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Junqueira’s Basic Histology
TEXT AND ATLAS
Anthony L. Mescher, PhD
Professor of Anatomy and Cell Biology
Indiana University School of Medicine Bloomington, Indiana
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1 Histology & Its Methods of Study 1
Preparation of Tissues for Study 1
Light Microscopy 4
Electron Microscopy 8
Autoradiography 9
Cell & Tissue Culture 10
Enzyme Histochemistry 10
Visualizing Specific Molecules 10
Interpretation of Structures in Tissue
Sections 14
Summary of Key Points 15
Assess Your Knowledge 16
2 The Cytoplasm 17
Cell Differentiation 17
The Plasma Membrane 17
Cytoplasmic Organelles 27
The Cytoskeleton 42
Inclusions 47
Summary of Key Points 51
Assess Your Knowledge 52
3 The Nucleus 53
Components of the Nucleus 53
The Cell Cycle 58
Mitosis 61
Stem Cells & Tissue Renewal 65
Meiosis 65
Apoptosis 67
Summary of Key Points 69
Assess Your Knowledge 70
4 Epithelial Tissue 71
Characteristic Features of Epithelial Cells 72
Specializations of the Apical Cell Surface 77
Types of Epithelia 80
Transport Across Epithelia 88
Renewal of Epithelial Cells 88
Summary of Key Points 90
Assess Your Knowledge 93
5 Connective Tissue 96
Cells of Connective Tissue 96
Fibers 103
Ground Substance 111
Types of Connective Tissue 114
Summary of Key Points 119
Assess Your Knowledge 120
6 Adipose Tissue 122
White Adipose Tissue 122
Brown Adipose Tissue 126
Summary of Key Points 127
Assess Your Knowledge 128
7 Cartilage 129
Hyaline Cartilage 129
Elastic Cartilage 133
Fibrocartilage 134
Cartilage Formation, Growth, & Repair 134
Summary of Key Points 136
Assess Your Knowledge 136
8 Bone 138
Bone Cells 138
Bone Matrix 143
Periosteum & Endosteum 143
Types of Bone 143
Osteogenesis 148
Bone Remodeling & Repair 152
Metabolic Role of Bone 153
Joints 155
Summary of Key Points 158
Assess Your Knowledge 159
9 Nerve Tissue & the Nervous System
161
Development of Nerve Tissue 161
Neurons 163
Glial Cells & Neuronal Activity 168
Central Nervous System 175
Peripheral Nervous System 182
Neural Plasticity & Regeneration 187
Summary of Key Points 190
Assess Your Knowledge 191
10 Muscle Tissue 193
Skeletal Muscle 193
Cardiac Muscle 207
Smooth Muscle 208
Regeneration of Muscle Tissue 213
Summary of Key Points 213
Assess Your Knowledge 214
11 The Circulatory System 215
Heart 215
Tissues of the Vascular Wall 219
Vasculature 220
Lymphatic Vascular System 231
Summary of Key Points 235
Assess Your Knowledge 235
12 Blood 237
Composition of Plasma 237
Blood Cells 239
Summary of Key Points 250
Assess Your Knowledge 252
13 Hemopoiesis 254
Stem Cells, Growth Factors, & Differentiation 254
Bone Marrow 255
Maturation of Erythrocytes 258
Maturation of Granulocytes 260
Maturation of Agranulocytes 263
Origin of Platelets 263
Summary of Key Points 265
Assess Your Knowledge 265
14 The Immune System & Lymphoid Organs 267
Innate & Adaptive Immunity 267
Cytokines 269
Antigens & Antibodies 270
Antigen Presentation 271
Cells of Adaptive Immunity 273
Thymus 276
Mucosa-Associated Lymphoid Tissue 281
Lymph Nodes 282
Spleen 286
Summary of Key Points 293
Assess Your Knowledge 294
15 Digestive Tract 295
General Structure of the Digestive Tract 295
Oral Cavity 298
Esophagus 305
Stomach 307
Small Intestine 314
Large Intestine 318
Summary of Key Points 326
Assess Your Knowledge 327
16 Organs Associated with the Digestive Tract 329
Salivary Glands 329
Pancreas 332
Liver 335
Biliary Tract & Gallbladder 345
Summary of Key Points 346
Assess Your Knowledge 348
17
The Respiratory System 349
Nasal Cavities 349
Pharynx 352
Larynx 352
Trachea 354
Bronchial Tree & Lung 354
Lung Vasculature & Nerves 366
Pleural Membranes 368
Respiratory Movements 368
Summary of Key Points 369
Assess Your Knowledge 369
18
Skin 371
Epidermis 372
Dermis 378
Subcutaneous Tissue 381
Sensory Receptors 381
Hair 383
Nails 384
Skin Glands 385
Skin Repair 388
Summary of Key Points 391
Assess Your Knowledge 391
19
The Urinary System 393
Kidneys 393
Blood Circulation 394
Renal Function: Filtration, Secretion, & Reabsorption 395
Ureters, Bladder, & Urethra 406
Summary of Key Points 411
Assess Your Knowledge 412
20 Endocrine Glands 413
Pituitary Gland (Hypophysis) 413
Adrenal Glands 423
Pancreatic Islets 427
Diffuse Neuroendocrine System 429
Thyroid Gland 429
Parathyroid Glands 432
Pineal Gland 434
Summary of Key Points 437
Assess Your Knowledge 437
21 The Male Reproductive System 439
Testes 439
Intratesticular Ducts 449
Excretory Genital Ducts 450
Accessory Glands 451
Penis 456
Summary of Key Points 457
Assess Your Knowledge 459
22 The Female Reproductive System 460
Ovaries 460
Uterine Tubes 470
Major Events of Fertilization 471
Uterus 471
Embryonic Implantation, Decidua, & the Placenta 478
Cervix 482
Vagina 483
External Genitalia 483
Mammary Glands 483
Summary of Key Points 488
Assess Your Knowledge 489
23 The Eye & Ear: Special Sense Organs 490
Eyes: The Photoreceptor System 490
Ears: The Vestibuloauditory System 509
Summary of Key Points 522
Assess Your Knowledge 522
APPENDIX 525
FIGURE CREDITS 527
INDEX 529
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Preface
With this 15th edition, Junqueira’s Basic Histology continues as the preeminent source of concise yet thorough information on human tissue structure and function. For over 45 years this educational resource has met the needs of learners for a well-organized and concise presentation of cell biology and histology that integrates the material with that of biochemistry, immunology, endocrinology, and physiology and provides an excellent foundation for subsequent studies in pathology. The text is prepared specifically for students of medicine and other health-related professions, as well as for advanced undergraduate courses in tissue biology. As a result of its value and appeal to students and instructors alike, Junqueira’s Basic Histology has been translated into a dozen different languages and is used by medical students throughout the world.
Unlike other histology texts and atlases, the present work again includes with each chapter a set of multiplechoice Self-Test Questions that allow readers to assess their comprehension and knowledge of important points in that chapter. At least a few questions in each set utilize clinical vignettes or cases to provide context for framing the medical relevance of concepts in basic science, as recommended by the US National Board of Medical Examiners. As with the last edition, each chapter also includes a Summary of Key Points designed to guide the students concerning what is clearly important and what is less so. Summary Tables in each chapter organize and condense important information, further facilitating efficient learning.
Each chapter has been revised and shortened, while coverage of specific topics has been expanded and updated as needed. Study is facilitated by modern page design. Inserted throughout each chapter are more numerous, short paragraphs that indicate how the information presented can be used medically and which emphasize the foundational relevance of the material learned.
The art and other figures are presented in each chapter, with the goal to simplify learning and integration with related material. The McGraw-Hill medical illustrations, now used throughout the text, are the most useful, thorough, and attractive of any similar medical textbook. Electron and light micrographs have been replaced throughout the book as needed, and they again make up a complete atlas of cell, tissue, and organ structures fully compatible with the students’ own collection of glass or digital slides. Health science students whose medical library offers AccessMedicine among its electronic resources (which includes more than 95% of
U.S. medical schools) can access a complete human histology Laboratory Guide linked to the virtual microscope at the URL given below. This digital Laboratory Guide, which is new with this edition of the text and unique among learning resources offered by any histology text and atlas, provides both links to the appropriate microscope slides needed for each topic and links to the correlated figures or tables in the text. Those without AccessMedicine will lack the digital Laboratory Guide, but may still study and utilize the 150 virtual microscope slides of all human tissues and organs, which are available at: http://medsci.indiana.edu/junqueira/virtual/junqueira.htm As with the previous edition, the book facilitates learning by its organization:
■ An opening chapter reviews the histological techniques that allow understanding of cell and tissue structure.
■ Two chapters then summarize the structural and functional organization of human cell biology, presenting the cytoplasm and nucleus separately.
■ The next seven chapters cover the four basic tissues that make up our organs: epithelia, connective tissue (and its major sub-types), nervous tissue, and muscle.
■ Remaining chapters explain the organization and functional significance of these tissues in each of the body’s organ systems, closing with up-to-date consideration of cells in the eye and ear.
For additional review of what’s been learned or to assist rapid assimilation of the material in Junqueira’s Basic Histology, McGraw-Hill has published a set of 200 full-color Basic Histology Flash Cards, also authored by Anthony Mescher. Each card includes images of key structures to identify, a summary of important facts about those structures, and a clinical comment. This valuable learning aid is available as a set of actual cards from Amazon.com, or as an app for smartphones or tablets from the online App Store. With its proven strengths and the addition of new features, I am confident that Junqueira’s Basic Histology will continue as one of the most valuable and most widely read educational resources in histology. Users are invited to provide feedback to the author with regard to any aspect of the book’s features.
Anthony L. Mescher Indiana University School of Medicine
mescher@indiana.edu
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Acknowledgments
I wish to thank the students at Indiana University School of Medicine and the undergraduates at Indiana University with whom I have studied histology and cell biology for over 35 years and from whom I have learned much about presenting basic concepts most effectively. Their input has greatly helped in the task of maintaining and updating the presentations in this classic textbook. As with the last edition, the help of Sue Childress and Dr. Mark Braun was invaluable in slide preparation and the virtual microscope for human histology respectively.
As with the last edition, the present text includes ten multiple-choice questions at the end of each chapter, aimed to test the learner’s retention and understanding of important points in that body of material. Many of these questions were used in my courses, but others are taken or modified from a few of the many excellent review books published by McGraw-Hill/Lange for students preparing to take the U.S. Medical Licensing Examination. These include Histology and Cell Biology: Examination and Board Review, by Douglas Paulsen; USMLE Road Map: Histology, by Harold Sheedlo; and Anatomy, Histology, & Cell Biology: PreTest Self-Assessment &
Review, by Robert Klein and George Enders. The use here of questions from these valuable resources is gratefully acknowledged. Students are referred to those review books for hundreds of additional self-assessment questions.
I am also grateful to my colleagues and reviewers from throughout the world who provided specialized expertise or original photographs, which are also acknowledged in figure captions. I thank those professors and students in the United States and countries throughout the world who provided useful suggestions that have improved the new edition of Junqueira’s Basic Histology. Finally, I am pleased to acknowledge the help and collegiality provided by the staff of McGraw-Hill, especially editors Michael Weitz and Brian Kearns, whose work made possible publication of this 15th edition of Junqueira’s Basic Histology.
Anthony L. Mescher Indiana University School of Medicine mescher@indiana.edu
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Histology & Its Methods of Study
Histology 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.
Tissues have two interacting components: cells and extracellular 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 contains the fluid transporting nutrients to the cells, and carrying away their wastes and secretory products. Cells produce the ECM locally and are in turn strongly influenced by matrix molecules. Many matrix components bind to specific cell surface receptors that span the cell membranes and connect to structural components inside the cells, forming a continuum in which cells and the ECM function together in a wellcoordinated manner.
During development, cells and their associated matrix become functionally specialized and give rise to fundamental types of tissues with characteristic structural features. Organs are formed by an orderly combination of these tissues, and their precise arrangement allows the functioning of each organ and of the organism as a whole.
The small size of cells and matrix components makes histology dependent on the use of microscopes and molecular methods of study. Advances in biochemistry, molecular biology, physiology, immunology,
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 microscopic approaches.
