most commonly. ding medium is trimmed and placed for sectioning in an Hematoxylin stains DNA in the cell nucleus, RNA-rich por- instrument called a microtome (see Figure 1–1). Paraffin sec- tions of the cytoplasm, and the matrix of cartilage, produc- tions are typically cut at 3-10 μm thickness for light microscopy, ing a dark blue or purple color. In contrast, eosin stains other but electron microscopy requires sections less than 1 μm thick. cytoplasmic structures and collagen pink (Figure 1–2a). Here One micrometer (1 μm) equals 1/1000 of a millimeter (mm) eosin is considered a counterstain, which is usually a single or 10−6 m. Other spatial units commonly used in microscopy dye applied separately to distinguish additional features of a are the nanometer (1 nm = 0.001 μm = 10−6 mm = 10−9 m) and tissue. More complex procedures, such as trichrome stains (eg, angstrom (1 Å = 0.1 nm or 10−4 μm). The sections are placed on Masson’s trichrome), allow greater distinctions among various glass slides and stained for light microscopy or on metal grids extracellular tissue components. for electron-microscopic staining and examination. The periodic acid–Schiff (PAS) reaction utilizes the hexose rings of polysaccharides and other carbohydrate-rich › ›› MEDICAL APPLICATION tissue structures and stains such macromolecules distinctly purple or magenta. Figure 1–2b shows an example of cells with Biopsies are tissue samples removed during surgery or routine carbohydrate-rich areas well-stained by the PAS reaction. The medical procedures. In the operating room, biopsies are fixed DNA of cell nuclei can be specifically stained using a modifica- in vials of formalin for processing and microscopic analysis in tion of the PAS procedure called the Feulgen reaction. a pathology laboratory. If results of such analyses are required Basophilic or PAS-positive material can be further identi- before the medical procedure is completed, for example to fied by enzyme digestion, pretreatment of a tissue section with know whether a growth is malignant before the patient is an enzyme that specifically digests one substrate. For example, closed, a much more rapid processing method is used. The pretreatment with ribonuclease will greatly reduce cytoplas- biopsy is rapidly frozen in liquid nitrogen, preserving cell mic basophilia with little overall effect on the nucleus, indicat- structures and at the same time making the tissue hard and ing the importance of RNA for the cytoplasmic staining. ready for sectioning. A microtome called a cryostat in a cabi- Lipid-rich structures of cells are revealed by avoiding the net at subfreezing temperature is used to section the block processing steps that remove lipids, such as treatment with with tissue, and the frozen sections are placed on slides for heat and organic solvents, and staining with lipid-soluble rapid staining and microscopic examination by a pathologist. dyes such as Sudan black, which can be useful in diagnosis Freezing of tissues is also effective in histochemical stud- of metabolic diseases that involve intracellular accumulations ies of very sensitive enzymes or small molecules because of cholesterol, phospholipids, or glycolipids. Less common freezing, unlike fixation, does not inactivate most enzymes. methods of staining can employ metal impregnation tech- Finally, because clearing solvents often dissolve cell lipids in niques, typically using solutions of silver salts to visual certain fixed tissues, frozen sections are also useful when structures ECM fibers and specific cellular elements in nervous tissue. containing lipids are to be studied histologically. The Appendix lists important staining procedures used for most of the light micrographs in this book. 01_Mescher_ch01_p001-016.indd 3 26/04/18 11:10 am  4 CHAPTER 1 Histology & Its Methods of Study FIGURE 1–2 Hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining. VetBooks.ir G G G L L G G G a b Micrographs of epithelium lining the small intestine, (a) stained lumen, where projecting microvilli have a prominent layer of with H&E, and (b) stained with the PAS reaction for glycoproteins. glycoproteins at the lumen (L) and in the mucin-rich secretory With H&E, basophilic cell nuclei are stained purple, while cyto- granules of goblet cells. Cell surface glycoproteins and mucin are plasm stains pink. Cell regions with abundant oligosaccharides PAS-positive because of their high content of oligosaccharides on glycoproteins, such as the ends of the cells at the lumen (L) and polysaccharides, respectively. The PAS-stained tissue was or the scattered mucus-secreting goblet cells (G), are poorly counterstained with hematoxylin to show the cell nuclei. stained. With PAS, however, cell staining is most intense at the (a. X400; b. X300) Slide preparation, from tissue fixation to observation (or ocular lens) further magnifying this image and projecting with a light microscope, may take from 12 hours to 2½ days, it onto the viewer’s retina or a charge-coupled device (CCD) depending on the size of the tissue, the embedding medium, highly sensitive to low light levels with a camera and monitor. and the method of staining. The final step before microscopic The total magnification is