Basic Histology Text and Atlas 11th Edition PDF

Table of Contents Cover......................................................................................................................................................................................................... 1 Contributors......................................................................................................................................................................................... 2 Preface..................................................................................................................................................................................................... 3 1. Histology and Its Methods of Study........................................................................................................................... 5 2. The Cytoplasm......................................................................................................................................................................... 35 3. The Cell Nucleus.................................................................................................................................................................... 73 4. Epithelial Tissue...................................................................................................................................................................... 94 5. Connective Tissue.............................................................................................................................................................. 126 6. Adipose Tissue...................................................................................................................................................................... 165 7. Cartilage..................................................................................................................................................................................... 172 8. Bone.............................................................................................................................................................................................. 181 9. Nerve Tissue and the Nervous System............................................................................................................ 207 10. Muscle Tissue..................................................................................................................................................................... 239 11. The Circulatory System............................................................................................................................................... 266 12. Blood Cells............................................................................................................................................................................ 289 13. Hematopoiesis................................................................................................................................................................... 309 14. Lymphoid Organs............................................................................................................................................................. 326 Get full version of Atop CHM to PDF Converter.............................................................................................. 356 basichistology11 Converted by Atop CHM to PDF Converter free version! http://www.chmconverter.com 1 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! C lose Window Contributors Paulo Alexandre Abrahamsohn, MD, PhD Professor Department of C ell and Developmental Biology Institute of Biomedical Sciences University of SÃ£o Paulo SÃ£o Paulo, Brazil Chapter 1: Histology & Its Methods of Study; Chapter 4: Epithelial Tissue; Chapter 14: Lymphoid Organs; Chapter 20: Endocrine Glands; Chapter 21: The Male Reproductive System; Chapter 22: The Female Reproductive System Marinilce Fagundes dos Santos, DDS, PhD Assistant Professor Department of C ell and Developmental Biology Institute of Biomedical Sciences University of SÃ£o Paulo SÃ£o Paulo, Brazil Chapter 15: Digestive Tract; Chapter 16: Organs Associated with the Digestive Tract Telma Maria TenÃ³rio Zorn, MD, PhD Professor Department of C ell and Developmental Biology Institute of Biomedical Sciences University of SÃ£o Paulo SÃ£o Paulo, Brazil Chapter 5: Connective Tissue; Chapter 11: The Circulatory System http://www.chmconverter.com 2 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! C lose Window Preface Preface The eleventh edition of Basic Histology continues to be a concise, well-illustrated exposition of the basic facts and interpretation of microscopic anatomy. The authors of this text recognize that students of biologic structure share a common goal, namely, to better understand how structure and function are integrated into the molecules, cells, tissues, and organs of a living creature. Histology is the branch of science that centers on the biology of cells and tissues within an organism and, as such, serves as the foundation on which pathology and pathophysiology are built. In this edition, we continue to emphasize the relationships and concepts that inextricably link cell and tissue structure with their functions as they constitute the fabric of a living organism. In revising Basic Histology, our intent is to provide our readers with the most contemporary and useful text possible. We do this in two ways: by describing the most important recent developments in the sciences basic to histology and by recognizing that our readers are faced with the task of learning an ever-increasing number of facts in an ever-decreasing period of time. Because of this, every attempt has been made to present the information as concisely as possible and organize it in a way that facilitates learning. INTENDED AUDIENCE This text is designed for students in professional schools of medicine, veterinary medicine, dentistry, nursing, and allied health sciences. It is also a useful, ready reference for both undergraduate students of microscopic anatomy and others in the structural biosciences. ORGANIZATION Because the study of histology requires a firm foundation in cell biology, Basic Histology begins with an accurate, up-to-date description of the structure and function of cells and their products and a brief introduction into the molecular biology of the cell. This foundation is followed by a description of the four basic tissues of the body, emphasizing how cells become specialized to perform the specific functions of these tissues. Finally, we devote individual chapters to each of the organs and organ systems of the human body. Here, the emphasis on spatial arrangements of the basic tissues provides the key to understanding the functions of each organ. Again, we emphasize cell biology as the most fundamental approach to the study of structure and function. As a further aid to learning, color photomicrographs and electron micrographs amplify the text and remind the reader of the laboratory basis of the study of histology. In addition, we place particular emphasis on full-color diagrams, three-dimensional illustrations, and charts to summarize morphologic and functional features of cells, tissues, and organs. FEATURES â€¢ All chapters have been revised to reflect new findings and interpretations, and the emphasis on human histology has been further strengthened. â€¢ The chapter on microscopy and techniques includes new information on methods that permit analysis of molecules, cells, and tissues. â€¢ New information on the molecular biology of the genome and its regulation is included in the chapter on the nucleus. â€¢ New information on the organization and molecular composition of the extracellular matrix has been included in the chapter on connective tissue. â€¢ A discussion of the mechanisms of signal transduction in intercellular communication that adds to the student's understanding of tissue organization has been included in the chapter on the cell. â€¢ The chapter on nerve tissue and the nervous system has been extensively rewritten to include contemporary concepts and information regarding neurons and glial cells and their interactions. â€¢ The chapter on the immune system has been further revised to include current information and to organize that material into a readily assimilated body of knowledge. â€¢ The 600+ illustrations that appear throughout the book include numerous color photomicrographs prepared from new tissue samples with distinctive labeling that clearly pinpoints the elements of interest in each figure. These new micrographs of resin-embedded specimens provide clearer detail of cell and tissue organization. â€¢ All existing diagrams have been revised in full color and new color figures have been added to enhance the usefulness of the text. A key points icon has been used to highlight the principal issues in each chapter. Medical Applications in each chapter illustrate the direct relevance of basic histologic information to the diagnosis, prognosis, pathobiology, and clinical aspects of disease. They, too, are highlighted in color with an accompanying icon. ACKNOWLEDGMENTS We wish to thank the following professors who critically read several parts of this book: Edna T. Kimura (thyroid gland), Nancy Amaral RebouÃ§as (in situ hybridization), Sirley Daffre (protein separation) Ises de Almeida Abrahamsohn (immune response), Antonio C arlos Bianco (thyroid), JosÃ© C ipolla Neto (epiphysis) and Wolfgang G. W. Zorn (blood vessels). We also extend our appreciation to the staff of McGraw-Hill, Janet Foltin, Harriet Lebowitz, C harissa Baker, Phil Galea, and Peter Boyle, as well as to Arline Keithe for her editorial expertise. We are pleased to announce that Italian, Spanish, Dutch, Indonesian, Japanese, Turkish, Korean, German, Serbo-C roatian, French, Portuguese, Greek, and C hinese translations of Basic Histology are now available. Luiz C arlos Junqueira, MD JosÃ© C arneiro, MD January 2005 http://www.chmconverter.com 3 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! http://www.chmconverter.com 4 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Print Close W indow Note: Large im ages and tables on this page m ay necessitate printing in landscape m ode. Copyright ©2007 The McGraw-Hill Companies. A ll rights reserved. Lange Histology > Chapter 1. Histology & Its Methods of Study > HIST OLOGY & IT S MET HODS OF ST UDY: INT RODUCT ION Histology (Gr. histo, w eb or tissue, + logos, study) is the study of the tissues of the body and of how these tissues are arranged to constitute organs. Four fundamental tissues are recognized: epithelial tissue, connective tissue, muscular tissue, and nervous tissue. Tissues are made of cells and extracellular matrix, tw o components that w ere formerly considered separate entities. The