› PREPARATION OF TISSUES FOR STUDY
The most common procedure used in histologic research is the preparation of tissue slices or “sections” that can be examined visually with transmitted light. Because most tissues and organs are too thick for light to pass through, thin translucent sections are cut from them and placed on glass slides for microscopic examination of the internal structures. The ideal microscopic preparation is preserved so that the tissue on the slide has the same structural features it had in the body. However, this is often not feasible because the preparation process can remove cellular lipid, with slight distortions of cell structure. The basic steps used in tissue preparation for light microscopy are shown in Figure 1–1.
Fixation
To preserve tissue structure and prevent degradation by enzymes released from the cells or microorganisms, pieces of
Most tissues studied histologically are prepared as shown, with this sequence of steps (a):
■ Fixation: Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes, which preserve cell and tissue structure.
■ Dehydration: The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water.
■ Clearing: Alcohol is removed in organic solvents in which both alcohol and paraffin are miscible.
■ Infiltration: The tissue is then placed in melted paraffin until it becomes completely infiltrated with this substance.
■ Embedding: The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden.
■ Trimming: The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome.
Drive wheel
Block holder
Paraffin block
Tissue
Steel
Similar steps are used in preparing tissue for transmission electron microscopy (TEM), except special fixatives and dehydrating solutions are used with smaller tissue samples and embedding involves epoxy resins which become harder than paraffin to allow very thin sectioning.
(b) A microtome is used for sectioning paraffin-embedded tissues for light microscopy. The trimmed tissue specimen is mounted in the paraffin block holder, and each turn of the drive wheel by the histologist advances the holder a controlled distance, generally from 1 to 10 μm. After each forward move, the tissue block passes over the steel knife edge and a section is cut at a thickness equal to the distance the block advanced. The paraffin sections are placed on glass slides and allowed to adhere, deparaffinized, and stained for light microscope study. For TEM, sections less than 1 μm thick are prepared from resin-embedded cells using an ultramicrotome with a glass or diamond knife.
organs are placed as soon as possible after removal from the body in solutions of stabilizing or cross-linking compounds called fixatives. Because a fixative must fully diffuse through the tissues to preserve all cells, tissues are usually cut into small fragments before fixation to facilitate penetration. To improve cell preservation in large organs, fixatives are often introduced via blood vessels, with vascular perfusion allowing fixation rapidly throughout the tissues.
One widely used fixative for light microscopy is formalin, a buffered isotonic solution of 37% formaldehyde. Both this compound and glutaraldehyde, a fixative used for electron
microscopy, react with the amine groups (NH2) of proteins, preventing their degradation by common proteases. Glutaraldehyde also cross-links adjacent proteins, reinforcing cell and ECM structures.
Electron microscopy provides much greater magnification and resolution of very small cellular structures, and fixation must be done very carefully to preserve additional “ultrastructural” detail. Typically in such studies, glutaraldehyde-treated tissue is then immersed in buffered osmium tetroxide, which preserves (and stains) cellular lipids as well as proteins.
knife
FIGURE 1–1 Sectioning fixed and embedded tissue.
Embedding & Sectioning
To permit thin sectioning, fixed tissues are infiltrated and embedded in a material that imparts a firm consistency. Embedding materials include paraffin, used routinely for light microscopy, and plastic resins, which are adapted for both light and electron microscopy.
Before infiltration with such media, the fixed tissue must undergo dehydration by having its water extracted gradually by transfers through a series of increasing ethanol solutions, ending in 100% ethanol. The ethanol is then replaced by an organic solvent miscible with both alcohol and the embedding medium, a step referred to as clearing because infiltration with the reagents used here gives the tissue a translucent appearance.
The fully cleared tissue is then placed in melted paraffin in an oven at 52°-60°C, which evaporates the clearing solvent and promotes infiltration of the tissue with paraffin, and then embedded by allowing it to harden in a small container of paraffin at room temperature. Tissues to be embedded with plastic resin are also dehydrated in ethanol and then infiltrated with plastic solvents that harden when cross-linking polymerizers are added. Plastic embedding avoids the higher temperatures needed with paraffin, which helps avoid tissue distortion.
The hardened block with tissue and surrounding embedding medium is trimmed and placed for sectioning in an instrument called a microtome (see Figure 1–1). Paraffin sections are typically cut at 3-10 μm thickness for light microscopy, but electron microscopy requires sections less than 1 μm thick. One micrometer (1 μm) equals 1/1000 of a millimeter (mm) or 10 6 m. Other spatial units commonly used in microscopy are the nanometer (1 nm = 0.001 μm = 10 6 mm = 10 9 m) and angstrom (1 Å = 0.1 nm or 10 4 μm). The sections are placed on glass slides and stained for light microscopy or on metal grids for electron-microscopic staining and examination.
› › MEDICAL APPLICATION
Biopsies are tissue samples removed during surgery or routine medical procedures. In the operating room, biopsies are fixed in vials of formalin for processing and microscopic analysis in a pathology laboratory. If results of such analyses are required before the medical procedure is completed, for example to know whether a growth is malignant before the patient is closed, a much more rapid processing method is used. The biopsy is rapidly frozen in liquid nitrogen, preserving cell structures and at the same time making the tissue hard and ready for sectioning. A microtome called a cryostat in a cabinet at subfreezing temperature is used to section the block with tissue, and the frozen sections are placed on slides for rapid staining and microscopic examination by a pathologist. Freezing of tissues is also effective in histochemical studies of very sensitive enzymes or small molecules because freezing, unlike fixation, does not inactivate most enzymes. Finally, because clearing solvents often dissolve cell lipids in fixed tissues, frozen sections are also useful when structures containing lipids are to be studied histologically.
Staining
Most cells and extracellular material are completely colorless, and to be studied microscopically tissue sections must be stained (dyed). Methods of staining have been devised that make various tissue components not only conspicuous but also distinguishable from one another. Dyes stain material more or less selectively, often behaving like acidic or basic compounds and forming electrostatic (salt) linkages with ionizable radicals of macromolecules in tissues. Cell components, such as nucleic acids with a net negative charge (anionic), have an affinity for basic dyes and are termed basophilic; cationic components, such as proteins with many ionized amino groups, stain more readily with acidic dyes and are termed acidophilic
Examples of basic dyes include toluidine blue, alcian blue, and methylene blue. Hematoxylin behaves like a basic dye, staining basophilic tissue components. The main tissue components that ionize and react with basic dyes do so because of acids in their composition (DNA, RNA, and glycosaminoglycans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain the acidophilic components of tissues such as mitochondria, secretory granules, and collagen.
Of all staining methods, the simple combination of hematoxylin and eosin (H&E) is used most commonly. Hematoxylin stains DNA in the cell nucleus, RNA-rich portions of the cytoplasm, and the matrix of cartilage, producing a dark blue or purple color. In contrast, eosin stains other cytoplasmic structures and collagen pink (Figure 1–2a). Here eosin is considered a counterstain, which is usually a single dye applied separately to distinguish additional features of a tissue. More complex procedures, such as trichrome stains (eg, Masson’s trichrome), allow greater distinctions among various extracellular tissue components.
The periodic acid–Schiff (PAS) reaction utilizes the hexose rings of polysaccharides and other carbohydrate-rich tissue structures and stains such macromolecules distinctly purple or magenta. Figure 1–2b shows an example of cells with carbohydrate-rich areas well-stained by the PAS reaction. The DNA of cell nuclei can be specifically stained using a modification of the PAS procedure called the Feulgen reaction.
Basophilic or PAS-positive material can be further identified by enzyme digestion, pretreatment of a tissue section with an enzyme that specifically digests one substrate. For example, pretreatment with ribonuclease will greatly reduce cytoplasmic basophilia with little overall effect on the nucleus, indicating the importance of RNA for the cytoplasmic staining.
Lipid-rich structures of cells are revealed by avoiding the processing steps that remove lipids, such as treatment with heat and organic solvents, and staining with lipid-soluble dyes such as Sudan black, which can be useful in diagnosis of metabolic diseases that involve intracellular accumulations of cholesterol, phospholipids, or glycolipids. Less common methods of staining can employ metal impregnation techniques, typically using solutions of silver salts to visual certain ECM fibers and specific cellular elements in nervous tissue. The Appendix lists important staining procedures used for most of the light micrographs in this book.
Micrographs of epithelium lining the small intestine, (a) stained with H&E, and (b) stained with the PAS reaction for glycoproteins. With H&E, basophilic cell nuclei are stained purple, while cytoplasm stains pink. Cell regions with abundant oligosaccharides on glycoproteins, such as the ends of the cells at the lumen (L) or the scattered mucus-secreting goblet cells (G), are poorly stained. With PAS, however, cell staining is most intense at the
Slide preparation, from tissue fixation to observation with a light microscope, may take from 12 hours to 2½ days, depending on the size of the tissue, the embedding medium, and the method of staining. The final step before microscopic observation is mounting a protective glass coverslip on the slide with clear adhesive.
› LIGHT MICROSCOPY
Conventional bright-field microscopy and more specialized applications like fluorescence, phase-contrast, confocal, and polarizing microscopy are all based on the interaction of light with tissue components and are used to reveal and study tissue features.
Bright-Field Microscopy
With the bright-field microscope, stained tissue is examined with ordinary light passing through the preparation. As shown in Figure 1–3, the microscope includes an optical system and mechanisms to move and focus the specimen. The optical components are the condenser focusing light on the object to be studied; the objective lens enlarging and projecting the image of the object toward the observer; and the eyepiece
lumen, where projecting microvilli have a prominent layer of glycoproteins at the lumen (L) and in the mucin-rich secretory granules of goblet cells. Cell surface glycoproteins and mucin are PAS-positive because of their high content of oligosaccharides and polysaccharides, respectively. The PAS-stained tissue was counterstained with hematoxylin to show the cell nuclei.
(a. X400; b. X300)
(or ocular lens) further magnifying this image and projecting it onto the viewer’s retina or a charge-coupled device (CCD) highly sensitive to low light levels with a camera and monitor. The total magnification is obtained by multiplying the magnifying power of the objective and ocular lenses.
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 seen as separate objects. The maximal resolving power of the light microscope is approximately 0.2 μm, which can permit clear images magnified 1000-1500 times. Objects smaller or thinner than 0.2 μm (such as a single ribosome or cytoplasmic microfilament) cannot be distinguished with this instrument. Likewise, two structures such as mitochondria will be seen as only one object if they are separated by less than 0.2 μm. The microscope’s resolving power determines the quality of the image, its clarity and richness of detail, and depends mainly on the quality of its objective lens. Magnification is of value only when accompanied by high resolution. Objective lenses providing higher magnification are designed to also have higher resolving power. The eyepiece lens only enlarges the image obtained by the objective and does not improve resolution.
Virtual microscopy, typically used for study of brightfield microscopic preparations, involves the conversion of a
FIGURE 1–2 Hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining.
Photograph of a bright-field light microscope showing its mechanical components and the pathway of light from the substage lamp to the eye of the observer. The optical system has three sets of lenses:
■ The condenser collects and focuses a cone of light that illuminates the tissue slide on the stage.
■ Objective lenses enlarge and project the illuminated image of the object toward the eyepiece. Interchangeable objectives with different magnifications routinely used in histology include X4 for observing a large area (field) of the tissue at low magnification; X10 for medium magnification of a smaller field; and X40 for high magnification of more detailed areas.
■ The two eyepieces or oculars magnify this image another X10 and project it to the viewer, yielding a total magnification of X40, X100, or X400.
(Used with permission from Nikon Instruments.)
stained tissue preparation to high-resolution digital images and permits study of tissues using a computer or other digital device, without an actual stained slide or a microscope. In this technique, regions of a glass-mounted specimen are captured digitally in a grid-like pattern at multiple magnifications using a specialized slide-scanning microscope and saved as thousands of consecutive image files. Software then converts 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 cost and ease of use, virtual microscopy is rapidly replacing light microscopes and collections of glass slides in histology laboratories for students.
Fluorescence Microscopy
When certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength— a phenomenon called fluorescence. In fluorescence microscopy, tissue sections are usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear bright on a dark background. For fluorescent microscopy, the instrument has a source of UV or other light and filters that select rays of different wavelengths emitted by the substances to be visualized.