obtained by multiplying the magni- observation is mounting a protective glass coverslip on the fying power of the objective and ocular lenses. slide with clear adhesive. The critical factor in obtaining a crisp, detailed image with a light microscope is its resolving power, defined as the smallest distance between two structures at which they can be ››LIGHT MICROSCOPY seen as separate objects. The maximal resolving power of the light microscope is approximately 0.2 μm, which can permit Conventional bright-field microscopy and more specialized clear images magnified 1000-1500 times. Objects smaller or applications like fluorescence, phase-contrast, confocal, and thinner than 0.2 μm (such as a single ribosome or cytoplasmic polarizing microscopy are all based on the interaction of light microfilament) cannot be distinguished with this instrument. with tissue components and are used to reveal and study tissue Likewise, two structures such as mitochondria will be seen as features. only one object if they are separated by less than 0.2 μm. The microscope’s resolving power determines the quality of the Bright-Field Microscopy image, its clarity and richness of detail, and depends mainly on With the bright-field microscope, stained tissue is examined the quality of its objective lens. Magnification is of value only with ordinary light passing through the preparation. As shown when accompanied by high resolution. Objective lenses pro- in Figure 1–3, the microscope includes an optical system and viding higher magnification are designed to also have higher mechanisms to move and focus the specimen. The optical resolving power. The eyepiece lens only enlarges the image components are the condenser focusing light on the object obtained by the objective and does not improve resolution. to be studied; the objective lens enlarging and projecting the Virtual microscopy, typically used for study of bright- image of the object toward the observer; and the eyepiece field microscopic preparations, involves the conversion of a 01_Mescher_ch01_p001-016.indd 4 26/04/18 11:10 am  Light Microscopy 5 FIGURE 1–3 Components and light path of a Fluorescence Microscopy C H A P T E R VetBooks.ir bright-field microscope. When certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength— Eyepiece Interpupillar adjustment a phenomenon called fluorescence. In fluorescence Binocular tubes Head microscopy, tissue sections are usually irradiated with ultra- violet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear bright on Stand a dark background. For fluorescent microscopy, the instru- 1 Measuring ment has a source of UV or other light and filters that select Histology & Its Methods of Study Light Microscopy graticule Beamsplitter rays of different wavelengths emitted by the substances to be Revolving nosepiece visualized. Specimen Objective Fluorescent compounds with affinity for specific cell holder macromolecules may be used as fluorescent stains. Acridine Mechanical stage orange, which binds both DNA and RNA, is an example. On/off switch When observed in the fluorescence microscope, these nucleic Condenser Illumination intensity acids emit slightly different fluorescence, allowing them to be Field lens control localized separately in cells (Figure 1–4a). Other compounds, Field such as DAPI and Hoechst, stain specifically bind DNA and diaphragm Collector are used to stain cell nuclei, emitting a characteristic blue fluo- lens rescence under UV. Another important application of fluores- cence microscopy is achieved by coupling compounds such as X-Y Base translation fluorescein to molecules that will specifically bind to certain Tungsten mechanism cellular components and thus allow the identification of these halogen lamp structures under the microscope (Figure 1–4b). Antibodies labeled with fluorescent compounds are extremely important Photograph of a bright-field light microscope showing its in immunohistologic staining. (See the section Visualizing mechanical components and the pathway of light from the Specific Molecules.) substage lamp to the eye of the observer. The optical system has three sets of lenses: Phase-Contrast Microscopy The condenser collects and focuses a cone of light that illuminates the tissue slide on the stage. Unstained cells and tissue sections, which are usually trans- Objective lenses enlarge and project the illuminated parent and colorless, can be studied with these modified image of the object toward the eyepiece. Interchangeable light microscopes. Cellular detail is normally difficult to see objectives with different magnifications routinely used in in unstained tissues because all parts of the specimen have histology include X4 for observing a large area (field) of the tissue at low magnification; X10 for medium magnification roughly similar optical densities. Phase-contrast micros- of a smaller field; and X40 for high magnification of more copy, however, uses a lens system that produces visible images detailed areas. from transparent objects and, importantly, can be used with The two eyepieces or oculars magnify this image another living, cultured cells (Figure 1–5). X10 and project it to the viewer, yielding a total magnifica- Phase-contrast microscopy