extracellular matrix consists of many kinds of molecules, some of w hich are highly organized and form complex structures, such as collagen fibrils and basement membranes. The main functions formerly attributed to the extracellular matrix w ere to furnish mechanical support for the cells, to transport nutrients to the cells, and to carry aw ay catabolites and secretory products. In addition to these functions, it is now know n that cells not only produce extracellular matrix components but are also influenced by them. There is thus an intense interaction betw een cells and matrix. Moreover, many molecules of the matrix are recognized by and attach to receptors present on cell surfaces. Most of these receptors are molecules that cross the cell membranes and connect to molecules w ithin the cytoplasm. Thus, cells and extracellular matrix form a continuum that functions together and reacts to stimuli and inhibitors together. Each of the fundamental tissues is formed by several types of cells and typically by specific associations of cells and extracellular matrix. These characteristic associations facilitate the recognition of the many subtypes of tissues by students. Most organs are formed by an orderly combination of several tissues, except the central nervous system, w hich is formed almost solely by nervous tissue. The precise combination of these tissues allow s the functioning of each organ and of the organism as a w hole. The small size of cells and matrix components makes histology dependent on the use of microscopes. Advances in chemistry, physiology, immunology, and pathology—and the interactions among these fields—are essential for a better know ledge of tissue biology. Familiarity w ith the tools and methods of any branch of science is essential for a proper understanding of the subject. This chapter review s some of the more common methods used to study cells and tissues and the principles involved in these methods. PREPARAT ION OF T ISSUES FOR MICROSCOPIC EXAMINAT ION The most common procedure used in the study of tissues is the preparation of histological sections that can be studied w ith the aid of the light microscope. Under the light microscope, tissues are examined via a light beam that is transmitted through the tissue. Because tissues and organs are usually too thick for light to pass through them, they must be sectioned to obtain thin, translucent sections. How ever, living cells, very thin layers of tissues, or transparent membranes of living animals (eg, the mesentery, the tail of a tadpole, the w all of a hamster's cheek pouch) can be observed directly in the microscope w ithout first being sectioned. It is then possible to study these structures for long periods and under varying physiological or experimental conditions. In most cases, how ever, tissues must be sliced into thin sections and attached on glass slides before they can be examined. These sections are precisely cut from tissues previously prepared for sectioning using fine cutting instruments called microtomes. The ideal microscope tissue preparation should be preserved so that the tissue on the slide has the same structure and molecular composition as it had in the body. This is sometimes possible but—as a practical matter—seldom feasible, and artifacts, distortions, and loss of components due to the preparation process are almost alw ays present. Fixation If a permanent section is desired, tissues must be fixed. To avoid tissue digestion by enzymes present w ithin the cells (autolysis) or by bacteria and to preserve the structure and molecular composition, pieces of organs should be promptly and adequately treated before or as soon as possible after removal from the animal's body. This treatment—fixation—can be done by chemical or, less frequently, physical methods. In chemical fixation, the tissues are usually immersed in solutions of stabilizing or cross-linking agents called fixatives. Because the fixative needs some time to fully diffuse into the tissues, the tissues are usually cut into small fragments before fixation to facilitate the penetration of the fixative and to guarantee preservation of the tissue. Intravascular perfusion of fixatives can be used. Because the fixative in this case rapidly reaches the tissues through the blood vessels, fixation is greatly improved. One of the best fixatives for routine light microscopy is a buffered isotonic solution of 4% formaldehyde. The chemistry of the process involved in fixation is complex and not alw ays w ell understood. Formaldehyde and glutaraldehyde, another w idely used fixative, are know n to react w ith the amine groups (NH2 ) of tissue proteins. In the case of glutaraldehyde, the fixing action is reinforced by virtue of its being a dialdehyde, w hich can cross-link proteins. In view of the high resolution afforded by the electron microscope, greater care in fixation is necessary to preserve ultrastructural detail. Tow ard that end, a double fixation procedure, using a buffered glutaraldehyde solution follow ed by a second fixation in buffered osmium tetroxide, has become a standard procedure in preparations for ultrastructural studies. The effect of osmium tetroxide is to preserve and stain lipids and proteins. http://www.chmconverter.com 5 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Embedding Tissues are usually embedded in a solid medium to facilitate sectioning. To obtain thin sections w ith the microtome, tissues must be infiltrated after fixation w ith embedding substances that impart a rigid consistency to the tissue. Embedding materials include paraffin and plastic resins. Paraffin is used routinely for light microscopy; resins are used for both light and electron microscopy. The process of paraffin embedding, or tissue impregnation, is ordinarily preceded by tw o main steps: dehydration and clearing. The w ater is first extracted from the fragments to be embedded by bathing them successively in a graded series of mixtures of ethanol and w ater (usually from 70% to 100% ethanol). The ethanol is then replaced w ith a solvent miscible w ith the embedding medium. In paraffin embedding, the solvent used is usually xylene. As the tissues are infiltrated w ith the solvent, they generally become transparent (clearing). Once the tissue is impregnated w ith the solvent, it is placed in melted paraffin in the oven, typically at 58–60°C. The heat causes the solvent to evaporate, and the spaces w ithin the tissues become filled w ith paraffin. The tissue together w ith its impregnating paraffin hardens after being taken out of the oven. Tissues to be embedded w ith plastic resin are also dehydrated in ethanol and—depending on the kind of resin used—subsequently infiltrated w ith plastic solvents. The ethanol or the solvents are later replaced by plastic solutions that are hardened by means of cross-linking polymerizers. Plastic embedding prevents the shrinking caused by the high temperatures needed for paraffin embedding and gives much better results. The hard blocks containing the tissues are then taken to a microtome (Figure 1–1) and are sectioned by the microtome's steel or glass blade to a thickness of 1–10 m. Remember that 1 micrometer (1 m) = 0.001 mm = 10 –6 m; 1 nanometer (1 nm) = 0.001 m = 10 –6 mm = 10 –9 m. The sections are floated on w ater and transferred to glass slides to be stained. Figure 1–1. Microtome for sectioning resin- and paraffin-embedded tissues for light microscopy. Rotation of the drive wheel moves the tissue-block holder up and down. Each turn of the drive wheel advances the specimen holder a controlled distance, generally between 1 and 10 m. After each forward move, the tissue block passes over the knife edge, which cuts the sections. (C ourtesy of Microm.) A completely different w ay to prepare tissue sections is to submit the tissues to rapid freezing. In this process, the tissues are fixed by freezing (physically, not chemically) and at the same time become hard and thus ready to be sectioned. A freezing microtome—the cryostat (Gr. kryos, cold, + statos, standing)—has been devised to section the frozen tissues. Because this method allow s stained sections to be prepared rapidly (w ithin a few minutes), it is routinely used in hospitals to study specimens during surgical procedures. Freezing of tissues is also effective in the histochemical study of very sensitive enzymes or small molecules, since freezing does not inactivate most enzymes. Because immersion of tissues in solvents such as xylene dissolves the tissue lipids, the use of frozen sections is advised w hen these compounds are to be studied. Staining To be studied microscopically most sections must be stained. W ith few exceptions, most tissues are colorless, so observing them unstained in the light microscope is useless. Methods of staining tissues have therefore been devised that not only make the various tissue components conspicuous but also permit distinctions to be made betw een them. The dyes stain tissue components more or less selectively. Most of these dyes behave like acidic or basic compounds and have a tendency to form electrostatic (salt) linkages w ith ionizable radicals of the tissues. Tissue components that stain more readily w ith basic dyes are termed basophilic (Gr. basis, base, + phileo, to love); those w ith an affinity for acid dyes are termed acidophilic. http://www.chmconverter.com 6 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Examples of basic dyes are toluidine blue and methylene blue. Hematoxylin behaves like a basic dye, that is, it stains the basophilic tissue components. The main tissue components that ionize and react w ith basic dyes do so because of acids in their composition (nucleic acids, glycosaminoglycans, and acid glycoproteins). Acid dyes (eg, orange G, eosin, acid fuchsin) stain the acidophilic components of tissues such as mitochondria, secretory granules, and collagen. Of all dyes, the combination of hematoxylin and eosin (H&E) is the most commonly used. Hematoxylin stains the cell nucleus and other acidic structures (such as RNA-rich portions of the cytoplasm and the matrix of hyaline cartilage) blue. In contrast, eosin stains the cytoplasm and collagen pink. Many other dyes, such as the trichromes (eg, Mallory's stain, Masson's stain), are used in different histological procedures. The trichromes, in addition to show ing the nuclei and cytoplasm very w ell, help to differentiate collagen from smooth muscle. A good technique for differentiating collagen is the use of picrosirius, especially w hen associated w ith polarized light (see Polarizing Microscopy). In many procedures (see Immunocytochemistry), the sections become labeled by a precipitate, but cells and cell limits are often not visible. In this case a counterstain, usually a single stain that is applied to a section to allow the recognition of nuclei or cytoplasm, is