Fluorescent compounds with affinity for specific cell macromolecules may be used as fluorescent stains. Acridine orange, which binds both DNA and RNA, is an example. When observed in the fluorescence microscope, these nucleic acids emit slightly different fluorescence, allowing them to be localized separately in cells (Figure 1–4a). Other compounds, such as DAPI and Hoechst, stain specifically bind DNA and are used to stain cell nuclei, emitting a characteristic blue fluorescence under UV. Another important application of fluorescence microscopy is achieved by coupling compounds such as fluorescein to molecules that will specifically bind to certain cellular components and thus allow the identification of these structures under the microscope (Figure 1–4b). Antibodies labeled with fluorescent compounds are extremely important in immunohistologic staining. (See the section Visualizing Specific Molecules.)
Phase-Contrast Microscopy
Unstained cells and tissue sections, which are usually transparent and colorless, can be studied with these modified light microscopes. Cellular detail is normally difficult to see in unstained tissues because all parts of the specimen have roughly similar optical densities. Phase-contrast microscopy, however, uses a lens system that produces visible images from transparent objects and, importantly, can be used with living, cultured cells (Figure 1–5).
Phase-contrast microscopy is based on the principle that light changes its speed when passing through cellular and 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. Because they allow the examination of cells without fixation or staining, phase-contrast microscopes are prominent tools in all cell culture laboratories. A modification of phase-contrast microscopy is differential interference contrast microscopy with Nomarski optics, which produces an image of living cells with a more apparent three-dimensional (3D) aspect (Figure 1–5c).
Confocal Microscopy
With a regular bright-field microscope, the beam of light is relatively large and fills the specimen. Stray (excess) light reduces contrast within the image and compromises the
FIGURE 1–3 Components and light path of a bright-field microscope.
FIGURE 1–4 Appearance of cells with fluorescent microscopy.
Components of cells are often stained with compounds visible by fluorescence microscopy.
(a) Acridine orange binds nucleic acids and causes DNA in cell nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to appear orange in these cells of a kidney tubule.
(b) Cultured cells stained with DAPI (4′,6-diamino-2-phenylindole) that binds DNA and with fluorescein phalloidin that binds actin
filaments show nuclei with blue fluorescence and actin filaments stained green. Important information such as the greater density of microfilaments at the cell periphery is readily apparent. (Both X500)
(Figure 1–4b, used with permission from Drs Claire E. Walczak and Rania Rizk, Indiana University School of Medicine, Bloomington. )
FIGURE 1–5 Unstained cells’ appearance in three types of light microscopy.
Living neural crest cells growing in culture appear differently with various techniques of light microscopy. Here the same field of unstained cells, including two differentiating pigment cells, is shown using three different methods (all X200):
(a) Bright-field microscopy: Without fixation and staining, only the two pigment cells can be seen.
(b) Phase-contrast microscopy: Cell boundaries, nuclei, and cytoplasmic structures with different refractive indices affect
in-phase light differently and produce an image of these features in all the cells.
(c) Differential interference contrast microscopy: Cellular details are highlighted in a different manner using Nomarski optics. Phase-contrast microscopy, with or without differential interference, is widely used to observe live cells grown in tissue culture.
(Used with permission from Dr Sherry Rogers, Department of Cell Biology and Physiology, University of New Mexico, Albuquerque, NM.)
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 confocal microscope. Light from a laser source hits the specimen and is reflected. A beam splitter directs the reflected light to a pinhole 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 resolution and sharp focus by using (1) a small point of highintensity light, often from a laser and (2) a plate with a pinhole aperture in front of the image detector. The point light source, the focal point of the lens, and the detector’s pinpoint aperture are all optically conjugated or aligned to each other in the focal plane (confocal), and unfocused light does not pass through the pinhole. This greatly improves resolution of the object in focus and allows the localization of specimen components with much greater precision than with the bright-field microscope.
Confocal microscopes include a computer-driven mirror system (the beam splitter) to move the point of illumination across the specimen automatically and rapidly. Digital images captured at many individual spots in a very thin plane of focus are used to produce an “optical section” of that plane. Creating
such optical sections at a series of focal planes through the specimen allows them to be digitally reconstructed into a 3D image.
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 vibrating in only one direction. If a second filter is placed in the microscope above the first one, with its main axis perpendicular to the first filter, no light passes through. If, however, tissue structures containing oriented macromolecules are located between the two polarizing filters, their repetitive 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 of vibration of polarized light is called birefringence and is
Polarizing light microscopy produces an image only of material having repetitive, periodic macromolecular structure; features without such structure are not seen. Pieces of thin, unsectioned mesentery were stained with red picrosirius, orcein, and hematoxylin, placed on slides and observed by bright-field (a) and polarizing (b) microscopy.
(a) With bright-field microscopy, collagen fibers appear red, with thin elastic fibers and cell nuclei darker. (X40)
(b) With polarizing microscopy, only the collagen fibers are visible and these exhibit intense yellow or orange birefringence. (a: X40; b: X100)
FIGURE 1–6 Principle of confocal microscopy.
FIGURE 1–7 Tissue appearance with bright-field and polarizing microscopy.
a feature of crystalline substances or substances containing highly oriented molecules, such as cellulose, collagen, microtubules, and actin filaments.
The utility of all light microscopic methods is greatly extended through the use of digital cameras. Many features of digitized histologic images can be analyzed quantitatively using appropriate software. Such images can also be enhanced to allow objects not directly visible through the eyepieces to be examined on a monitor.
› ELECTRON MICROSCOPY
Transmission and scanning electron microscopes are based on the interaction of tissue components with beams of electrons.
Objective lens
Intermediate lens
Projector lens Image on viewing screen
Electron detector with CCD camera
Electron gun
The wavelength in an electron beam is much shorter than that of light, allowing a 1000-fold increase in resolution.
Transmission Electron Microscopy
The transmission electron microscope (TEM) is an imaging system that permits resolution around 3 nm. This high resolution allows isolated particles magnified as much as 400,000 times to be viewed in detail. Very thin (40-90 nm), resin-embedded tissue sections are typically studied by TEM at magnifications up to approximately 120,000 times.
Figure 1–8a indicates the components of a TEM and the basic principles of its operation: a beam of electrons focused using electromagnetic “lenses” passes through the tissue section to produce an image with black, white, and intermediate
Electron gun Copper gr id with three sections
Electron microscopes are large instruments generally housed in a specialized EM facility.
(a) Schematic view of the major components of a transmission electron microscope (TEM), which is configured rather like an upsidedown light microscope. With the microscope column in a vacuum, a metallic (usually tungsten) filament (cathode) at the top emits electrons that travel to an anode with an accelerating voltage between 60 and 120 kV. Electrons passing through a hole in the anode form a beam that is focused electromagnetically by circular electric coils in a manner analogous to the effect of optical lenses on light. The first lens is a condenser focusing the beam on the section. Some electrons interact with atoms in the section, being absorbed or scattered to different extents, while others are simply transmitted through the specimen with no interaction. Electrons reaching the objective lens form an image that is then magnified and finally projected on a fluorescent screen or a charge-coupled device (CCD) monitor and camera.
(b) Scanning electron microscope
In a TEM image areas of the specimen through which electrons passed appear bright (electron lucent), while denser areas or those that bind heavy metal ions during specimen preparation absorb or deflect electrons and appear darker (electron dense). Such images are therefore always black, white, and shades of gray.
(b) The scanning electron microscope (SEM) has many similarities to a TEM. However, here the focused electron beam does not pass through the specimen, but rather is moved sequentially (scanned) from point to point across its surface similar to the way an electron beam is scanned across a television tube or screen. For SEM specimens are coated with metal atoms with which the electron beam interacts, producing reflected electrons and newly emitted secondary electrons. All of these are captured by a detector and transmitted to amplifiers and processed to produce a black-and-white image on the monitor. The SEM shows only surface views of the coated specimen but with a striking 3D, shadowed quality. The inside of organs or cells can be analyzed after sectioning to expose their internal surfaces.
FIGURE 1–8 Electron microscopes.
Cathode
(a) Transmission electron microscope
shades of gray regions. These regions of an electron micrograph correspond to tissue areas through which electrons passed readily (appearing brighter or electron-lucent) and areas where electrons were absorbed or deflected (appearing darker or more electron-dense). To improve contrast and resolution in TEM, compounds with heavy metal ions are often added to the fixative or dehydrating solutions used for tissue preparation. These include osmium tetroxide, lead citrate, and uranyl compounds, which bind cellular macromolecules, increasing their electron density and visibility.
Cryofracture and freeze etching are techniques that allow TEM study of cells without fixation or embedding and have been particularly useful in the study of membrane structure. In these methods, very small tissue specimens are rapidly frozen in liquid nitrogen and then cut or fractured with a knife. A replica of the frozen exposed surface is produced in a vacuum by applying thin coats of vaporized platinum or other metal atoms. After removal of the organic material, the replica of the cut surface can be examined by TEM. With membranes the random fracture planes often split the lipid bilayers, exposing protein components whose size, shape, and distribution are difficult to study by other methods.
Scanning Electron Microscopy
Scanning electron microscopy (SEM) provides a highresolution view of the surfaces of cells, tissues, and organs. Like the TEM, this microscope produces and focuses a very narrow beam of electrons, but in this instrument the beam does not pass through the specimen (Figure 1–8b). Instead, the surface of the specimen is first dried and spray-coated with a very thin
Autoradiographs are tissue preparations in which particles called silver grains indicate the cells or regions of cells in which specific macromolecules were synthesized just prior to fixation. Shown here are autoradiographs from the salivary gland of a mouse injected with 3H-fucose 8 hours before tissue fixation. Fucose was incorporated into oligosaccharides, and the free 3H-fucose was removed during fixation and sectioning of the gland. Autoradiographic processing and microscopy reveal locations of newly synthesized glycoproteins containing that sugar.
layer of heavy metal (often gold) that reflects electrons in a beam scanning the specimen. The reflected electrons are captured by a detector, producing signals that are processed to produce a black-and-white image. SEM images are usually easy to interpret because they present a three-dimensional view that appears to be illuminated in the same way that large objects are seen with highlights and shadows caused by light.
› AUTORADIOGRAPHY
Microscopic autoradiography is a method of localizing newly synthesized macromolecules in cells or tissue sections. Radioactively labeled metabolites (nucleotides, amino acids, sugars) provided to the living cells are incorporated into specific macromolecules (DNA, RNA, protein, glycoproteins, and polysaccharides) and emit weak radiation that is restricted to those regions where the molecules are located. Slides with radiolabeled cells or tissue sections are coated in a darkroom with photographic emulsion in which silver bromide crystals act as microdetectors of the radiation in the same way that 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 radiation produce small black grains of metallic silver, which under either the light microscope or TEM indicate the locations of radiolabeled macromolecules in the tissue (Figure 1–9).
Much histological information becomes available by autoradiography. If a radioactive precursor of DNA (such as tritium-labeled thymidine) is used, it is possible to know which cells in a tissue (and how many) are replicating DNA
(a) Black grains of silver from the light-sensitive material coating the specimen are visible over cell regions with secretory granules and the duct indicating glycoprotein locations. (X1500)
(b) The same tissue prepared for TEM autoradiography shows silver grains with a coiled or amorphous appearance again localized mainly over the granules (G) and in the gland lumen (L). (X7500) (Figure 1–9b, used with permission from Drs Ticiano G. Lima and A. Antonio Haddad, School of Medicine, Ribeirão Preto, Brazil.)
FIGURE 1–9 Microscopic autoradiography.
and preparing to divide. Dynamic events may also be analyzed. For example, if one wishes to know where in the cell protein is produced, if it is secreted, and its path in the cell before being secreted, several animals are injected with a radioactive amino acid and tissues collected at different times after the injections. Autoradiography of the tissues from the sequential times will indicate the migration of the radioactive proteins.
› CELL & TISSUE CULTURE
Live cells and tissues can be maintained and studied outside the body in culture (in vitro). In the organism (in vivo), cells are bathed in fluid derived from blood plasma and containing many different molecules required for survival and growth. Cell culture allows the direct observation of cellular behavior under a phase-contrast microscope, and many experiments technically impossible to perform in the intact animal can be accomplished in vitro.