is based on the principle that tion of X40, X100, or X400. light changes its speed when passing through cellular and (Used with permission from Nikon Instruments.) extracellular structures with different refractive indices. These changes are used by the phase-contrast system to cause the structures to appear lighter or darker in relation to each other. stained tissue preparation to high-resolution digital images Because they allow the examination of cells without fixation or and permits study of tissues using a computer or other digi- staining, phase-contrast microscopes are prominent tools in tal device, without an actual stained slide or a microscope. In all cell culture laboratories. A modification of phase-contrast this technique, regions of a glass-mounted specimen are cap- microscopy is differential interference contrast micros- tured digitally in a grid-like pattern at multiple magnifications copy with Nomarski optics, which produces an image of liv- using a specialized slide-scanning microscope and saved as ing cells with a more apparent three-dimensional (3D) aspect thousands of consecutive image files. Software then converts (Figure 1–5c). this dataset for storage on a server using a format that allows access, visualization, and navigation of the original slide with common web browsers or other devices. With advantages in Confocal Microscopy cost and ease of use, virtual microscopy is rapidly replacing With a regular bright-field microscope, the beam of light light microscopes and collections of glass slides in histology is relatively large and fills the specimen. Stray (excess) light laboratories for students. reduces contrast within the image and compromises the 01_Mescher_ch01_p001-016.indd 5 26/04/18 11:10 am  6 CHAPTER 1 Histology & Its Methods of Study FIGURE 1–4 Appearance of cells with fluorescent microscopy. VetBooks.ir N N R a b Components of cells are often stained with compounds visible by filaments show nuclei with blue fluorescence and actin filaments fluorescence microscopy. stained green. Important information such as the greater density (a) Acridine orange binds nucleic acids and causes DNA in cell of microfilaments at the cell periphery is readily apparent. (Both nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to X500) appear orange in these cells of a kidney tubule. (Figure 1–4b, used with permission from Drs Claire E. Walczak and Rania Rizk, Indiana University School of Medicine, (b) Cultured cells stained with DAPI (4′,6-diamino-2-phenylindole) Bloomington.) that binds DNA and with fluorescein phalloidin that binds actin FIGURE 1–5 Unstained cells’ appearance in three types of light microscopy. a b c Living neural crest cells growing in culture appear differently in-phase light differently and produce an image of these features with various techniques of light microscopy. Here the same field in all the cells. of unstained cells, including two differentiating pigment cells, is (c) Differential interference contrast microscopy: Cellular details shown using three different methods (all X200): are highlighted in a different manner using Nomarski optics. (a) Bright-field microscopy: Without fixation and staining, only Phase-contrast microscopy, with or without differential interfer- the two pigment cells can be seen. ence, is widely used to observe live cells grown in tissue culture. (b) Phase-contrast microscopy: Cell boundaries, nuclei, and (Used with permission from Dr Sherry Rogers, Department of Cell cytoplasmic structures with different refractive indices affect Biology and Physiology, University of New Mexico, Albuquerque, NM.) 01_Mescher_ch01_p001-016.indd 6 26/04/18 11:10 am  Light Microscopy 7 such optical sections at a series of focal planes through the FIGURE 1–6 Principle of confocal microscopy. specimen allows them to be digitally reconstructed into a C H A P T E R VetBooks.ir 3D image. Laser Polarizing Microscopy Polarizing microscopy allows the recognition of stained or unstained structures made of highly organized subunits. When normal light passes through a polarizing filter, it exits 1 Scanner vibrating in only one direction. If a second filter is placed in Histology & Its Methods of Study Light Microscopy the microscope above the first one, with its main axis per- Detector pendicular to the first filter, no light passes through. If, how- ever, tissue structures containing oriented macromolecules are located between the two polarizing filters, their repeti- tive structure rotates the axis of the light emerging from the polarizer and they appear as bright structures against a dark background (Figure 1–7). The ability to rotate the direction Plate with of vibration of polarized light is called birefringence and is pinhole Beam splitter FIGURE 1–7 Tissue appearance with bright-field and polarizing microscopy. Lens Other Focal plane out-of-focus Specimen planes Although a very small spot of light originating from one plane of the section crosses the pinhole and reaches the detector, rays originating from other planes are blocked by the blind. Thus, only one very thin plane of the specimen is focused at a time. The diagram shows the practical arrangement of a confo- a cal microscope. Light from a laser source