used. Although most stains are useful in visualizing the various tissue components, they usually provide no insight into the chemical nature of the tissue being studied. In addition to tissue staining w ith dyes, impregnation w ith metals such as silver and gold is a common method, especially in studies of the nervous system. The w hole procedure, from fixation to observing a tissue in a light microscope, may take from 12 h to 21⁄2 days, depending on the size of the tissue, the fixative, and the embedding medium. LIGHT MICROSCOPY Conventional light, phase-contrast, differential interference, polarizing, confocal, and fluorescence microscopy are all based on the interaction of light and tissue components. W ith the light microscope, stained preparations are usually examined by means of light that passes through the specimen. The microscope is composed of mechanical and optical parts (Figure 1–2). The optical components consist of three systems of lenses: condenser, objective, and eyepiece. The condenser collects and focuses light, producing a cone of light that illuminates the object to be observed. The objective lenses enlarge and project the illuminated image of the object in the direction of the eyepiece. The eyepiece further magnifies this image and projects it onto the view er's retina, a photographic plate, or (to obtain a digital image) a detector such as a charged coupled device camera. The total magnification is obtained by multiplying the magnifying pow er of the objective and eyepiece. Figure 1–2. http://www.chmconverter.com 7 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Schematic drawing of a light microscope showing its main components and the pathway of light from the substage lamp to the eye of the observer. (C ourtesy of C arl Zeiss C o.) Resolution The critical factor in obtaining a crisp, detailed image w ith the microscope is its resolving power, that is, the smallest distance betw een tw o particles at w hich they can be seen as separate objects. The maximal resolving pow er of the light microscope is approximately 0.2 m; this pow er permits good images magnified 1000–1500 times. Objects smaller than 0.2 m (such as a membrane or a filament of actin) cannot be distinguished w ith this instrument. Likew ise, tw o objects, such as tw o mitochondria or tw o lysosomes, w ill be seen as only one object if they are separated by less than 0.2 m. The quality of the image—its clarity and richness of detail—depends on the microscope's resolving pow er. The magnification is of value only w hen accompanied by high resolution. The resolving pow er of a microscope depends mainly on the quality of its objective lens. The eyepiece lens enlarges only the image obtained by the objective; it does not improve resolution. For this reason, w hen comparing objectives of different magnifications, those that provide higher magnification also have higher resolving pow er. Highly sensitive video cameras enhance the pow er of the light microscope and allow the capture of digitized images that can be fed into computers for quantitative image analysis and printing. The frontiers of light microscopy have been redefined by the use of video cameras highly sensitive to light. W ith cameras and image-enhancement programs, objects that may not be visible w hen view ed directly through the eyepiece may be made visible in the video screen. These video systems are also useful for studying living cells for long periods of time, because they use low - intensity light and thus avoid the cellular damage that can result from intense illumination. The electronic images from video cameras can be easily digitized and adapted to the specific requirements of an experiment through computer programming. For example, contrast enhancement is an important computer-assisted technique that may reveal to the investigator a structural image not immediately seen w hen the specimen is observed directly in the microscope. Softw are developed for image analysis allow s the measurement of microscopic structures. PHASE-CONT RAST MICROSCOPY & DIFFERENT IAL INT ERFERENCE MICROSCOPY Some optical arrangements allow the observation of unstained cells and tissue sections. Unstained biological specimens are usually transparent and difficult to view in detail, since all parts of the specimen have almost the same optical density. Phase-contrast microscopy, how ever, uses a lens system that produces visible images from transparent objects (Figure 1–3). Figure 1–3. http://www.chmconverter.com 8 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! C ultured neural crest cells seen with different optical techniques. The cells are unstained, and the same cells appear in all photographs. Two pigmented cells are used for orientation in each image. A: C onventional light microscopy. B: Phase-contrast microscopy. C: Nomarski differential interference microscopy. High magnification. (C ourtesy of S Rogers.) Phase-contrast microscopy is based on the principle that light changes speed w hen passing through cellular and extracellular structures w ith different refractive indices. These changes are used by the phase-contrast system to cause the structures to appear lighter or darker relative to each other, w hich makes this kind of microscopy a pow erful tool w ith w hich to observe living cells. Another w ay to observe unstained cells or tissue sections is Nomarski differential interference microscopy, w hich produces an apparently three-dimensional image (Figure 1–3). POLARIZING MICROSCOPY Polarizing microscopy allow s structures made of highly organized molecules to be recognized. W hen normal light passes through a polarizing filter (such as a Polaroid), it exits vibrating in only one direction. If a second filter is placed in the microscope above the first one, w ith its main axis perpendicular to the first filter, no light passes through. If, how ever, tissue structures containing oriented molecules (such as cellulose, collagen, microtubules, and microfilaments) are located betw een the tw o polarizing filters, their repetitive, oriented molecular structure rotates the axis of the light emerging from the polarizer. http://www.chmconverter.com 9 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! the tw o polarizing filters, their repetitive, oriented molecular structure rotates the axis of the light emerging from the polarizer. Consequently, they appear as bright structures against a dark background (Figure 1–4). The ability to rotate the direction of vibration of polarized light is called birefringence and is a feature of crystalline substances or substances containing highly oriented molecules. Figure 1–4. Polarized light microscopy. A small piece of rat mesentery was stained with the picrosirius method, which stains collagen fibers. The mesentery was then placed on the slide and observed by transparency. Under polarized light, collagen fibers exhibit intense birefringence and appear brilliant or yellow. Medium magnification. CONFOCAL MICROSCOPY The depth of focus in the regular light microscope is relatively long, especially w hen small magnification objectives are used. This means that a rather w ide extent of the specimen is seen in focus simultaneously, causing superimposition of the image of a three-dimensional object. W ith confocal microscopy, on the other hand, only a very thin plane of the specimen is seen in focus at one time. There are tw o principles on w hich this is based: (1) the specimen is illuminated by a very small beam of light (w hereas in the common light microscope, a large beam of light floods the specimen) and (2) the image collected from the specimen must pass through a small pinhole. The result is that only the image originating from the focused plane reaches the detector w hereas the images in front of and behind this plane are blocked (Figure 1–5). The harmful glare of the out-of- focus objects is lost, and the definition of the focused object becomes better and allow s the localization of any specimen component w ith much greater precision than in the common light microscope. Figure 1–5. http://www.chmconverter.com 10 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Principle of confocal microscopy. While 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 plate. Thus, only one very thin plane of the specimen is focused at a time. For practical reasons, the follow ing arrangement is used in most confocal microscopes (Figure 1–6): (1) the illumination is provided by a laser source; (2) because it is a very small point, it must be moved over the specimen (scanned) to allow the observation of a larger area of the specimen; (3) the component of the specimen that is of interest must be labeled w ith a fluorescent molecule (meaning that a routine section cannot be studied); (4) the light that is reflected by the specimen is used to form an image; (5) because the reflected light is captured by a detector, the signal can be electronically enhanced to be seen in a monitor. Figure 1–6. 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 are blocked by the plate. The laser scans the specimen so that a larger area of the specimen can be observed. http://www.chmconverter.com 11 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Because only a very thin focal plane (also called an optical section) is focused at a time, it is possible to reunite several focused planes of one specimen and reconstruct them into a three-dimensional image. To accomplish the reconstruction and many of its other features, the confocal microscope depends on heavy computing capacity. FLUORESCENCE MICROSCOPY W hen certain substances are irradiated by light of a certain w avelength, they emit light w ith a longer w avelength. This phenomenon is called fluorescence. In fluorescence microscopy, tissue sections are irradiated w ith either ultraviolet (UV) light or laser, and the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant or colored on a dark background. Fluorescent compounds that have an affinity for cell macromolecules may be used as fluorescent stains. Acridine orange, w hich can combine w ith DNA and RNA, is an example. W hen observed in the fluorescence microscope, the DNA–acridine orange complex emits a yellow ish-green light, and the RNA–acridine orange complex emits a reddish-orange light. It is thus possible to identify and localize nucleic acids in the cells (Figure 1–7). Another important application of fluorescence microscopy is achieved by coupling fluorescent substances (such as fluorescein isothiocyanate, FITC) to molecules that w ill specifically bind to components of the tissues and w ill thus allow the identification of these components under the microscope (see Detection Methods Using High-Affinity Interactions betw een Molecules). Figure 1–7. Photomicrograph of kidney cells in culture, stained with acridine orange. Under a fluorescence