The cells and tissues are grown in complex solutions of known composition (salts, amino acids, vitamins) to which serum or specific growth factors are added. Cells to be cultured are dispersed mechanically or enzymatically from a tissue or organ and placed with sterile procedures in a clear dish to which they adhere, usually as a single layer (see Figure 1–5). Such preparations are called primary cell cultures. Some cells can be maintained in vitro for long periods because they become immortalized and constitute a permanent cell line. Most cells obtained from normal tissues have a finite, genetically programmed life span. However, certain changes (some related to oncogenes; see Chapter 3) can promote cell immortality, a process called transformation, and are similar to the initial changes in a normal cell’s becoming a cancer cell. Improvements in culture technology and use of specific growth factors now allow most cell types to be maintained in vitro.
As shown in Chapter 2, incubation of living cells in vitro with a variety of new fluorescent compounds that are sequestered and metabolized in specific compartments of the cell provides a new approach to understanding these compartments both structurally and physiologically. Other histologic techniques applied to cultured cells have been particularly important for understanding the locations and functions of microtubules, microfilaments, and other components of the cytoskeleton.
› › MEDICAL APPLICATION
Cell culture is very widely used to study molecular changes that occur in cancer; to analyze infectious viruses, mycoplasma, and some protozoa; and for many routine genetic or chromosomal analyses. Cervical cancer cells from a patient later identified as Henrietta Lacks, who died from the disease in 1951, were used to establish one of the first cell lines, called HeLa cells, which are still used in research on cellular structure and function throughout the world.
› ENZYME HISTOCHEMISTRY
Enzyme histochemistry (or cytochemistry) is a method for localizing cellular structures using a specific enzymatic activity present in those structures. To preserve the endogenous enzymes, histochemical procedures usually use unfixed or mildly fixed tissue, which is sectioned on a cryostat to avoid adverse effects of heat and organic solvents on enzymatic activity. For enzyme histochemistry: (1) tissue sections are immersed in a solution containing the substrate of the enzyme to be localized; (2) the enzyme is allowed to act on its substrate; (3) the section is then put in contact with a marker compound that reacts with a product of the enzymatic action on the substrate; and (4) the final product from the marker, which must be insoluble and visible by light or electron microscopy, precipitates over the site of the enzymes, identifying their location.
Examples of enzymes that can be detected histochemically include the following:
■ Phosphatases, which remove phosphate groups from macromolecules (Figure 1–10).
■ Dehydrogenases, which transfer hydrogen ions from one substrate to another, such as many enzymes of the citric acid (Krebs) cycle, allowing histochemical identification of such enzymes in mitochondria.
■ Peroxidase, which promotes the oxidation of substrates with the transfer of hydrogen ions to hydrogen peroxide.
› › MEDICAL APPLICATION
Many enzyme histochemical procedures are used in the medical laboratory, including Perls’ Prussian blue reaction for iron (used to diagnose the iron storage diseases, hemochromatosis and hemosiderosis), the PAS-amylase and alcian blue reactions for polysaccharides (to detect glycogenosis and mucopolysaccharidosis), and reactions for lipids and sphingolipids (to detect sphingolipidosis).
› VISUALIZING SPECIFIC MOLECULES
A specific macromolecule present in a tissue section may also be identified by using tagged compounds or macromolecules that bind specifically with the molecule of interest. The compounds that interact with the molecule must be visible with the light or electron microscope, often by being tagged with a detectible label. The most commonly used labels are fluorescent compounds, radioactive atoms that can be detected with autoradiography, molecules of peroxidase or other enzymes that can be detected with histochemistry, and metal (usually gold) particles that can be seen with light and electron microscopy. These methods can be used to detect and localize specific sugars, proteins, and nucleic acids.
(a) Micrograph of cross sections of kidney tubules treated histochemically to demonstrate alkaline phosphatases (with maximum activity at an alkaline pH) showing strong activity of this enzyme at the apical surfaces of the cells at the lumens (L) of the tubules. (X200)
(b) TEM image of a kidney cell in which acid phosphatase has been localized histochemically in three lysosomes (Ly) near the nucleus (N). The dark material within these structures is lead phosphate that precipitated in places with acid phosphatase activity. (X25,000)
(Figure 1–10b, used with permission from Dr Eduardo Katchburian, Department of Morphology, Federal University of São Paulo, Brazil.)
Examples of molecules that interact specifically with other molecules include the following:
■ Phalloidin, a compound extracted from mushroom, Amanita phalloides, interacts strongly with the actin protein of microfilaments.
■ Protein A, purified from Staphylococcus aureus bacteria, binds to the Fc region of antibody molecules, and can therefore be used to localize naturally occurring or applied antibodies bound to cell structures.
■ Lectins, glycoproteins derived mainly from plant seeds, bind to carbohydrates with high affinity and specificity. Different lectins bind to specific sugars or sequences of sugar residues, allowing fluorescently labeled lectins to be used to stain specific glycoproteins or other macromolecules bearing specific sequences of sugar residues.
Immunohistochemistry
A highly specific interaction between macromolecules is that between an antigen and its antibody. For this reason, labeled antibodies are routinely used in immunohistochemistry to identify and localize many specific proteins, not just those with enzymatic activity that can be demonstrated by histochemistry.
The body’s immune cells interact with and produce antibodies against other macromolecules—called antigens—that are recognized as “foreign,” not a normal part of the organism, and potentially dangerous. Antibodies belong to the immunoglobulin family of glycoproteins and are secreted by lymphocytes. These molecules normally bind specifically to their provoking antigens and help eliminate them.
Widely applied for both research and diagnostic purposes, every immunohistochemical technique requires an antibody against the protein that is to be detected. This means that the protein must have been previously purified using biochemical or molecular methods so that antibodies against it can be produced. To produce antibodies against protein x of a certain animal species (eg, a human or rat), the isolated protein is injected into an animal of another species (eg, a rabbit or a goat). If the protein’s amino acid sequence is sufficiently different for this animal to recognize it as foreign—that is, as an antigen—the animal will produce antibodies against the protein.
Different groups (clones) of lymphocytes in the injected animal recognize different parts of protein x and each clone produces an antibody against that part. These antibodies are collected from the animal’s plasma and constitute a mixture of polyclonal antibodies, each capable of binding a different region of protein x
It is also possible, however, to inject protein x into a mouse and a few days later isolate the activated lymphocytes and place them into culture. Growth and activity of these cells can be prolonged indefinitely by fusing them with lymphocytic tumor cells to produce hybridoma cells. Different hybridoma clones produce different antibodies against the several parts of
FIGURE 1–10 Enzyme histochemistry.
protein x, and each clone can be isolated and cultured separately so that the different antibodies against protein x can be collected separately. Each of these antibodies is a monoclonal antibody. An advantage to using a monoclonal antibody rather than polyclonal antibodies is that it can be selected to be highly specific and to bind strongly to the protein to be detected, with less nonspecific binding to other proteins that are similar to the one of interest.
In immunohistochemistry, a tissue section that one believes contains the protein of interest is incubated in a solution containing antibody (either monoclonal or polyclonal) against this protein. The antibody binds specifically to the protein and after a rinse the protein’s location in the tissue or cells can be seen with either the light or electron microscope by visualizing the antibody. Antibodies are commonly tagged with fluorescent compounds, with peroxidase or alkaline phosphatase for histochemical detection, or with electrondense gold particles for TEM.
As Figure 1–11 indicates, there are direct and indirect methods of immunocytochemistry. The direct method just involves a labeled antibody that binds the protein of interest.
Indirect immunohistochemistry involves sequential application of two antibodies and additional washing steps. The (primary) antibody specifically binding the protein of interest is not labeled. The detectible tag is conjugated to a secondary antibody made in an animal species different (“foreign”) from that which made the primary antibody. For example, primary antibodies made by mouse lymphocytes (such as most monoclonal antibodies) are specifically recognized and bound by antibodies made in a rabbit or goat injected with mouse antibody immunoglobulin.
The indirect method is used more widely in research and pathologic tests because it is more sensitive, with the extra level of antibody binding serving to amplify the visible signal. Moreover, the same preparation of labeled secondary antibody can be used in studies with different primary antibodies (specific for different antigens) as long as all these are made in the same species. There are other indirect methods that involve the use of other intermediate molecules, such as the biotin-avidin technique, which are also used to amplify detection signals. Examples of indirect immunocytochemistry are shown in Figure 1–12, demonstrating the use of this method with cells in culture or after tissue sectioning for both light microscopy and TEM.
› › MEDICAL APPLICATION
Because cells in some diseases, including many cancer cells, often produce proteins unique to their pathologic condition, immunohistochemistry can be used by pathologists to diagnose many diseases, including certain types of tumors and some virus-infected cells. Table 1–1 shows some applications of immunocytochemistry routinely used in clinical practice.
Hybridization Techniques
Hybridization usually implies the specific binding between two single strands of nucleic acid, which occurs under appropriate conditions if the strands are complementary. The greater the similarities of their nucleotide sequences, the more readily the complementary strands form “hybrid” double-strand molecules. Hybridization at stringent conditions allows the
Immunocytochemistry (or immunohistochemistry) can be direct or indirect. Direct immunocytochemistry (left) uses an antibody made against the tissue protein of interest and tagged directly with a label such as a fluorescent compound or peroxidase. When placed with the tissue section on a slide, these labeled antibodies bind specifically to the protein (antigen) against which they were produced and can be visualized by the appropriate method. Indirect immunocytochemistry (right) uses first a primary antibody made against the protein (antigen) of interest and applied to the tissue section to bind its specific antigen. Then a
labeled secondary antibody is obtained that was (1) made in another species against immunoglobulin proteins (antibodies) from the species in which the primary antibodies were made and (2) labeled with a fluorescent compound or peroxidase. When the labeled secondary antibody is applied to the tissue section, it specifically binds the primary antibodies, indirectly labeling the protein of interest on the slide. Because more than one labeled secondary antibody can bind each primary antibody molecule, labeling of the protein of interest is amplified by the indirect method.
FIGURE 1–11 Immunocytochemistry techniques.
FIGURE 1–12
Cells and tissues stained by immunohistochemistry.
Immunocytochemical methods to localize specific proteins can be applied to either light microscopic or TEM preparations using a variety of labels.
(a) A single cultured uterine cell stained fluorescently to reveal a meshwork of intermediate filaments (green) throughout
the cytoplasm. Primary antibodies against the filament protein desmin and fluorescein isothiocyanate (FITC)-labeled secondary antibodies were used in the indirect staining technique, with the nucleus counterstained blue with DAPI. (X650)
(b) A section of small intestine treated with an antibody against the enzyme lysozyme. The secondary antibody labeled with peroxidase was then applied and the localized brown color produced histochemically with the peroxidase substrate 3,3′-diamino-azobenzidine (DAB). The method demonstrates lysozyme-containing structures in scattered macrophages and in the large clusters of cells. Nuclei were counterstained with hematoxylin. (X100)
(c) A section of pancreatic cells in a TEM preparation incubated with an antibody against the enzyme amylase and then with protein A coupled with gold particles. Protein A has high affinity toward antibody molecules and the resulting image reveals the presence of amylase with the gold particles localized as very small black dots over dense secretory granules and developing granules (left). With specificity for immunoglobulin molecules, labeled protein A can be used to localize any primary antibody. (X5000)
(Figure 1–12c, used with permission from Dr Moise Bendayan, Departments of Pathology and Cell Biology, University of Montreal, Montreal, Canada.)
TABLE 1–1 Examples of specific antigens with diagnostic importance.
Antigens Diagnosis
Specific cytokeratins
Protein and polypeptide hormones
Carcinoembryonic antigen (CEA)
Steroid hormone receptors
Antigens produced by viruses
Tumors of epithelial origin
Certain endocrine tumors
Glandular tumors, mainly of the digestive tract and breast
Breast duct cell tumors
Specific virus infections
specific identification of sequences in genes or RNA. This can occur with cellular DNA or RNA when nucleic acid sequences in solution are applied directly to prepared cells and tissue sections, a procedure called in situ hybridization (ISH).