hits the specimen and is reflected. A beam splitter directs the reflected light to a pin- hole and a detector. Light from components of the specimen that are above or below the focused plane is blocked by the blind. The laser scans the specimen so that a larger area of the specimen can be observed. resolving power of the objective lens. Confocal microscopy (Figure 1–6) avoids these problems and achieves high reso- lution and sharp focus by using (1) a small point of high- intensity light, often from a laser and (2) a plate with a pinhole aperture in front of the image detector. The point b light source, the focal point of the lens, and the detector’s pinpoint aperture are all optically conjugated or aligned to Polarizing light microscopy produces an image only of material each other in the focal plane (confocal), and unfocused light having repetitive, periodic macromolecular structure; features does not pass through the pinhole. This greatly improves without such structure are not seen. Pieces of thin, unsec- resolution of the object in focus and allows the localization tioned mesentery were stained with red picrosirius, orcein, and of specimen components with much greater precision than hematoxylin, placed on slides and observed by bright-field (a) with the bright-field microscope. and polarizing (b) microscopy. Confocal microscopes include a computer-driven mirror (a) With bright-field microscopy, collagen fibers appear red, with thin elastic fibers and cell nuclei darker. (X40) system (the beam splitter) to move the point of illumination across the specimen automatically and rapidly. Digital images (b) With polarizing microscopy, only the collagen fibers are visible and these exhibit intense yellow or orange birefrin- captured at many individual spots in a very thin plane of focus gence. (a: X40; b: X100) are used to produce an “optical section” of that plane. Creating 01_Mescher_ch01_p001-016.indd 7 26/04/18 11:10 am  8 CHAPTER 1 Histology & Its Methods of Study a feature of crystalline substances or substances containing The wavelength in an electron beam is much shorter than that highly oriented molecules, such as cellulose, collagen, micro- of light, allowing a 1000-fold increase in resolution. VetBooks.ir tubules, and actin filaments. The utility of all light microscopic methods is greatly Transmission Electron Microscopy extended through the use of digital cameras. Many features The transmission electron microscope (TEM) is an imag- of digitized histologic images can be analyzed quantitatively ing system that permits resolution around 3 nm. This high using appropriate software. Such images can also be enhanced resolution allows isolated particles magnified as much as to allow objects not directly visible through the eyepieces to be 400,000 times to be viewed in detail. Very thin (40-90 nm), examined on a monitor. resin-embedded tissue sections are typically studied by TEM at magnifications up to approximately 120,000 times. ››ELECTRON MICROSCOPY Figure 1–8a indicates the components of a TEM and the basic principles of its operation: a beam of electrons focused Transmission and scanning electron microscopes are based on using electromagnetic “lenses” passes through the tissue sec- the interaction of tissue components with beams of electrons. tion to produce an image with black, white, and intermediate FIGURE 1–8 Electron microscopes. Electron gun Electron gun Cathode Cathode 3 mm Anode Anode Copper grid Condensor lens with three sections Lens Specimen Column Objective lens holder Lens Column Scanner Intermediate lens Electron detector TEM image Lens SEM image Projector lens Image on viewing screen Specimen Electron detector with CCD camera (a) Transmission electron microscope (b) Scanning electron microscope Electron microscopes are large instruments generally housed in a In a TEM image areas of the specimen through which electrons specialized EM facility. passed appear bright (electron lucent), while denser areas or (a) Schematic view of the major components of a transmission elec- those that bind heavy metal ions during specimen preparation tron microscope (TEM), which is configured rather like an upside- absorb or deflect electrons and appear darker (electron dense). down light microscope. With the microscope column in a vacuum, a Such images are therefore always black, white, and shades of gray. metallic (usually tungsten) filament (cathode) at the top emits elec- (b) The scanning electron microscope (SEM) has many similarities trons that travel to an anode with an accelerating voltage between to a TEM. However, here the focused electron beam does not pass 60 and 120 kV. Electrons passing through a hole in the anode form through the specimen, but rather is moved sequentially (scanned) a beam that is focused electromagnetically by circular electric from point to point across its surface similar to the way an electron coils in a manner analogous to the effect of optical lenses on light. beam is scanned across a television tube or screen. For SEM speci- The first lens is a condenser focusing the beam on the section. mens are coated with metal atoms with which the electron beam Some electrons interact with atoms in the section, being absorbed interacts, producing reflected electrons and newly emitted secondary or scattered to different extents, while others are simply transmit- electrons. All of these are captured by a detector and transmitted to ted through the specimen with no interaction. Electrons reaching amplifiers and processed to produce a black-and-white image on the the objective lens form an image that is then magnified and finally monitor. The SEM shows only surface views of the coated specimen projected on a fluorescent screen or a charge-coupled device but with a striking 3D, shadowed quality. The inside of organs or cells (CCD) monitor and camera. can be analyzed after sectioning to expose their internal surfaces. 