microscope, DNA (within the nuclei) emits yellow light, and the RNA-rich cytoplasm appears reddish or orange. (C ourtesy of A Geraldes and JMV C osta.) ELECT RON MICROSCOPY Transmission and scanning electron microscopes are based on the interaction betw een electrons and tissue components. Transmission Electron Microscopy The transmission electron microscope is an imaging system that theoretically permits very high resolution (0.1 nm) (Figure 1–8). In practice, how ever, the resolution obtained by most good instruments is around 3 nm. This high resolution allow s magnifications of up to 400,000 times to be view ed w ith detail. Unfortunately, this level of magnification applies only to isolated molecules or particles. Very thin tissue sections can be observed w ith detail at magnifications of up to about 120,000 times. Figure 1–8. http://www.chmconverter.com 12 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Photograph of the JEM-1230 transmission electron microscope. (C ourtesy of JEOL USA, Inc., Peabody, MA.) The transmission electron microscope functions on the principle that a beam of electrons can be deflected by electromagnetic fields in a manner similar to light deflection in glass lenses. In the electron microscope, electrons are released by heating a very thin metallic (usually tungsten) filament (the cathode) in a vacuum. The electrons released are then submitted to a voltage difference of 60–120 kV betw een the cathode and the anode, w hich is a metallic plate w ith a hole in its center (Figure 1–9). Electrons are thus attracted to the anode and accelerated to high speeds. They pass through the central opening in the anode, forming a constant stream (or beam) of electrons that penetrates the tube of the microscope. The beam passes inside electric coils and is deflected in a w ay roughly analogous to w hat occurs in optical lenses, because electrons change their path w hen submitted to electromagnetic fields. For this reason, the electric coils of electron microscopes are called electromagnetic lenses. Figure 1–9. http://www.chmconverter.com 13 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Schematic view of a transmission electron microscope with its lenses and the pathway of the electrons. C C D, charged coupled device. The configuration of the electron microscope is very similar to that of the optical microscope, although the optics of the electron microscope are usually placed upside dow n (Figure 1–9). The first lens is a condenser that focuses the beam of electrons on the section. Some electrons interact w ith atoms of the section and continue their course, w hereas others simply cross the specimen w ithout interacting. Most electrons reach the objective lens, w hich forms a magnified image that is then projected through other magnifying lenses. Because the human eye is not sensitive to electrons, the image is finally projected on a fluorescent screen or is registered by photographic plates or a charged coupled device camera. Because most of the image in the transmission electron microscope is produced by the balance betw een the electrons that hit the fluorescent screen (or the photographic plate) and the electrons that are retained in the tube of the microscope, the resulting image is alw ays in black and w hite. Dark areas of an electron micrograph are usually called electron dense, w hereas light areas are called electron lucent. To provide a good interaction betw een the specimen and the electrons, electron microscopy requires very thin sections (40–90 nm); therefore, embedding is performed w ith a resin that becomes very hard. The blocks thus obtained are so hard that glass or diamond knives are usually necessary to section them. The extremely thin sections are collected on small metal grids and transferred to the interior of the microscope to be analyzed. Freezing techniques allow the examination of tissues by electron microscopy w ithout the need for fixation and embedding. There are few er artifacts than w ith conventional tissue preparation, although the technique is usually arduous. Frozen tissues may be sectioned and submitted to cytochemistry or immunocytochemistry or may be fractured (cryofracture, freeze fracture) to reveal details of the internal structure of the membranes. Scanning Electron Microscopy Scanning electron microscopy permits pseudo-three-dimensional view s of the surfaces of cells, tissues, and organs. This electron microscope produces a very narrow electron beam that is moved sequentially (scanned) from point to point across the specimen. Unlike the electrons in the transmission electron microscope, those in the scanning electron microscope do not pass through the specimen (Figure 1–10). The electron beam interacts w ith a very thin metal coating previously applied to the specimen and produces reflected or emitted electrons. These electrons are captured by a detector that transmits them to amplifiers and other devices so that in the end the signal is projected into a cathode ray tube (a monitor), resulting in a black- and-w hite image. The resulting photographs are easily understood, since they present a view that appears to be illuminated from above, just as our ordinary macroscopic w orld is filled w ith highlights and shadow s caused by illumination from above. The scanning electron microscope show s only surface view s. The inside of organs can be analyzed by freezing the organs and fracturing them to expose their internal surfaces. Examples of scanning electron microscopy can be seen in Figures 12–3 and 12 –4. Figure 1–10. http://www.chmconverter.com 14 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Schematic view of a scanning electron microscope. AUT ORADIOGRAPHY OF T ISSUE SECT IONS Autoradiography is the study of biological events in tissue sections using radioactivity. Autoradiography permits the localization of radioactive substances in tissues by means of emitted radiation effects on photographic emulsions. Silver bromide crystals present in the emulsion act as microdetectors of radioactivity in the same w ay that they respond to light in common photography. The first step of autoradiography is to deliver a radioactive compound to the cells. A variety of molecules, including radioactive amino acids, radioactive nucleotides, and radioactive sugars, can be used, depending on the purpose of the study. These molecules are called precursors, because they may be used by the cells to synthesize larger molecules, such as proteins, nucleic acids, or polysaccharides and glycoproteins. The tissue sections are prepared and are covered w ith photographic emulsion. The slides are kept in light-proof boxes; after an adequate exposure time they are developed photographically and examined. W hen the silver bromide crystals present in the photographic emulsion are hit by radiation they are transformed into small black granules of metallic silver, thus revealing the existence of radioactivity in the tissue. The structures that contain radioactive molecules become covered by these granules. This procedure can be used in both light and electron microscopy (Figure 1–11). Figure 1–11. http://www.chmconverter.com 15 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Autoradiographs from the submandibular gland of a mouse injected with [3 H] fucose 8 h before being killed. A: With a light microscope it is possible to observe black silver grains indicating radioactive regions in the cells. Most radioactivity is in the granules of the cells of the granular ducts of the gland. High magnification. B: The same tissue prepared for electron microscope autoradiography. The silver grains in this enlargement appear as coiled structures localized mainly over the granules (G) and in the gland lumen (L). High magnification. (C ourtesy of TG Lima and A Haddad.) Much information becomes available by localizing radioactivity in tissue components. Thus, if a radioactive amino acid is used, it is possible to know w hich cells in a tissue produce more protein and w hich cells produce less, because the number of silver granules formed over the cells is proportional to the intensity of protein synthesis. If a radioactive precursor of DNA (such as radioactive thymidine) is used, it is possible to know w hich cells in a tissue (and how many) are preparing to divide. Dynamic events may also be analyzed. For instance, to determine w here in the cell a protein is produced, if it is secreted, and w hich path it follow s in the cell before being secreted, several animals are injected w ith a radioactive amino acid and are killed at different times after the injection. Autoradiographs of the sections, taken at various times throughout the experiment, w ill show the migration of the radioactive proteins. To determine w here new cells are produced in an organ and w here they migrate, several animals are injected w ith radioactive thymidine and are killed at different times after the injection. Autoradiographs of the sections w ill show w here the cells divide and w here (or if) they migrate (Figure 1–12). Figure 1–12. http://www.chmconverter.com 16 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Autoradiographs of tissue sections from a mouse that was injected with [3 H] thymidine 1 h before the organs were collected. Because the autoradiographs were exposed for a very long time, the radioactive nuclei became heavily labeled and appear covered by clouds of dark granules. A: Many cells were dividing at the base of the intestinal glands (arrowheads), but no cells were dividing along the villi (arrows). Low magnification. B: A section of a lymph node shows that cell division occurs mostly at the germinal centers of this structure (arrow). Low magnification. (C ourtesy of TMT Zorn, M Soto-Suazo, C MR Pellegrini, and WE Stumpf.) CELL & T ISSUE CULT URE Live cells and tissues can be maintained and studied outside the body. In a complex organism, tissues and organs are formed by several kinds of cells. These cells are bathed in blood plasma, w hich contains hundreds of different molecules. Cell and tissue culture has been very helpful in isolating the effect of a single molecule on one type of cell or tissue. It also allow s the direct observation of the behavior of living cells under a microscope. Several experiments that cannot be performed in the living animal can be reproduced in v itro. The cells and tissues are grow n in complex solutions of know n composition (salts, amino acids, vitamins) to w hich serum components are frequently added. In preparing cultures from a tissue or organ, cells must be initially dispersed either mechanically or by treating the tissue w ith enzymes. Once isolated, the cells can be cultivated in a suspension or spread out on a Petri dish or glass slide, to w hich they adhere, usually as a single layer of cells (Figure 1–3). Cultures of cells that are isolated in this w ay