This technique is ideal for (1) determining if a cell has a specific sequence of DNA, such as a gene or part of a gene (Figure 1–13), (2) identifying the cells containing specific messenger RNAs (mRNAs) (in which the corresponding gene is being transcribed), or (3) determining the localization of a gene in a specific chromosome. DNA and RNA of the cells must be initially denatured by heat or other agents to become completely single-stranded, and the nucleotide sequences of interest are detected with probes consisting of single-stranded complementary DNA (cDNA). The probe may be obtained by cloning, by polymerase chain reaction (PCR) amplification of the target sequence, or by chemical synthesis if the desired sequence is short. The probe is tagged with nucleotides containing a radioactive isotope (localized by autoradiography) or modified with a small compound such as digoxigenin (identified by immunocytochemistry). A solution containing the probe is placed over the specimen under conditions allowing hybridization and after the excess unbound probe is washed off, the localization of the hybridized probe is revealed through its label.
In situ hybridization of this tissue section with probes for the human papillomavirus (HPV) reveals the presence of many cells containing the virus. The section was incubated with a solution containing a digoxigenin-labeled complementary DNA (cDNA) probe for the HPV DNA. The probe was then visualized by direct immunohistochemistry using peroxidaselabeled antibodies against digoxigenin. This procedure stains brown only those cells containing HPV. (X400; H&E) (Used with permission from Dr Jose E. Levi, Virology Lab, Institute of Tropical Medicine, University of São Paulo, Brazil.)
› › MEDICAL APPLICATION
Warts on the skin of the genitals and elsewhere are due to infection with the human papillomavirus (HPV) which causes the characteristic benign proliferative growth. As shown in Figure 1–12, such virus-infected cells can often be demonstrated by ISH. Certain cancer cells with unique or elevated expression of specific genes are also localized in tumors and studied microscopically by ISH.
› INTERPRETATION OF STRUCTURES IN TISSUE SECTIONS
In studying and interpreting stained tissue sections, it is important to remember that microscopic preparations are the end result of a series of processes that began with collecting the tissue and ended with mounting a coverslip on the slide. Certain steps in this procedure may distort the tissues slightly, producing minor structural abnormalities called artifacts not present in the living tissue.
One such distortion is minor shrinkage of cells or tissue regions produced by the fixative, by the ethanol, or by the heat needed for paraffin embedding. Shrinkage can create artificial spaces between cells and other tissue components. Such spaces can also result from the loss of lipids or low-molecular-weight substances not preserved by the fixative or removed by the dehydrating and clearing fluids. Slight cracks in sections may also appear as large spaces in the tissue.
Other artifacts may include small wrinkles in the section (which the novice may confuse with linear structures in tissue) and precipitates from the stain (which may be confused with cellular structures such as cytoplasmic granules). Students must be aware of the existence of artifacts and able to recognize them.
Another difficulty in the study of histologic sections is the impossibility of differentially staining all tissue components on one slide. A single stain can seldom demonstrate well nuclei, mitochondria, lysosomes, basement membranes, elastic fibers, etc. With the light microscope, it is necessary to examine preparations stained by different methods before an idea of the whole composition and structure of a cell or tissue can be obtained. The TEM allows the observation of cells with all its internal structures and surrounding ECM components, but only a few cells in a tissue can be conveniently studied in these very small samples.
Finally, when a structure’s three-dimensional volume is cut into very thin sections, the sections appear microscopically to have only two dimensions: length and width. When examining a section under the microscope, the viewer must always keep in mind that components are missing in front of and behind what is being seen because many tissue structures are thicker than the section. Round structures seen microscopically may actually be portions of spheres or tubes. Because structures in a tissue have different orientations, their two-dimensional (2D) appearance will also vary depending on the plane of section. A single convoluted tube will appear in a tissue section as many separate rounded or oval structures (Figure 1–14).
FIGURE 1–13 In situ hybridization (ISH).
FIGURE 1–14
In thin sections 3D structures appear to have only two dimensions. Such images must be interpreted correctly to understand the actual structure of tissue and organ components. For example, blood vessels and other tubular structures appear in sections as round or oval shapes whose size and shape depend on the transverse or oblique angle of the cut. A highly coiled tube will appear as several round and oval structures. In TEM sections of cells, round structures may represent spherical organelles or transverse cuts through tubular organelles such as mitochondria. It is important to develop such interpretive skill to understand tissue and cell morphology in microscopic preparations.
Histology & Its Methods of Study SUMMA RY OF KEY POINTS
Preparation of Tissues for Study
■ Chemical fixatives such as formalin are used to preserve tissue structure by cross-linking and denaturing proteins, inactivating enzymes, and preventing cell autolysis or self-digestion.
■ Dehydration of the fixed tissue in alcohol and clearing in organic solvents prepare it for embedding and sectioning.
■ Embedding in paraffin wax or epoxy resin allows the tissue to be cut into very thin sections (slices) with a microtome.
■ Sections are mounted on glass slides for staining, which is required to reveal specific cellular and tissue components with the microscope.
■ The most commonly used staining method is a combination of the stains H&E, which act as basic and acidic dyes, respectively.
■ Cell substances with a net negative (anionic) charge, such as DNA and RNA, react strongly with hematoxylin and basic stains; such material is said to be “basophilic.”
■ Cationic substances, such as collagen and many cytoplasmic proteins react with eosin and other acidic stains and are said to be “acidophilic.”
Light Microscopy
Autoradiography
■ This process localizes cell components synthesized using radioactive precursors by detecting silver grains produced by weakly emitted radiation in a photographic emulsion coating the tissue section or cells.
■ With either light microscopy or TEM, autoradiography permits unique studies of processes such as tissue growth (using radioactive DNA precursors) or cellular pathways of macromolecular synthesis.
Cell & Tissue Culture
■ Cells can be grown in vitro from newly explanted tissues (primary cultures) or as long-established cell lines and can be examined in the living state by phase-contrast light microscopy.
Enzyme Histochemistry
■ Histochemical (or cytochemical) techniques use specific enzymatic activities in lightly fixed or unfixed tissue sections to produce visible products in the specific enzyme locations.
■ Fixation and paraffin embedding denatures most enzymes, so histochemistry usually uses frozen tissue sectioned with a cryostat
■ Enzyme classes for which histochemical study is useful include phosphatases, dehydrogenases, and peroxidases, with peroxidase often conjugated to antibodies used in immunohistochemistry.
■ Bright-field microscopy, the method most commonly used by both students and pathologists, uses ordinary light and the colors are imparted by tissue staining.
■ Fluorescence microscopy uses UV light, under which only fluorescent molecules are visible, allowing localization of fluorescent probes which can be much more specific than routine stains.
■ Phase-contrast microscopy uses the differences in refractive index of various natural cell and tissue components to produce an image without staining, allowing observation of living cells.
■ Confocal microscopy involves scanning the specimen at successive focal planes with a focused light beam, often from a laser, and produces a 3D reconstruction from the images.
Visualizing Specific Molecules
■ Some substances specifically bind certain targets in cells.
■ Immunohistochemistry is based on specific reactions between an antigen and antibodies labeled with visible markers, often fluorescent compounds or peroxidase for light microscopy and gold particles for TEM.
■ If the cell or tissue antigen of interest is detected by directly binding a labeled primary antibody specific for that antigen, the process is considered direct immunohistochemistry
■ Indirect immunohistochemistry uses an unlabeled primary antibody that is detected bound to its antigen with labeled secondary antibodies.
■ The indirect immunohistochemical method is more commonly used because the added level of antibody binding amplifies the signal detected and provides greater technical flexibility.
■ Specific gene sequences or mRNAs of cells can be detected microscopically using labeled cDNA probes in a technique called in situ hybridization (ISH).
Interpretation of Structures in Tissue Sections
■ Many steps in tissue processing, slide preparation, and staining can introduce minor artifacts such as spaces and precipitates that are not normally present in the living tissue and must be recognized.
■ Sections of cells or tissues are essentially 2D planes through 3D structures, and understanding this fact is important for their correct interpretation and study.
Histology & Its Methods of Study ASS ESS YOUR KNO WLEDGE
1. In preparing tissue for routine light microscopic study, which procedure immediately precedes clearing the specimen with an organic solvent?
a. Dehydration
b. Fixation
c. Staining
d. Clearing
e. Embedding
2. Which of the following staining procedures relies on the cationic and anionic properties of the material to be stained?
a. Enzyme histochemistry
b. PAS reaction
c. H&E staining
d. Immunohistochemistry
e. Metal impregnation techniques
3. In a light microscope used for histology, resolution and magnification of cells are largely dependent on which component?
a. Condenser
b. Objective lens
c. Eyepieces or ocular lenses
d. Specimen slide
e. The control for illumination intensity
4. Cellular storage deposits of glycogen, a free polysaccharide, could best be detected histologically using what procedure?
a. Autoradiography
b. Electron microscopy
c. Enzyme histochemistry
d. H&E staining
e. PAS reaction
5. Adding heavy metal compounds to the fixative and ultrathin sectioning of the embedded tissue with a glass knife are techniques used for which histologic procedure?
a. Scanning electron microscopy
b. Fluorescent microscopy
c. Enzyme histochemistry
d. Confocal microscopy
e. TEM
6. Resolution in electron microscopy greatly exceeds that of light microscopy due to which of the following?
a. The wavelength of the electrons in the microscope beam is shorter than that of a beam of light.
b. The lenses of an electron microscope are of greatly improved quality.
c. For electron microscopy the tissue specimen does not require staining.
d. The electron microscope allows much greater magnification of a projected image than a light microscope provides.
e. An electron microscope can be much more finely controlled than a light microscope.
7. Microscopic autoradiography uses radioactivity and can be employed to study what features in a tissue section?
a. The types of enzymes found in various cell locations
b. Cellular sites where various macromolecules are synthesized
c. The sequences of mRNA made in the cells
d. The dimensions of structures within the cells
e. The locations of genes transcribed for specific mRNA
8. To identify and localize a specific protein within cells or the ECM, one would best use what approach?
a. Autoradiography
b. Enzyme histochemistry
c. Immunohistochemistry
d. TEM
e. Polarizing microscopy
9. In situ hybridization is a histologic technique used to visualize what type of macromolecule?
a. Proteins
b. Carbohydrates
c. Certain enzymes
d. Nucleic acids
e. Lipids
10. Hospital laboratories frequently use unfixed, frozen tissue specimens sectioned with a cryostat for rapid staining, microscopic examination, and diagnosis of pathologic conditions. Besides saving much time by avoiding fixation and procedures required for paraffin embedding, frozen sections retain and allow study of what macromolecules normally lost in the paraffin procedure?
a. Carbohydrates
b. Small mRNA
c. Basic proteins
d. Acidic proteins
e. Lipids
The Cytoplasm
Cells and extracellular material together comprise the tissues that make up animal organs. In all tissues, cells are the basic structural and functional units, the smallest living parts of the body. Animal cells are enclosed by cell membranes and are eukaryotic, each with a distinct, membrane-enclosed nucleus surrounded by cytoplasm, fluid containing a system of membranous organelles, nonmembranous molecular assemblies, and a cytoskeleton. In contrast, the smaller prokaryotic cells of bacteria typically have a cell wall and lack nuclei and membranous cytoplasmic structures.
› CELL DIFFERENTIATION
The average adult human body consists of nearly 40 trillion cells, according to the best available estimate. These cells exist as hundreds of histologically distinct cell types, all derived from the zygote, and the single cell formed by the merger of a spermatozoon with an oocyte at fertilization. The first zygotic cellular divisions produce cells called blastomeres, and as part of the early embryo’s inner cell mass blastomeres give rise to all tissue types of the fetus. Explanted to tissue culture cells of the inner cell mass are called embryonic stem cells Most cells of the fetus undergo a specialization process called differentiation in which they predominantly express sets of genes that mediate specific cytoplasmic activities, becoming efficiently organized in tissues with specialized functions and usually changing their shape accordingly. For example, muscle cell precursors elongate into long, fiber-like cells containing large arrays of actin and myosin. All animal cells contain actin filaments and myosins, but muscle cells are specialized for
using these proteins to convert chemical energy into forceful contractions.