01_Mescher_ch01_p001-016.indd 8 26/04/18 11:10 am  Autoradiography 9 shades of gray regions. These regions of an electron micro- layer of heavy metal (often gold) that reflects electrons in a graph correspond to tissue areas through which electrons beam scanning the specimen. The reflected electrons are cap- C H A P T E R VetBooks.ir passed readily (appearing brighter or electron-lucent) and tured by a detector, producing signals that are processed to pro- areas where electrons were absorbed or deflected (appearing duce a black-and-white image. SEM images are usually easy to darker or more electron-dense). To improve contrast and reso- interpret because they present a three-dimensional view that lution in TEM, compounds with heavy metal ions are often appears to be illuminated in the same way that large objects are added to the fixative or dehydrating solutions used for tissue seen with highlights and shadows caused by light. preparation. These include osmium tetroxide, lead citrate, 1 and uranyl compounds, which bind cellular macromolecules, ››AUTORADIOGRAPHY Histology & Its Methods of Study Autoradiography increasing their electron density and visibility. Cryofracture and freeze etching are techniques that Microscopic autoradiography is a method of localizing allow TEM study of cells without fixation or embedding and newly synthesized macromolecules in cells or tissue sections. have been particularly useful in the study of membrane struc- Radioactively labeled metabolites (nucleotides, amino acids, ture. In these methods, very small tissue specimens are rap- sugars) provided to the living cells are incorporated into spe- idly frozen in liquid nitrogen and then cut or fractured with a cific macromolecules (DNA, RNA, protein, glycoproteins, and knife. A replica of the frozen exposed surface is produced in a polysaccharides) and emit weak radiation that is restricted vacuum by applying thin coats of vaporized platinum or other to those regions where the molecules are located. Slides with metal atoms. After removal of the organic material, the replica radiolabeled cells or tissue sections are coated in a darkroom of the cut surface can be examined by TEM. With membranes with photographic emulsion in which silver bromide crystals the random fracture planes often split the lipid bilayers, expos- ing protein components whose size, shape, and distribution act as microdetectors of the radiation in the same way that are difficult to study by other methods. they respond to light in photographic film. After an adequate exposure time in lightproof boxes, the slides are developed photographically. Silver bromide crystals reduced by the radia- Scanning Electron Microscopy tion produce small black grains of metallic silver, which under Scanning electron microscopy (SEM) provides a high- either the light microscope or TEM indicate the locations of resolution view of the surfaces of cells, tissues, and organs. Like radiolabeled macromolecules in the tissue (Figure 1–9). the TEM, this microscope produces and focuses a very narrow Much histological information becomes available by beam of electrons, but in this instrument the beam does not autoradiography. If a radioactive precursor of DNA (such pass through the specimen (Figure 1–8b). Instead, the surface as tritium-labeled thymidine) is used, it is possible to know of the specimen is first dried and spray-coated with a very thin which cells in a tissue (and how many) are replicating DNA FIGURE 1–9 Microscopic autoradiography. L G G a b Autoradiographs are tissue preparations in which particles called (a) Black grains of silver from the light-sensitive material coating silver grains indicate the cells or regions of cells in which specific the specimen are visible over cell regions with secretory granules macromolecules were synthesized just prior to fixation. Shown and the duct indicating glycoprotein locations. (X1500) here are autoradiographs from the salivary gland of a mouse (b) The same tissue prepared for TEM autoradiography shows sil- injected with 3H-fucose 8 hours before tissue fixation. Fucose was ver grains with a coiled or amorphous appearance again localized incorporated into oligosaccharides, and the free 3H-fucose was mainly over the granules (G) and in the gland lumen (L). (X7500) removed during fixation and sectioning of the gland. Autoradio- (Figure 1–9b, used with permission from Drs Ticiano G. Lima and graphic processing and microscopy reveal locations of newly syn- A. Antonio Haddad, School of Medicine, Ribeirão Pret