are called primary cell cultures. Many cell types w ere once isolated in this w ay from normal or pathological tissue and have been maintained in vitro ever since because they have been immortalized and now constitute a permanent cell line. Most cells obtained from normal tissues have a finite, genetically programmed life span. Certain changes, how ever (mainly related to oncogenes; see Chapter 3: The Cell Nucleus), can promote cell immortality, a process called transformation, w hich http://www.chmconverter.com may be a first step in transforming a normal cell into a cancer cell. Because of transformation and other improvements in culture 17 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! may be a first step in transforming a normal cell into a cancer cell. Because of transformation and other improvements in culture technology, most cell types can now be maintained in the laboratory indefinitely. All procedures w ith living cells and tissues must be performed in a sterile area, using sterile solutions and equipment. MEDICAL APPLICATION Cell culture has been w idely used for the study of the metabolism of normal and cancerous cells and for the development of new drugs. This technique is also useful in the study of parasites that grow only w ithin cells, such as viruses, mycoplasma, and some protozoa (Figure 1–13). In cytogenetic research, determination of human karyotypes (the number and morphology of an individual's chromosomes) is accomplished by the short-term cultivation of blood lymphocytes or of skin fibroblasts. By examining cells during mitotic division in tissue cultures, it is possible to detect anomalies in the number and morphology of the chromosomes that have been show n to be related and are diagnostic of numerous diseases collectively called genetic disorders. In addition, cell culture is central to contemporary techniques of molecular biology and recombinant DNA technology. Figure 1–13. Photomicrograph of chicken fibroblasts that were grown in tissue culture and infected by the protozoan Trypanosoma cruzi. Although the borders of the cells are not readily visible, their nuclei (N) can be easily seen. Many trypanosomes are present within each cell (arrows). High magnification. (C ourtesy of S Yoneda.) CELL FRACT IONAT ION Organelles and other components of cells and tissues can be isolated by cell fractionation. This is the physical process by w hich centrifugal force is used to separate organelles and cellular components as a function of their sedimentation coefficients. The sedimentation coefficient of a particle depends on its size, form, and density and on the viscosity of the medium (Figure 1–14). The organelles obtained w ith these techniques can be analyzed for purity in the electron microscope (Figure 1–15), and their chemical composition and functions can be studied in vitro. Figure 1–14. http://www.chmconverter.com 18 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! C ell fractionation allows the isolation of cell constituents by differential centrifugation. The drawings at the right show the cellular organelles at the bottom of each tube after centrifugation. C entrifugal force is expressed by g, which is equivalent to the force of gravity. (1) A fragment of tissue is minced with razor blades or scissors and dissociated with a homogenizer or by ultrasound. (2) The dissociated tissue is left standing for about 20 min. C lumps of cells and fibers of extracellular matrix precipitate to the bottom. (3) The supernatant is centrifuged at 1000 g for 20 min. Nuclei precipitate. (4) The supernatant is centrifuged at 10,000 g for 20 min. Mitochondria and lysosomes precipitate. (5) The supernatant is centrifuged at 105,000 g for 120 min. Microsomes precipitate. (6) If the supernatant is first treated with sodium deoxycholate and then centrifuged at 105,000 g for 120 min, the microsomes dissociate and precipitate separately as endoplasmic reticulum membranes and ribosomes. (Redrawn and reproduced, with permission, from Bloom W, Fawcett DW: A Textbook of Histology, 9th ed. Saunders, 1968.) Figure 1–15. http://www.chmconverter.com 19 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Electron micrographs of three cell fractions isolated by density gradient centrifugation. A: Mitochondrial fraction, contaminated with microsomes. B: Microsomal fraction. C: Lysosomal fraction. High magnifications. (C ourtesy of P Baudhuin.) HIST OCHEMIST RY & CYT OCHEMIST RY The terms histochemistry and cytochemistry are used to indicate methods for localizing substances in tissue sections. Several procedures are used to obtain this type of information, most of them based on specific chemical reactions or on high-affinity interactions betw een macromolecules. These methods usually produce insoluble colored or electron-dense compounds that enable the localization of specific substances by means of light or electron microscopy. Ions Several ions (eg, calcium, iron, phosphate) have been localized in tissues w ith these methods, using chemical reactions that produce a dark insoluble product (Figure 1–16). Figure 1–16. Photomicrograph of a bone section treated with a histochemical technique to demonstrate calcium ions. The dark precipitate indicates the presence of calcium phosphate in calcified bone and cartilage. Noncalcified cartilage tissue (stained in pink) is in the upper portion of the figure. Medium magnification. (C ourtesy of PA Abrahamsohn.) Nucleic Acids DNA can be identified and quantified in cell nuclei using the Feulgen reaction, w hich produces a red color in DNA. DNA and RNA can also be analyzed by staining cells or tissue sections w ith a basic stain. Proteins http://www.chmconverter.com 20 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Although there are general methods to detect proteins in tissue sections, the histochemical methods usually do not permit identification of specific proteins in cells and tissues. Immunocytochemistry, presented later in this chapter, can do so. Several histochemical methods, how ever, can be used to reveal, more or less specifically, a large group of proteins, the enzymes. These methods usually make use of the capacity of the enzymes to react w ith specific chemical bonds. Most histoenzymatic methods w ork in the follow ing w ay: (1) tissue sections are immersed in a solution that contains the substrate of the enzyme intended for study; (2) the enzyme is allow ed to act on its substrate; (3) at this stage or at a later stage in the method, the section is put in contact w ith a marker compound; (4) this compound reacts w ith a molecule that results from the degradation or transformation of the substrate; (5) the final reaction product, w hich must be insoluble and visible by light or electron microscopy, precipitates over the sites that contain the enzyme. W hen examining such a section in the microscope, it is possible to see the cells (or organelles) covered w ith a colored or electron-dense material. Examples of enzymes that can be detected include the follow ing: Phosphatases are enzymes w idely found in the body. They split the bond betw een a phosphate group and an alcohol residue of phosphorylated molecules. The colored insoluble reaction product of phosphatases is usually lead phosphate or lead sulfide. Alkaline phosphatases, w hich have their maximum activity at an alkaline pH, can be detected (Figure 1–17). Acid phosphatases are frequently used to demonstrate lysosomes, cytoplasmic organelles that contain acid phosphatase (Figure 1–18). Figure 1–17. Photomicrograph of a rat kidney section treated by the Gomori method to demonstrate the enzyme alkaline phosphatase. The sites where this enzyme is present are covered by a black precipitate (arrows). Medium magnification. Figure 1–18. http://www.chmconverter.com 21 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Detection of acid phosphatase. Electron micrograph of a rat kidney cell showing three lysosomes (ly) close to the nucleus (N). The dark material on the lysosomes is lead phosphate that precipitated on places where acid phosphatase was present. (C ourtesy of E Katchburian.) Dehydrogenases remove hydrogen from one substrate and transfer it to another. There are many dehydrogenases in the body, and they play an important role in several metabolic processes. Dehydrogenases are detected histochemically by incubating nonfixed tissue sections in a substrate solution containing a molecule that receives hydrogen and precipitates as an insoluble colored compound. By this method, succinate dehydrogenase—a key enzyme in the citric acid (Krebs) cycle—can be localized in mitochondria. Peroxidase, w hich is present in several types of cells, is an enzyme that promotes the oxidation of certain substrates w ith the transfer of hydrogen ions to hydrogen peroxide, forming molecules of w ater. In this method, sections of adequately fixed tissue are incubated in a solution containing hydrogen peroxide and 3,3'- diaminoazobenzidine. The latter compound is oxidized in the presence of peroxidase, resulting in an insoluble, brow n, electron- dense precipitate that permits the localization of peroxidase activity by light and electron microscopy. Peroxidase activity in blood cells, w hich is important in the diagnosis of leukemias, can be detected by this method. Because peroxidase is extremely active and produces an appreciable amount of insoluble precipitate in a short time, it has also been used for an important practical application: tagging other compounds. Molecules of peroxidase can be purified, isolated, and coupled w ith another molecule. Later in this chapter, applications of tagging molecules w ith peroxidase are presented. Polysaccharides & Oligosaccharides Polysaccharides in the body occur either in a free state or combined w ith proteins and lipids. In the combined state, they constitute an extremely complex heterogeneous group. Polysaccharides can be demonstrated by the periodic acid–Schiff (PAS) reaction, w hich is based on the transformation into aldehyde of 1,2-glycol groups present in the sugar molecules. These aldehydes are then revealed by Schiff's reagent, w hich produces a purple or magenta color in areas of the section w ith an accumulation of polysaccharides. A ubiquitous free polysaccharide in the body is glycogen, w hich can be demonstrated by the PAS reaction in liver, striated muscle, and other tissues w here it accumulates. Glycoproteins are protein molecules associated w ith small, branched chains of sugars (oligosaccharides). The protein chain predominates in w eight and volume over the oligosaccharide chain. Because both glycogen and neutral glycoproteins are PAS positive, the specificity of the PAS reaction can be improved by comparing the staining of regular sections w ith that of sections pretreated w ith an enzyme that breaks glycogen (eg, amylase present in the saliva). Structures