Major cellular functions performed by specialized cells in the body are listed in Table 2–1. It is important to understand that the functions listed there can be performed by most cells of the body; specialized cells have greatly expanded their capacity for one or more of these functions during differentiation. Changes in cells’ microenvironments under normal and pathologic conditions can cause the same cell type to have variable features and activities. Cells that appear similar structurally often have different families of receptors for signaling molecules such as hormones and extracellular matrix (ECM) components, causing them to behave differently. For example, because of their diverse arrays of receptors, breast fibroblasts and uterine smooth muscle cells are exceptionally sensitive to female sex hormones, while most other fibroblasts and smooth muscle cells are insensitive.
› THE PLASMA MEMBRANE
The plasma membrane (cell membrane or plasmalemma) that envelops every eukaryotic cell consists of phospholipids, cholesterol, and proteins, with oligosaccharide chains covalently linked to many of the phospholipids and proteins. This limiting membrane functions as a selective barrier regulating the passage of materials into and out of the cell and facilitating the transport of specific molecules. One important role of the cell membrane is to keep constant the ion content of cytoplasm, which differs from that of the extracellular fluid. Membrane proteins also perform a number of specific recognition and signaling functions, playing a key role in the interactions of the cell with its environment.
TABLE 2–1 Differentiated cells typically specialize in one activity.
General Cellular Activity Specialized Cell(s)
Movement Muscle and other contractile cells
Form adhesive and tight junctions between cells
Synthesize and secrete components of the extracellular matrix
Convert physical and chemical stimuli into action potentials
Synthesis and secretion of degradative enzymes
Synthesis and secretion of glycoproteins
Synthesis and secretion of steroids
Ion transport
Intracellular digestion
Epithelial cells
Fibroblasts, cells of bone and cartilage
Neurons and sensory cells
Cells of digestive glands
Cells of mucous glands
Certain cells of the adrenal gland, testis, and ovary
Cells of the kidney and salivary gland ducts
Macrophages and neutrophils
Lipid storage Fat cells
Metabolite absorption
Cells lining the intestine
Although the plasma membrane defines the outer limit of the cell, a continuum exists between the interior of the cell and extracellular macromolecules. Certain plasma membrane proteins, the integrins, are linked to both the cytoskeleton and ECM components and allow continuous exchange of influences, in both directions, between the cytoplasm and material in the ECM.
Membranes range from 7.5 to 10 nm in thickness and consequently are visible only in the electron microscope. The line between adjacent cells sometimes seen faintly with the light microscope consists of plasma membrane proteins plus extracellular material, which together can reach a dimension visible by light microscopy.
Membrane phospholipids are amphipathic, consisting of two nonpolar (hydrophobic or water-repelling) long-chain fatty acids linked to a charged polar (hydrophilic or waterattracting) head that bears a phosphate group (Figure 2–1a). Phospholipids are most stable when organized into a double layer (bilayer) with the hydrophobic fatty acid chains located in a middle region away from water and the hydrophilic polar head groups contacting the water (Figure 2–1b). Molecules of cholesterol, a sterol lipid, insert at varying densities among the closely-packed phospholipid fatty acids, restricting their movements and modulating the fluidity of all membrane components. The phospholipids in each half of the bilayer are different. For example, in the well-studied membranes of red blood cells, phosphatidylcholine and sphingomyelin are more
abundant in the outer half, while phosphatidylserine and phosphatidylethanolamine are more concentrated in the inner layer. Some of the outer layer’s lipids, known as glycolipids, include oligosaccharide chains that extend outward from the cell surface and contribute to a delicate cell surface coating called the glycocalyx (Figures 2–1b and 2–2). With the transmission electron microscope (TEM) the cell membrane—as well as all cytoplasmic membranes—may exhibit a trilaminar appearance after fixation in osmium tetroxide; osmium binds the polar heads of the phospholipids and the oligosaccharide chains, producing the two dark outer lines that enclose the light band of osmium-free fatty acids (Figure 2–1b).
Proteins are major constituents of membranes (~50% by weight in the plasma membrane). Integral proteins are incorporated directly within the lipid bilayer, whereas peripheral proteins are bound to one of the two membrane surfaces, particularly on the cytoplasmic side (Figure 2–2). Peripheral proteins can be extracted from cell membranes with salt solutions, whereas integral proteins can be extracted only by using detergents to disrupt the lipids. The polypeptide chains of many integral proteins span the membrane, from one side to the other, several times and are accordingly called multipass proteins. Integration of the proteins within the lipid bilayer is mainly the result of hydrophobic interactions between the lipids and nonpolar amino acids of the proteins.
Freeze-fracture electron-microscope studies of membranes show that parts of many integral proteins protrude from both the outer or inner membrane surface (Figure 2–2b). Like those of glycolipids, the carbohydrate moieties of glycoproteins project from the external surface of the plasma membrane and contribute to the glycocalyx (Figure 2–3). They are important components of proteins acting as receptors, which participate in important interactions such as cell adhesion, cell recognition, and the response to protein hormones. As with lipids, the distribution of membrane polypeptides is different in the two surfaces of the cell membranes. Therefore, all membranes in the cell are asymmetric.
Studies with labeled membrane proteins of cultured cells reveal that many such proteins are not bound rigidly in place and are able to move laterally (Figure 2–4). Such observations as well as data from biochemical, electron microscopic, and other studies showed that membrane proteins comprise a moveable mosaic within the fluid lipid bilayer, the wellestablished fluid mosaic model for membrane structure (Figure 2–2a). Unlike the lipids, however, lateral diffusion of many membrane proteins is often restricted by their cytoskeletal attachments. Moreover, in most epithelial cells tight junctions between the cells (see Chapter 4) also restrict lateral diffusion of unattached transmembrane proteins and outer layer lipids, producing different domains within the cell membranes.
Membrane proteins that are components of large enzyme complexes are also usually less mobile, especially those involved in the transduction of signals from outside the cell. Such protein complexes are located in specialized membrane
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nothing more than the result of the conception of the parts and of their connection with each other. Suppose now that the poet should lead us in proper order from one part of the object to the other; suppose he should succeed in making the connection of these parts perfectly clear to us; how much time will he have consumed?
The details, which the eye takes in at a glance, he enumerates slowly one by one, and it often happens that, by the time he has brought us to the last, we have forgotten the first. Yet from these details we are to form a picture. When we look at an object the various parts are always present to the eye. It can run over them again and again. The ear, however, loses the details it has heard, unless memory retain them. And if they be so retained, what pains and effort it costs to recall their impressions in the proper order and with even the moderate degree of rapidity necessary to the obtaining of a tolerable idea of the whole.
Let us take an example which may be called a masterpiece of its kind.
Dort ragt das hohe Haupt vom edeln Enziane Weit übern niedern Chor der Pöbelkräuter hin, Ein ganzes Blumenvolk dient unter seiner Fahne, Sein blauer Bruder selbst bückt sich und ehret ihn.
Der Blumen helles Gold, in Strahlen umgebogen, Thürmt sich am Stengel auf, und krönt sein grau Gewand, Der Blätter glattes Weiss mit tiefem Grün durchzogen, Strahlt von dem bunten Blitz von feuchtem Diamant. Gerechtestes Gesetz! dass Kraft sich Zier vermähle, In einem schönen Leib wohnt eine schön’re Seele.
Hier kriecht ein niedrig Kraut, gleich einem grauen Nebel, Dem die Natur sein Blatt im Kreuze hingelegt, Die holde Blume zeigt die zwei vergöldten Schnäbel, Die ein von Amethyst gebildter Vogel trägt.
Dort wirft ein glänzend Blatt, in Finger ausgekerbet, Auf einen hellen Bach den grünen Wiederschein; Der Blumen zarten Schnee, den matter Purpur färbet, Schliesst ein gestreifter Stern in weisse Strahlen ein. Smaragd und Rosen blühn auch auf zertretner Heide, Und Felsen decken sich mit einem Purpurkleide.[106]
The learned poet is here painting plants and flowers with great art and in strict accordance with nature, but there is no illusion in his
picture. I do not mean that a person who had never seen these plants and flowers could form little or no idea of them from his description. Perhaps all poetical pictures require a previous knowledge of their subject. Neither would I deny that a person possessing such knowledge might derive from the poet a more vivid idea of certain details. I only ask how it is with a conception of the whole. If that is to become more vivid, none of the separate details must stand in undue prominence, but the new illumination must be equally shared by all. Our imagination must be able to embrace them all with equal rapidity in order to form from them in an instant that one harmonious whole which the eye takes in at a glance. Is that the case here? If not, how can it be said, “that the most exact copy produced by a painter is dull and faint compared with this poetical description?”[107] It is far inferior to what lines and colors can produce on canvas. The critic who bestowed upon it this exaggerated praise must have regarded it from an entirely false point of view. He must have looked at the foreign graces which the poet has woven into his description, at his idealization of vegetable life, and his development of inward perfections, to which outward beauty serves but as the shell. These he was considering, and not beauty itself or the degree of resemblance and vividness of the image, which painter and poet respectively can give us. Upon this last point every thing depends, and whoever maintains that the lines,
Der Blumen helles Gold in Strahlen umgebogen, Thürmt sich am Stengel auf, und krönt sein grau Gewand,
Der Blätter glattes Weiss, mit tiefem Grün durchzogen, Strahlt von dem bunten Blitz von feuchtem Diamant, can vie in vividness of impression with a flowerpiece by a Huysum, must either never have analyzed his own sensations, or must wilfully ignore them. It might be very pleasant to hear the lines read if we had the flowers in our hand; but, taken by themselves, they say little or nothing. I hear in every word the laborious poet, but the thing itself I am unable to see.
Once more, then, I do not deny that language has the power of describing a corporeal whole according to its parts. It certainly has, because its signs, although consecutive, are nevertheless arbitrary. But I deny that this power exists in language as the instrument of poetry. For illusion, which is the special aim of poetry, is not
produced by these verbal descriptions of objects, nor can it ever be so produced. The coexistence of the body comes into collision with the sequence of the words, and although while the former is getting resolved into the latter, the dismemberment of the whole into its parts is a help to us, yet the reunion of these parts into a whole is made extremely difficult, and not infrequently impossible.
Where the writer does not aim at illusion, but is simply addressing the understanding of his readers with the desire of awakening distinct and, as far as possible, complete ideas, then these descriptions of corporeal objects, inadmissible as they are in poetry, are perfectly appropriate. Not only the prose writer, but the didactic poet (for in as far as he is didactic he is no poet) may use them with good effect. Thus Virgil, in his Georgics, describes a cow fit for breeding:—
Optima torvæ
Forma bovis, cui turpe caput, cui plurima cervix, Et crurum tenus a mento palearia pendent.
Tum longo nullus lateri modus: omnia magna: Pes etiam, et camuris hirtæ sub cornibus aures. Nec mihi displiceat maculis insignis et albo, Aut juga detractans interdumque aspera cornu, Et faciem tauro propior; quæque ardua tota, Et gradiens ima verrit vestigia cauda.[108]
Or a handsome colt:—
Illi ardua cervix,
Argutumque caput, brevis alvus, obesaque terga, Luxuriatque toris animosum pectus, &c.[109]
Here the poet is plainly concerned more with the setting forth of the separate parts than with the effect of the whole. His object is to tell us the characteristics of a handsome colt and a good cow, so that we may judge of their excellence according to the number of these characteristics which they possess. Whether or not all these can be united into a vivid picture was a matter of indifference to him.
Except for this purpose, elaborate pictures of bodily objects, unless helped out by the above-mentioned Homeric device of making an actual series out of their coexistent parts, have always been considered by the best critics as ineffective trifles, requiring little or
no genius. “When a poetaster,” says Horace, “can do nothing else, he falls to describing a grove, an altar, a brook winding through pleasant meadows, a rushing river, or a rainbow.”
Lucus et ara Dianæ, Et properantis aquæ per amœnos ambitus agros, Aut flumen Rhenum, aut pluvius describitur arcus.[110]
Pope, when a man, looked back with contempt on the descriptive efforts of his poetic childhood. He expressly enjoined upon every one, who would not prove himself unworthy the name of poet, to abandon as early as possible this fondness for description. A merely descriptive poem he declared to be a feast made up of sauces.[111]
Herr Von Kleist, I know, prided himself very little on his “Spring.” Had he lived, he would have refashioned it altogether. He wanted to introduce into it some plan, and was meditating how he could best make the crowd of pictures, which seemed to have been drawn at random from the whole vast range of fresh creation, rise in some natural order and follow each other in fitting sequence. He would, at the same time, have done what Marmontel, doubtless with reference to his Eclogues, recommended to several German poets. He would have converted a series of pictures scantily interwoven with mental emotions, into a series of emotions sparingly interspersed with images.[112]
XVIII.