that stain intensely w ith the PAS reaction but do not stain after treatment w ith amylase contain glycogen. Figure 1–19 show s examples of structures stained by the PAS reaction. Figure 1–19. http://www.chmconverter.com 22 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Photomicrograph of an intestinal villus stained by PAS. Staining is intense in the cell surface brush border (arrows) and in the secretory product of goblet cells (G) because of their high content of polysaccharides. The counterstain was hematoxylin. High magnification. Glycosaminoglycans are strongly anionic, unbranched long-chain polysaccharides containing aminated monosaccharides (amino sugars). The complex molecules formed by the attachment of glycosaminoglycan chains to a protein core constitute the proteoglycans. Some of the significant constituents of connective tissue matrices are proteoglycans (see Chapter 5: Connective Tissue and Chapter 7: Cartilage). Unlike the glycoproteins, the carbohydrate chains in proteoglycans constitute the major component of the molecule. Glycosaminoglycans and acidic glycoproteins are strongly anionic because of their high content of carboxyl and sulfate groups. For this reason, they react strongly w ith the alcian blue dye. Lipids Lipids are best revealed w ith dyes that are soluble in lipids. Frozen sections are immersed in alcohol solutions saturated w ith the dye. Sudan IV and Sudan black are the most commonly used dyes. The dye dissolves in the cellular lipid droplets, w hich become stained in red or black. Additional methods used for the localization of cholesterol and its esters, phospholipids, and glycolipids are useful in diagnosing metabolic diseases in w hich there are intracellular accumulations of different kinds of lipids. MEDICAL APPLICATION Several histochemical procedures are frequently used in laboratory diagnosis of diseases that result in the storage of iron, glycogen, glycosaminoglycans, and other substances. Examples are Perls' reaction for iron (eg, hemochromatosis, hemosiderosis), the PAS-amylase reaction for glycogen (glycogenosis), alcian blue staining for glycosaminoglycans (mucopolysaccharidosis), and lipid staining (sphingolipidosis). DET ECT ION MET HODS USING HIGH-AFFINIT Y INT ERACT IONS BET WEEN MOLECULES A molecule present in a tissue section may be identified by using compounds that specifically interact w ith the molecule. The compounds that w ill interact w ith the molecule must be tagged w ith a label that can be detected under the light or electron microscope (Figure 1–20). The most commonly used labels are fluorescent compounds (w hich can be seen w ith a fluorescence or laser microscope), radioactive atoms (w hich can be detected w ith autoradiography), molecules of peroxidase (w hich can be detected after demonstration of the enzyme w ith hydrogen peroxide and 3,3'-diaminoazobenzidine) or other enzymes (w hich can be detected w ith their respective substrates), and metal (usually gold) particles that can be observed w ith light and electron microscopy. These methods are mainly used for detecting sugars, proteins, and nucleic acids. Figure 1–20. http://www.chmconverter.com 23 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! C ompounds that have affinity toward another molecule can be tagged with a label and used to identify that molecule. (1) Molecule A has a high and specific affinity toward a portion of molecule B. (2) When A and B are mixed, A binds to the portion of B it recognizes. (3) Molecule A may be tagged with a label that can be visualized with a light or electron microscope. The label can be a fluorescent compound, an enzyme such as peroxidase, a gold particle, or a radioactive atom. (4) If molecule B is present in a cell or extracellular matrix that is incubated with labeled molecule A, molecule B can be detected. Phalloidin, protein A, lectins, and antibodies are examples of compounds that interact specifically w ith other molecules. Phalloidin, w hich is extracted from a mushroom (Amanita phalloides), interacts strongly w ith actin and is usually labeled w ith fluorescent dyes to demonstrate actin filaments. Protein A is a protein obtained from Staphylococcus aureus that binds to the Fc region of immunoglobulin (antibody) molecules. W hen protein A is tagged w ith a label, immunoglobulins can be detected. Lectins are proteins or glycoproteins that are derived mainly from plant seeds and that bind w ith high affinity and specificity to carbohydrates. Different lectins bind to specific sequences of sugar molecules. They may bind to glycoproteins, proteoglycans, and glycolipids and are w idely used to characterize membrane molecules containing defined sequences of sugars. Immunocytochemistry A highly specific interaction betw een molecules is that betw een an antigen and its antibody. For this reason, methods using labeled antibodies have proved most useful in identifying and localizing specific proteins and glycoproteins. The body has cells that are able to distinguish its ow n molecules (self) from foreign ones. W hen exposed to foreign molecules —called antigens—the body may respond by producing proteins—antibodies—that react specifically and bind to the antigen, thus helping to eliminate the foreign substance. Antibodies are proteins of a large family, the immunoglobulin family. In immunocytochemistry, a tissue section (or cells in culture) that may contain a certain protein is incubated in a solution containing an antibody to this protein. The antibody binds specifically to the protein, w hose location can then be seen w ith either the light or electron microscope, depending on the type of compound used to label the antibody. One of the most important requirements for immunocytochemistry is the availability of an antibody against the protein that is to be detected. This means that the protein must have been previously purified and isolated so that antibodies can be produced. Some methods for protein isolation can be seen in Figures 1–21 and 1–22. Figure 1–21. http://www.chmconverter.com 24 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Ultracentrifugation (A) and chromatography (B): methods of protein isolation. A: A mixture of proteins obtained from homogenized cells or tissues is submitted to centrifugation at high speed for several hours. The proteins separate into several bands, depending on the size and density of the protein molecules. The ultracentrifugation medium is drained and collected in several fractions that contain different proteins, which can be analyzed further. B: A solution containing a mixture of proteins obtained from homogenized cells or tissues is added to a column filled with beads that have different chemical properties. For instance, the beads may have different electrostatic charges (attracting proteins according to their charge) or different sizes of pores (acting as sieves for different-sized molecules). As the proteins migrate through the column, their movement is slowed according to their interaction with the particles. When the effluent is recovered, the different groups of proteins may be collected separately. Figure 1–22. http://www.chmconverter.com 25 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Gel electrophoresis: a method of protein isolation. A: Isolation of proteins. (1) Mixtures of proteins are obtained from homogenized cells or tissues. They are usually treated with a strong detergent (sodium dodecyl sulfate) and with mercaptoethanol to unfold and separate the protein subunits. (2) The samples are put on top of a slab of polyacrylamide gel, which is submitted to an electrical field. The proteins migrate along the gel according to their size and shape. (3) A mixture of proteins of known molecular mass is added to the gel as a reference to identify the molecular mass of the other proteins. B: Detection and identification of the proteins. (1) Staining. All proteins will stain the same color. The color intensity is proportional to the protein concentration. (2) Autoradiography. Radioactive proteins can be detected by autoradiography. An x-ray film is apposed to the gel for a certain time and then developed. Radioactive proteins will appear as dark bands in the film. (3) Immunoblotting. The proteins can be transferred from the gel to a nitrocellulose membrane. The membrane is incubated with a labeled antibody made against proteins that may be present in the sample. POLY CLONAL AND MONOCLONAL ANTIBODIES Let us suppose that our objective is to produce antibodies against protein x of a certain animal species (eg, a rat or a human). If protein x is already isolated, it is injected into an animal of another species (eg, a rabbit or a goat). If the protein is sufficiently different for this animal to recognize it as foreign—that is, as an antigen—the animal w ill produce antibodies against the protein (eg, rabbit antibody against rat x or goat antibody against human x). These antibodies are collected from the animal's plasma and used for immunocytochemistry. Several groups (clones) of lymphocytes of the animal that w as injected w ith protein x may recognize different parts of protein x and each group produces an antibody against each part. These antibodies constitute a mixture of polyclonal antibodies. It is possible, how ever, to furnish protein x for lymphocytes maintained in cell culture (actually, lymphocytes fused w ith tumor cells). The different clones of lymphocytes w ill produce different antibodies against the several parts of protein x. Each clone can http://www.chmconverter.com 26 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! cells). The different clones of lymphocytes w ill produce different antibodies against the several parts of protein x. 