And shall Homer nevertheless have fallen into those barren descriptions of material objects?
Let us hope that only a few such passages can be cited. And even those few, I venture to assert, will be found really to confirm the rule, to which they appear to form an exception.
The rule is this, that succession in time is the province of the poet, coexistence in space that of the artist.
To bring together into one and the same picture two points of time necessarily remote, as Mazzuoli does the rape of the Sabine women and the reconciliation effected by them between their husbands and relations; or as Titian does, representing in one piece the whole story of the Prodigal Son,—his dissolute life, his misery, and repentance,— is an encroachment of the painter on the domain of the poet, which good taste can never sanction.
To try to present a complete picture to the reader by enumerating in succession several parts or things which in nature the eye necessarily takes in at a glance, is an encroachment of the poet on the domain of the painter, involving a great effort of the imagination to very little purpose.
Painting and poetry should be like two just and friendly neighbors, neither of whom indeed is allowed to take unseemly liberties in the heart of the other’s domain, but who exercise mutual forbearance on the borders, and effect a peaceful settlement for all the petty encroachments which circumstances may compel either to make in haste on the rights of the other.
I will not bring forward in support of this the fact that, in large historical pictures the single moment of time is always somewhat extended, and that perhaps no piece, very rich in figures, can be
found, in which every character has exactly the motion and attitude proper to him at that particular moment. The position of some belongs to a preceding point of time, that of others to a later. This is a liberty which the painter must justify by certain subtleties of arrangement, such as placing his figures more in the foreground or background, and thus making them take a more or less immediate interest in what is going on. I will merely quote, in favor of my view, a criticism of Mengs on Raphael’s drapery.[113] “There is a reason for all his folds, either in the weight of the material or the tension of the limbs. We can often infer from their present condition what they had been previously. Raphael indeed aimed at giving them significance in this way. We can judge from the folds whether, previously to the present posture, a leg or an arm had been more in front or more behind, whether a limb had been bent and is now straightening itself, or whether it had been outstretched and is now bending.” Here unquestionably the artist unites into one two distinct points of time. For, since the foot in its motion forward is immediately followed by that portion of the garment which rests upon it,—unless indeed the garment be of exceedingly stiff material, in which case it is ill adapted to painting,—there can be no moment at which the drapery assumes in the least degree any other fold than the present posture of the limb demands. If any other be represented, then the fold is that of the preceding moment while the position of the foot is that of the present. Few, however, will be inclined to deal thus strictly with the artist who finds it for his interest to bring these two moments of time before us at once. Who will not rather praise him for having had the wisdom and the courage to commit a slight fault for the sake of greater fulness of expression?
A similar indulgence is due to the poet. The continuity of his imitation permits him, strictly speaking, to touch at one moment on only a single side, a single property of his corporeal objects. But if the happy construction of his language enables him to do this with a single word, why should he not sometimes be allowed to add a second such word? why not a third, if it be worth his while, or even a fourth? As I have said, a ship in Homer is either simply the black ship, or the hollow ship, or the swift ship; at most the well-manned black ship. That is true of his style in general. Occasionally a passage occurs where he adds a third descriptive epithet:[114] Καμπύλα κύκλα, χάλκεα, ὀκτάκνημα, “round, brazen, eight-spoked wheels.” Even a
fourth: ἀσπίδα πάντοσε ἐΐσην, καλήν, χαλκείην, ἐξήλατον,[115] “a uniformly smooth, beautiful, brazen, wrought shield.” Who will not rather thank than blame him for this little luxuriance, when we perceive its good effect in a few suitable passages?
The true justification of both poet and painter shall not, however, be left to rest upon this analogy of two friendly neighbors. A mere analogy furnishes neither proof nor justification. I justify them in this way. As in the picture the two moments of time follow each other so immediately that we can without effort consider them as one, so in the poem the several touches answering to the different parts and properties in space are so condensed, and succeed each other so rapidly, that we seem to catch them all at once.
Here, as I have said, Homer is greatly aided by his admirable language. It not only allows him all possible freedom in multiplying and combining his epithets, but enables him to arrange them so happily that we are relieved of all awkward suspense with regard to the subject. Some of the modern languages are destitute of one or more of those advantages. Those which, like the French, must have recourse to paraphrase, and convert the καμπύλα κύκλα, χάλκεα, ὀκτάκνημα of Homer into “the round wheels which were of brass and had eight spokes,” give the meaning, but destroy the picture. The sense is here, however, nothing; the picture every thing. The one without the other turns the most graphic of poets into a tiresome tattler. This fate has often befallen Homer under the pen of the conscientious Madame Dacier. The German language can generally render the Homeric adjectives by equally short equivalents, but it cannot follow the happy arrangement of the Greek. It can say, indeed, “the round, brazen, eight-spoked;” but “wheels” comes dragging after. Three distinct predicates before any subject make but a confused, uncertain picture. The Greek joins the subject with the first predicate and lets the others follow. He says, “round wheels, brazen, eight-spoked.” Thus we know at once of what he is speaking, and learn first the thing and then its accidents, which is the natural order of our thoughts. The German language does not possess this advantage. Or shall I say, what really amounts to the same thing, that, although possessing it, the language can seldom use it without ambiguity? For if adjectives be placed after the subject (runde Räder, ehern und achtspeichigt) they are indeclinable, differing in nothing
from adverbs, and if referred, as adverbs, to the first verb that is predicated of the subject, the meaning of the whole sentence becomes always distorted, and sometimes entirely falsified.
But I am lingering over trifles and seem to have forgotten the shield of Achilles, that famous picture, which more than all else, caused Homer to be regarded among the ancients as a master of painting.[116] But surely a shield, it may be said, is a single corporeal object, the description of which according to its coexistent parts cannot come within the province of poetry. Yet this shield, its material, its form, and all the figures which occupied its enormous surface, Homer has described, in more than a hundred magnificent lines, so circumstantially and precisely that modern artists have found no difficulty in making a drawing of it exact in every detail.
My answer to this particular objection is, that I have already answered it. Homer does not paint the shield finished, but in the process of creation. Here again he has made use of the happy device of substituting progression for coexistence, and thus converted the tiresome description of an object into a graphic picture of an action. We see not the shield, but the divine master-workman employed upon it. Hammer and tongs in hand he approaches the anvil; and, after having forged the plates from the rough metal, he makes the pictures designed for its decoration rise from the brass, one by one, under his finer blows. Not till the whole is finished do we lose sight of him. At last it is done; and we wonder at the work, but with the believing wonder of an eyewitness who has seen it a-making.
The same cannot be said of the shield of Æneas in Virgil. The Roman poet either failed to see the fineness of his model, or the things which he wished to represent upon his shield seemed to him not of such a kind as to allow of their being executed before our eyes. They were prophecies, which the god certainly could not with propriety have uttered in our presence as distinctly as the poet explains them in his work. Prophecies, as such, require a darker speech, in which the names of those persons to come, whose fortunes are predicted, cannot well be spoken. In these actual names, however, lay, it would seem, the chief point of interest to the poet and courtier.[117] But this, though it excuse him, does not do away with the disagreeable effect of his departure from the Homeric method, as all readers of taste will admit. The preparations made by
Vulcan are nearly the same in Homer as in Virgil. But while in Homer we see, besides the preparations for the work, the work itself, Virgil, after showing us the god at work with his Cyclops,
Ingentem clypeum informant ...
... Alii ventosis follibus auras
Accipiunt, redduntque; alii stridentia tingunt
Æra lacu. Gemit impositis incudibus antrum.
Illi inter sese multa vi brachia tollunt
In numerum, versantque tenaci forcipe massam,[118]
suddenly drops the curtain and transports us to a wholly different scene. We are gradually led into the valley where Venus appears, bringing Æneas the arms that in the mean while have been finished. She places them against the trunk of an oak; and, after the hero has sufficiently stared at them, and wondered over them, and handled them, and tried them, the description or picture of the shield begins, which grows so cold and tedious from the constantly recurring “here is,” and “there is,” and “near by stands,” and “not far from there is seen,” that all Virgil’s poetic grace is needed to prevent it from becoming intolerable. Since, moreover, this description is not given by Æneas, who delights in the mere figures without any knowledge of their import,
... rerumque ignarus imagine gaudet,
nor by Venus, although she might be supposed to know as much about the fortunes of her dear grandson as her good-natured husband, but by the poet himself, the action meanwhile necessarily remains at a stand-still. Not a single one of the characters takes part; nor is what follows in the least affected by the representations on the shield. The subtle courtier, helping out his material with every manner of flattering allusion, is apparent throughout; but no trace do we see of the great genius, who trusts to the intrinsic merit of his work, and despises all extraneous means of awakening interest. The shield of Æneas is therefore, in fact, an interpolation, intended solely to flatter the pride of the Romans; a foreign brook with which the poet seeks to give fresh movement to his stream. The shield of Achilles, on the contrary, is the outgrowth of its own fruitful soil. For a shield was needed; and, since even what is necessary never comes from the hands of deity devoid of beauty, the shield had to be
ornamented. The art was in treating these ornamentations as such, and nothing more; in so weaving them into the material that when we look at that we cannot but see them. This could be accomplished only by the method which Homer adopted. Homer makes Vulcan devise decorations, because he is to make a shield worthy of a divine workman. Virgil seems to make him fashion the shield for the sake of the decorations, since he deems these of sufficient importance to deserve a special description long after the shield is finished.
XIX.
The objections brought against Homer’s shield by the elder Scaliger, Perrault, Terrasson, and others, are well known, as are also the answers of Madame Dacier, Boivin, and Pope. But these latter, it seems to me, have gone somewhat too far, and confiding in the justness of their cause have asserted things incorrect in themselves and contributing little to the poet’s justification.
In answer to the chief objection, that Homer had burdened his shield with more figures than there could possibly have been room for, Boivin undertook to show in a drawing how the necessary space might be obtained. His idea of the various concentric circles was very ingenious, although there is no foundation for it in the poet’s words and nothing anywhere to indicate that shields divided in this way were known to the ancients. Since Homer calls it (σάκος
δεδαιλωμένον) a shield, artistically wrought on all sides, I should prefer to gain the required space by turning to account the concave surface. A proof that the old artists did not leave this empty is furnished in the shield of Minerva by Phidias.[119] But not only does Boivin fail to seize this advantage, but, by separating into two or three pictures what the poet evidently meant for one, he unnecessarily multiplies the representations while diminishing the space by one-half. I know the motive which led him to this, but it was one by which he should not have allowed himself to be influenced. He should have shown his opponents the unreasonableness of their demands, instead of trying to satisfy them.
An example will make my meaning clear. When Homer says of one of the two cities:[120]
he refers, as I understand him, to but a single picture, that of a public lawsuit about the contested payment of a considerable fine for the committal of a murder. The artist, who is to execute this design, can use but a single moment of the action,—that of the accusation, of the examination of witnesses, of the pronouncing of the sentence, or any other preceding or following or intervening moment which may seem to him most fitting. This single moment he makes as pregnant as possible, and reproduces it with all that power of illusion which in the presentation of visible objects art possesses above poetry. Left far behind in this respect, what remains to the poet, if his words are to paint the same design with any degree of success, but to avail himself of his peculiar advantages? These are the liberty of extending his representation over what preceded, as well as what was to follow, the artist’s single point of time, and the power of showing not only what the artist shows, but what he has to leave to our imagination. Only by using these advantages can the poet raise himself to a level with the artist. Their works most resemble each other when their effect is equally vivid; not when one brings before the imagination through the ear neither more nor less than the other presents to the eye. Had Boivin defended the passage in Homer according to this principle, he would not have divided it into as many separate pictures as he thought he detected distinct points of time. All that Homer relates could not, indeed, be united in a single picture. The accusation and the denial, the summoning of the witnesses and the shouts of the divided populace, the efforts of the heralds to quiet the tumult and the sentence of the judges, are things successive in time, not coexistent in space. But what is not actually in the picture is there virtually, and the only true way of representing an actual picture in words is to combine what virtually exists in it with what is absolutely visible. The poet who allows himself to be bound by the limits of art may furnish data for a picture, but can never create one of his own.
The picture of the beleaguered city[121] Boivin divides likewise into three. He might as well have made twelve out of it as three. For since he has once for all failed to grasp the spirit of the poet, and requires him to be bound by the unities of a material picture, he might have discovered many more violations of these unities. In fact he ought almost to have devoted a separate space on the shield to every separate touch of the poet. In my opinion Homer has but ten different pictures on the whole shield, every one of which he introduces with ἐν μὲν ἔτευξε, or ἐν δὲ ποίησε, or ἐν δ’ ἐτίθει, or ἐν δὲ πόικιλλε Ἀμφιγυήεις, “on it he wrought,” “on it he placed,” “on it he formed,” “on it Vulcan skilfully fashioned.”[122] In the absence of these introductory words we have no right to suppose a distinct picture. On the contrary every thing which they cover must be regarded as a single whole, wanting in nothing but the arbitrary concentration into one moment of time, which the poet was in no way bound to observe. Had he observed this, and, by strictly limiting himself to it, excluded every little feature which in the material representation would have been inconsistent with this unity of time; had he in fact done what his cavillers require,—these gentlemen would indeed have had no fault to find with him, but neither would any person of taste have found aught to admire.
Pope not only accepted Boivin’s drawing, but thought he was doing a special service by showing that every one of these mutilated pieces was in accordance with the strictest rules of painting, as laid down at the present day. Contrast, perspective, the three unities, he found, were all observed in the best possible manner. And although well aware that, according to the testimony of good and trustworthy witnesses, painting at the time of the Trojan war was still in its cradle, he supposes either that Homer, instead of being bound by the achievements of painting at that time or in his own day, must in virtue of his godlike genius have anticipated all that art should in future be able to accomplish, or else that the witnesses could not have been so entirely worthy of faith that the direct testimony of this artistic shield should not be preferred to theirs. Whoever will, may accept the former supposition: the latter, surely, no one will be persuaded to adopt who knows any thing more of the history of art than the date of the historians. That painting in the time of Homer was still in its infancy he believes, not merely on the authority of
Pliny, or some other writer, but chiefly because, judging from the works of art mentioned by the ancients, he sees that even centuries later no great progress had been made. The pictures of Polygnotus, for instance, by no means stand the test which Pope thinks can be successfully applied to Homer’s shield. The two great works by this master at Delphi, of which Pausanias has left a circumstantial description,[123] were evidently wholly wanting in perspective. The ancients had no knowledge of this branch of art, and what Pope adduces as proof that Homer understood it, only proves that he has a very imperfect understanding of it himself.[124]
“That Homer,” he says, “was not a stranger to aerial perspective appears in his expressly marking the distance of object from object. He tells us, for instance, that the two spies lay a little remote from the other figures, and that the oak under which was spread the banquet of the reapers stood apart. What he says of the valley sprinkled all over with cottages and flocks appears to be a description of a large country in perspective. And, indeed, a general argument for this may be drawn from the number of figures on the shield, which could not be all expressed in their full size; and this is therefore a sort of proof that the art of lessening them according to perspective was known at that time.” The mere representing of an object at a distance as smaller than it would be if nearer the eye, by no means constitutes perspective in a picture. Perspective requires a single point of view; a definite, natural horizon; and this was wanting in the old pictures. In the paintings of Polygnotus the ground, instead of being level, rose so decidedly at the back that the figures which were meant to stand behind seemed to be standing above one another. If this was the usual position of the various figures and groups,—and that it was so may fairly be concluded from the old basreliefs, where those behind always stand higher than those in front, and look over their heads,—then we may reasonably take it for granted in Homer, and should not unnecessarily dismember those representations of his, which according to this treatment might be united in a single picture. The double scene in the peaceful city, through whose streets a joyous marriage train was moving at the same time that an important trial was going on in the market-place, requires thus no double picture. Homer could very well think of it as one, since he imagined himself to be overlooking the city from such a height as to command at once a view of the streets and the market.
My opinion is that perspective in pictures came incidentally from scene-painting, which was already in its perfection. But the applications of its rules to a single smooth surface was evidently no easy matter; for, even in the later paintings found among the antiquities of Herculaneum, there are many and various offences against perspective, which would now hardly be excusable even in a beginner.[125]
But I will spare myself the labor of collecting my desultory observations on a point whereon I may hope to receive complete satisfaction from Winkelmann’s promised “History of Art.”[126]
XX.
To return, then, to my road, if a saunterer can be said to have a road.
What I have been saying of bodily objects in general applies with even more force to those which are beautiful.
Physical beauty results from the harmonious action of various parts which can be taken in at a glance. It therefore requires that these parts should lie near together; and, since things whose parts lie near together are the proper subjects of painting, this art and this alone can imitate physical beauty.
The poet, who must necessarily detail in succession the elements of beauty, should therefore desist entirely from the description of physical beauty as such. He must feel that these elements arranged in a series cannot possibly produce the same effect as in juxtaposition; that the concentrating glance which we try to cast back over them immediately after their enumeration, gives us no harmonious picture; and that to conceive the effect of certain eyes, a certain mouth and nose taken together, unless we can recall a similar combination of such parts in nature or art, surpasses the power of human imagination.
Here again Homer is the model of all models. He says, Nireus was fair; Achilles was fairer; Helen was of godlike beauty. But he is nowhere betrayed into a more detailed description of these beauties. Yet the whole poem is based upon the loveliness of Helen. How a modern poet would have revelled in descriptions of it!
Even Constantinus Manasses sought to adorn his bald chronicle with a picture of Helen. I must thank him for the attempt, for I really should not know where else to turn for so striking an example of the folly of venturing on what Homer’s wisdom forbore to undertake. When I read in him:[127]
it is like seeing stones rolled up a mountain,[128] on whose summit they are to be built into a gorgeous edifice; but which all roll down of themselves on the other side. What picture does this crowd of words leave behind? How did Helen look? No two readers out of a thousand would receive the same impression of her.
But political verses by a monk are, it is true, no poetry. Let us hear Ariosto describe his enchantress Alcina:[129]
Di persona era tanto ben formata,
Quanto mai finger san pittori industri.
Con bionda chioma, lunga e annodata, Oro non è, che piu risplenda e lustri.
Spargeasi per la guancia delicata
Misto color di rose e di ligustri.
Di terso avorio era la fronte lieta,
Che lo spazio finia con giusta meta.
Sotto due negri, e sottilissimi archi
Son due negri, occhi, anzi due chiari soli
Pietosi a riguardar, a mover parchi,
Intorno a cui par ch’ Amor scherzi, e voli,
E ch’ indi tutta la faretra scarchi,
E che visibilmente i cori involi.
Quindi il naso per mezzo il viso scende
Che non trova l’ invidia ove l’ emende.
Sotto quel sta, quasi fra due vallette, La bocca sparsa di natio cinabro,
Quivi due filze son di perle elette,
Che chiude, ed apre un bello e dolce labro;
Quindi escon le cortesi parolette,
Da render molle ogni cor rozzo e scabro;
Quivi si forma quel soave riso, Ch’ apre a sua posta in terra il paradiso.
Bianca neve è il pel collo, e ’l petto latte, Il collo è tondo, il petto colmo e largo; Due pome acerbe, e pur d’ avorio fatte, Vengono e van, come onda al primo margo,
Quando piacevole aura il mar combatte.
Non potria l’ altre parti veder Argo, Ben si può giudicar, che corrisponde, A quel ch’ appar di fuor, quel che s ’ asconde.
Mostran le braccia sua misura giusta, Et la candida man spesso si vede, Lunghetta alquanto, e di larghezza angusta, Dove nè nodo appar, nè vena eccede. Si vede al fin de la persona augusta Il breve, asciutto, e ritondetto piede.
Gli angelici sembianti nati in cielo
Non si ponno celar sotto alcun velo.
Milton, speaking of Pandemonium, says:—
The work some praise, and some the architect.
Praise of one, then, is not always praise of the other. A work of art may merit great approbation without redounding much to the credit of the artist; and, again, an artist may justly claim our admiration, even when his work does not entirely satisfy us. By bearing this in mind we can often reconcile contradictory judgments, as in the present case. Dolce, in his dialogues on painting, makes Aretino speak in terms of the highest praise of the above-quoted stanzas,[130] while I select them as an instance of painting without picture. We are both right. Dolce admires the knowledge of physical beauty which the poet shows: I consider only the effect which this knowledge, conveyed in words, produces on my imagination. Dolce concludes from this knowledge that good poets are no less good painters: I, judging from the effect, conclude that what painters can best express by lines and colors is least capable of expression in words. Dolce recommends Ariosto’s description to all painters as a perfect model of a beautiful woman: I recommend it to all poets as the most instructive of warnings not to attempt, with still greater want of success, what could not but fail when tried by an Ariosto.
It may be that when the poet says,—
Di persona era tanto ben formata,
Quanto mai finger san pittori industri,
he proves himself to have had a complete knowledge of the laws of perspective, such as only the most industrious artist can acquire from a study of nature and of ancient art.[131]
In the words,—
Spargeasi per la guancia delicata
Misto color di rose e di ligustri,
he may show himself to be a perfect master of color,—a very Titian. [132] His comparing Alcina’s hair to gold, instead of calling it golden hair, may be taken as proof that he objected to the use of actual gold in coloring.[133] We may even discover in the descending nose the profile of those old Greek noses, afterwards borrowed by Roman artists from the Greek masterpieces.[134] Of what use is all this insight and learning to us readers who want to fancy we are looking at a beautiful woman, and desire to feel that gentle quickening of the pulses which accompanies the sight of actual beauty? The poet may know the relations from which beauty springs, but does that make us know them? Or, if we know them, does he show them to us here? or does he help us in the least to call up a vivid image of them?
A brow that forms a fitting bound,
Che lo spazio finia con giusta meta;
A nose where envy itself finds nothing to amend, Che non trova l’ invidia, ove l’ emende;
A hand, narrow, and somewhat long, Lunghetta alquanto, e di larghezza angusta;
what sort of a picture do these general formulæ give us? In the mouth of a drawing-master, directing his pupils’ attention to the beauties of the academic model, they might have some meaning. For the students would have but to look at the model to see the fitting bounds of the gay forehead, the fine cut of the nose, and the slenderness of the pretty hand. But in the poem I see nothing, and am only tormented by the futility of all my attempts to see any thing.
In this respect Virgil, by imitating Homer’s reticence, has achieved tolerable success. His Dido is only the most beautiful (pulcherrima) Dido. Any further details which he may give, have reference to her rich ornaments and magnificent dress.
Tandem progreditur ...
Sidoniam picto chlamydem circumdata limbo: Cui pharetra ex auro, crines nodantur in aurum, Aurea purpuream subnectit fibula vestem.[135]
If, on this account, any should apply to him what the old artist said to one of his pupils who had painted a gayly decked Helen,—“Since you could not paint her beautiful, you have painted her rich,”—Virgil would answer: “I am not to blame that I could not paint her beautiful. The fault lies in the limits of my art, within which it is my merit to have kept.”
I must not forget here the two odes of Anacreon wherein he analyzes the beauty of his mistress and of Bathyllus.[136] The device which he uses entirely justifies the analysis. He imagines that he has before him a painter who is working from his description. “Thus paint me the hair,” he says; “thus the brow, the eyes, the mouth; thus the neck and bosom, the thighs and hands.” As the artist could execute but one detail at a time, the poet was obliged to give them to him thus piecemeal. His object is not to make us see and feel, in these spoken directions to the painter, the whole beauty of the beloved object. He is conscious of the inadequacy of all verbal expression; and for that reason summons to his aid the expression of art, whose power of illusion he so extols, that the whole song seems rather a eulogium of art than of his lady. He sees not the picture but herself, and fancies she is about to open her mouth to speak.
So, too, in his ode to Bathyllus, the praises of the beautiful boy are so mingled with praises of art and the artist, that we are in doubt in whose honor the song was really written. He selects the most beautiful parts from various pictures, the parts for which the pictures were remarkable. He takes the neck from an Adonis, breast and hands from a Mercury, the thighs from a Pollux, the belly from a