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. There are several advantages to using a monoclonal antibody rather than a polyclonal antibody: for instance, a monoclonal antibody can be selected to be highly specific and to bind strongly to the protein to be detected. Therefore, there w ill be less nonspecific binding to other proteins similar to the one being looked for. In the direct method of immunocytochemistry, the antibody (either monoclonal or polyclonal) must be tagged w ith an appropriate label. A tissue section is incubated w ith the antibody for some time so that the antibody interacts w ith and binds to protein x. The section is then w ashed to remove the unbound antibody (Figure 1–23). Depending on the label that w as used (fluorescent compound, enzyme, gold particles), the section can be observed w ith a light or electron microscope. If peroxidase or another enzyme w as used as a label, the enzyme must be detected before the tissue section is observed in the microscope (see Histochemistry & Cytochemistry). The areas of the tissue section that contain protein x w ill become fluorescent or w ill be covered by gold particles or by a dark precipitate if an enzyme w as used as a marker. Figure 1–23. Direct method of immunocytochemistry. (1) Immunoglobulin molecule (Ig). (2) Production of a polyclonal antibody. Protein x from a rat is injected into a rabbit. Several rabbit Igs are produced against protein x. (3) Labeling the antibody. The rabbit Igs are tagged with a label. (4) Immunocytochemical reaction. The rabbit Igs recognize and bind to different parts of protein x. The indirect method of immunocytochemistry is more sensitive but requires more steps. Let us suppose that our objective is to detect protein x, present in rats. Before proceeding to the immunochemical reaction, tw o procedures are needed: (1) antibodies (polyclonal or monoclonal) to rat protein x must first be produced in an animal of another species (eg, a rabbit); (2) in a parallel procedure, immunoglobulin from a normal (noninjected) rabbit must be injected into an animal of a third species (eg, a goat). Rabbit immunoglobulins are considered foreign by a goat and are thus capable of inducing the production of an antibody (an antiantibody or antiimmunoglobulin) in that animal. Indirect immunocytochemical detection is performed by initially incubating a section of a rat tissue believed to contain protein x w ith rabbit anti-x antibody. After w ashing, the tissue sections are incubated w ith labeled goat antibody against rabbit antibodies. The antiantibodies w ill bind to the rabbit antibody that had previously recognized protein x (Figure 1–24). Protein x can then be detected by using a microscopic technique appropriate for the label used in the secondary antibody. There are other indirect methods that involve the use of other intermediate molecules, such as the biotin-avidin technique. Figure 1–24. Indirect method of immunocytochemistry. (1) Production of a primary polyclonal antibody. Protein x from a rat is injected into a rabbit. Several rabbit immunoglobulins (Ig) are produced against protein x. (2) Production of secondary antibody. Ig from a nonimmune (normal) rabbit is injected into a goat. Goat Igs against rabbit Ig are produced. The goat Igs are then isolated and tagged with a label. (3) First27 http://www.chmconverter.com step / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! rabbit is injected into a goat. Goat Igs against rabbit Ig are produced. The goat Igs are then isolated and tagged with a label. (3) First step of the immunocytochemical reaction. The rabbit Igs recognize and bind to different parts of protein x. (4) Second step of the immunocytochemical reaction. Labeled goat Igs recognize and bind to different parts of rabbit immunoglobulin molecules, therefore labeling protein x. MEDICAL APPLICATION Immunocytochemistry has contributed significantly to research in cell biology and to the improvement of medical diagnostic procedures. Figures 1–25, 1–26, 1–27, and 1–28 show examples of immunocytochemical detection of molecules. Table 1–1 show s some of the routine applications of immunocytochemical procedures in clinical practice. Table 1–1. Commonly Used Proteins (Antigens) Important for Immunocytochemical Diagnosis and Treatment of Disease. Antigens Diagnosis Intermediate filament proteins Cytokeratins Tumors of epithelial origin Glial fibrillary acid protein Tumors of some glial cells Vimentin Tumors of connective tissue Desmin Tumors of muscle Other proteins Protein and polypeptide hormones Protein or polypeptide hormone–producing tumors Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract and breast Steroid hormone receptors Breast duct cell tumors Antigens produced by viruses Specific virus infections Figure 1–25. Photomicrograph of a mouse decidual cell grown in vitro. The protein desmin, which forms intermediate filaments, was detected with an indirect immunofluorescence technique. A mesh of fluorescent intermediate filaments occupies most of the cytoplasm. The nucleus (N) is stained blue. High magnification. (C ourtesy of FG C osta.) Figure 1–26. http://www.chmconverter.com 28 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Photomicrograph of a section of small intestine in which an antibody against the enzyme lysozyme was applied to demonstrate lysosomes in macrophages and Paneth cells. The brown color, indicating the presence of lysozyme, results from the reaction done to show peroxidase, which was linked to the secondary antibody. Nuclei were counterstained with hematoxylin. Medium magnification. Figure 1–27. http://www.chmconverter.com 29 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! C arcinoembryonic antigen is a protein present in several malignant tumors mainly of the breast and intestines. This photomicrograph is an immunocytochemical demonstration of carcinoembryonic antigen in a section of large intestine adenocarcinoma. The antibody was labeled with peroxidase and the brown precipitate indicates tumor cells. The counterstain was hematoxylin. Medium magnification. Figure 1–28. Electron micrograph showing a section of a pancreatic acinar cell that was incubated with antiamylase antibody and stained by protein A coupled with gold particles. Protein A has high affinity toward antibody molecules. The gold particles appear as very small black dots over the secretory granules. (C ourtesy of M Bendayan.) Hybridization Techniques The central challenge in modern cell biology is to understand the w orkings of the cell in molecular detail. This goal requires techniques that permit analysis of the molecules involved in the process of information flow from DNA to protein. Many techniques are based on hybridization. Hybridization is the binding betw een tw o single strands of nucleic acids (DNA w ith DNA, RNA w ith RNA, or RNA w ith DNA) that recognize each other if the strands are complementary. The greater the similarities of the http://www.chmconverter.com 30 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! RNA w ith RNA, or RNA w ith DNA) that recognize each other if the strands are complementary. The greater the similarities of the sequences, the more readily complementary strands form "hybrid" double-stranded molecules. Hybridization thus allow s the specific identification of sequences of DNA or RNA. IN SITU HY BRIDIZATION W hen applied directly to cells and tissue sections, smears, or chromosomes of squashed mitotic cells, the technique is called in situ hybridization. This technique is ideal for determining if a cell has a specific sequence of DNA (such as a gene or part of a gene), for identifying the cells in w hich a specific gene is being transcribed, or for determining the localization of a gene in a specific chromosome. The DNA inside the cell must be initially denatured by heat or by denaturing agents so that both strands of the DNA separate. They are then ready to be hybridized w ith a segment of single-stranded DNA or RNA that is complementary to the sequence to be detected. This sequence is called a probe. The probe may be obtained by cloning, by polymerase chain reaction (PCR) amplification of the target sequence, or by synthesis if the desired sequence is short. The probe must be tagged w ith a label, usually a radioactive isotope (w hich can be localized by autoradiography) or a modified nucleotide (digoxygenin), w hich can be identified by immunocytochemistry. In in situ hybridization, the tissue section, cultured cells, smears, or chromosomes of squashed mitotic cells must first be heated to separate the double strands of their DNA. A solution containing the probe is then placed over the specimen for a period of time necessary for hybridization. After w ashing off the excess probe, the localization of the bound probe is revealed through its label (Figure 1–29). Figure 1–29. Tissue section of a benign epithelial tumor (condyloma) submitted to in situ hybridization. The brown areas are places where DNA of human papillomavirus type 2 is present. The counterstain was hematoxylin. Medium magnification. (C ourtesy of JE Levi.) Hybridization can also be performed w ith purified DNA or RNA in solid supports. Mixtures of DNA or RNA are separated by electrophoresis in an agarose gel or a polyacrylamide gel. After electrophoresis, the fragments of nucleic acids are transferred to a nylon or nitrocellulose sheet by solvent drag: a buffer flow s through the gel and membrane by capillarity, carrying the nucleic acid molecules that bind strongly to the nylon or nitrocellulose sheet, w here the nucleic acids can be further analyzed. This technique of DNA identification is called Southern blotting. W hen electrophoresis of RNA is performed, the technique is called Northern blotting. Hybridization techniques are highly specific and are routinely used in research, clinical diagnosis, and forensic medicine. PROBLEMS IN T HE INT ERPRET AT ION OF T ISSUE SECT IONS Distortions & Artifacts Caused by Tissue Processing A key point to be remembered in studying and interpreting stained tissue sections in microscope preparations is that the observed product is the end result of a series of processes that begins w ith fixation and finishes w ith the staining of the section. Several steps of this procedure may distort the tissues, delivering an image that may differ from how the structures appeared w hen they w ere alive. One cause of distortion is the shrinkage produced by the fixative, by the ethanol, and by the heat needed for paraffin embedding. Shrinkage is decreased w hen specimens are embedded in resin. http://www.chmconverter.com 31 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! A consequence of shrinkage is the appearance of artificial spaces betw een cells and other tissue components. Another source of artificial spaces is the loss of molecules that w ere not properly kept in the tissues by the fixative or that w ere removed by the dehydrating and clearing fluids. Glycogen and lipids are often lost during tissue preparation. All these artificial spaces and other distortions caused by the section preparation procedure are called artifacts. Other artifacts may include w rinkles of the section (w hich may be confused w ith blood capillaries), precipitates of stain (w hich may be confused w ith cytoplasmic granules), and many more. Students must be aw are of the existence of artifacts and try to recognize them so as not to be confused by these distortions. Totality of the Tissue Another difficulty in the study of histological sections is the impossibility of differentially staining all tissue components on only one slide. Thus, w hen observing cells under a light microscope, it is almost impossible to see the nuclei, mitochondria, lysosomes, and peroxisomes, surrounded by a basement membrane as w ell as by collagen and elastic and reticular fibers. It is necessary to examine several preparations, each one stained by a different method, before an idea of the w hole composition and structure of a tissue can be obtained. The transmission electron microscope, on the other hand, allow s the observation of a cell w ith all its organelles and inclusions surrounded by the components of the extracellular matrix. Two Dimensions & Three Dimensions W hen a three-dimensional volume is cut into very thin sections, the sections seem to have only tw o dimensions: length and w idth. This often leads observers to err if they do not realize that a sectioned ball looks like a circle and that a sectioned tube looks like a ring (Figure 1–30). W hen a section is observed under the microscope, the student must alw ays imagine that something may be missing in front of or behind that section, because many structures are thicker than the section. It must also be remembered that the structures w ithin a tissue are usually sectioned randomly. Figure 1–30. http://www.chmconverter.com 32 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! How different three-dimensional structures may appear when thin sectioned. A: Different sections through a hollow ball and a hollow tube. B: A section through a single coiled tube may appear as sections of many separate tubes. C: Sections through a solid ball (above) and sections through a solid cylinder (below) may be very similar. To understand the architecture of an organ, it is necessary to study sections made in different planes. Sometimes only the study of serial sections and their reconstruction into a three-dimensional volume make it possible to understand a complex organ. REFERENCES Alberts B et al: Molecular Biology of the Cell, 3rd ed. Garland, 1994. Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, 1990. Cuello ACC: Immunocytochemistry. W iley, 1983. Darnell J, Lodish H, Baltimore D: Molecular Cell Biology, 2nd ed. Scientific American Books, 1990. Hayat MA: Stains and Cytochemical Methods. Plenum, 1993. James J: Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff, 1976. Junqueira LCU et al: Differential staining of collagen types I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 1978;41:267. [PMID: 82432] http://www.chmconverter.com 33 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Meek GA: Practical Electron Microscopy for Biologists. W iley, 1976. Pease AGE: Histochemistry: Theoretical and Applied, 4th ed. Churchill Livingstone, 1980. Rochow TG, Tucker PA: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Plenum, 1994. Rogers AW: Techniques of Autoradiography, 3rd ed. Elsevier, 1979. Rubbi CP: Light Microscopy. Essential Data. W iley, 1994. Spencer M: Fundamentals of Light Microscopy. Cambridge University Press, 1982. Stow ard PJ, Polak JM (editors): Histochemistry: The Widening Horizons of Its Applications in Biological Sciences. W iley, 1981. Copyright ©2007 The McGraw-Hill Com panies. All rights reserved. Privacy Notice. Any use is subject to the Term s of Use and Notice. Additional Credits and Copyright Inform ation. http://www.chmconverter.com 34 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! Print Close W indow Note: Large im ages and tables on this page m ay necessitate printing in landscape m ode. Copyright ©2007 The McGraw-Hill Companies. A ll rights reserved. Lange Histology > Chapter 2. The Cytoplasm > T HE CYT OPLASM: INT RODUCT ION Cells are the structural units of all living organisms. There are tw o fundamentally different types of cells, but so many biochemical similarities exist betw een them that some investigators have postulated that one group evolved from the other. Prokaryotic (Gr. pro, before, + karyon, nucleus) cells are found only in bacteria. These cells are small (1–5 m long), typically have a cell w all outside the plasmalemma, and lack a nuclear envelope separating the genetic material (DNA) from other cellular constituents. In addition, prokaryotes have no histones (specific basic proteins) bound to their DNA and usually no membranous organelles. In contrast, eukaryotic (Gr. eu, good, + karyon, nucleus) cells are larger and have a distinct nucleus surrounded by a nuclear envelope (Figure 2–1). Histones are associated w ith the genetic material, and numerous membrane-limited organelles are found in the cytoplasm. This book is concerned exclusively w ith eukaryotic cells. Figure 2–1. The ultrastructure and molecular organization (right) of the cell membrane. The dark lines at the left represent the two dense layers observed in the electron microscope; these are caused by the deposit of osmium in the hydrophilic portions of the phospholipid molecules. CELLULAR DIFFERENT IAT ION The human organism has about 200 different cell types, all derived from the zygote, a single cell formed by fertilization of an oocyte by a spermatozoon. The first cellular divisions of the zygote originate cells called blastomeres, w hich are able to form all cell types of the adult. Through this process, called cell differentiation, the cells synthesize specific proteins, change their shape, and become very efficient in specialized functions. For example, muscle cell precursors elongate into spindle-shaped cells that synthesize and accumulate myofibrillar proteins (actin, myosin). The resulting cell efficiently converts chemical energy into contractile force. The main cellular functions performed by specialized cells in the body are listed in Table 2–1. Table 2–1. Cellular Functions in Some Specialized Cells. Function Specialized Cell(s) Movement Muscle cell Synthesis and secretion of enzymes Pancreatic acinar cells Synthesis and secretion of mucous substances Mucous-gland cells Synthesis and secretion of steroids Some adrenal gland, testis, and ovary cells Ion transport Cells of the kidney and salivary gland ducts Intracellular digestion Macrophages and some w hite blood cells Transformation of physical and chemical stimuli into nervous impulses Sensory cells Metabolite absorption Cells of the intestine CELL ECOLOGY Because the body experiences considerable environmental diversity (eg, normal and pathological conditions), the same cell type can exhibit different characteristics and behaviors in different regions and circumstances. Thus, macrophages and neutrophils (both of w hich are phagocytic defense cells) w ill shift from oxidative metabolism to glycolysis in an anoxic, inflammatory environment. Cells that appear to be structurally similar may react in different w ays because they have different families of receptors for signaling molecules (such as hormones and extracellular matrix macromolecules). For example, because of their http://www.chmconverter.com 35 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! receptors for signaling molecules (such as hormones and extracellular matrix macromolecules). For example, because of their diverse library of receptors, breast fibroblasts and uterine smooth muscle cells are exceptionally sensitive to female sex hormones. CELL COMPONENT S The cell is composed of tw o basic parts: cytoplasm (Gr. kytos, cell, + plasma, thing formed) and nucleus (L. nux, nut). Individual cytoplasmic components are usually not clearly distinguishable in common hematoxylin and eosin-stained preparations; the nucleus, how ever, appears intensely stained dark blue or black. Cytoplasm The outermost component of the cell, separating the cytoplasm from its extracellular environment, is the plasma membrane (plasmalemma). How ever, even if the plasma membrane is the external limit of the cell, there is a continuum betw een the interior of the cell and extracellular macromolecules. The plasma membrane contains proteins called integrins that are linked to cytoplasmic cytoskeletal filaments and to extracellular molecules. Through these linkages there is a constant exchange of influence, in both w ays, betw een the extracellular matrix and the cytoplasm. The cytoplasm is composed of a matrix, or cytosol, in w hich are embedded the organelles, the cytoskeleton, and deposits of carbohydrates, lipids, and pigments. The cytoplasm of eukaryotic cells is divided into several distinct compartments by membranes that regulate the intracellular traffic of ions and molecules. These compartments concentrate enzymes and the respective substrates, thus increasing the efficiency of the cell. Plasma Membrane All eukaryotic cells are enveloped by a limiting membrane composed of phospholipids, cholesterol, proteins, and chains of oligosaccharides covalently linked to phospholipids and protein molecules. The cell, or plasma, membrane functions as a selective barrier that regulates the passage of certain materials into and out of the cell and facilitates the transport of specific molecules. One important role of the cell membrane is to keep constant the intracellular milieu, w hich is different from the extracellular fluid. Membranes also carry out a number of specific recognition and regulatory functions (to be discussed later), playing an important role in the interactions of the cell w ith its environment. Membranes range from 7.5 to 10 nm in thickness and consequently are visible only in the electron microscope. The line sometimes seen on the light microscope betw een adjacent cells is formed by the membranes of the tw o cells plus extracellular molecules. These three components together reach a dimension visible on the light microscope. Electron micrographs reveal that the plasmalemma—and, for that matter, all other organellar membranes—exhibit a trilaminar structure after fixation in osmium tetroxide (Figure 2–1). Because all membranes have this appearance, the three-layered structure has been designated the unit membrane (Figure 2–2). The three layers seen in the electron microscope are apparently produced by the deposit of reduced osmium on the hydrophilic groups present on each side of the lipid bilayer. Figure 2–2. Electron micrograph of a section of the surface of an epithelial cell, showing the unit membrane with its two dark lines enclosing a clear band. The granular material on the surface of the membrane is the glycocalyx. x100,000. Membrane phospholipids, such as phosphatidylcholine (lecithin) and phosphatidylethanolamine (cephalin), consist of tw o long, nonpolar (hydrophobic) hydrocarbon chains linked to a charged (hydrophilic) head group. Cholesterol is also a constituent of cell membranes. W ithin the membrane, phospholipids are most stable w hen organized into a double layer w ith their hydrophobic (nonpolar) chains directed tow ard the center of the membrane and their hydrophilic (charged) heads directed outw ard (Figure 2 –1). Cholesterol breaks up the close packing of the phospholipid long chains, and this disruption makes the membrane more http://www.chmconverter.com 36 / 356 basichistology11 Converted by Atop CHM to PDF Converter free version! –1). Cholesterol breaks up the close packing of the phospholipid long chains, and this disruption makes the membrane more fluid. The cell controls the fluidity of the membranes through the amount of cholesterol present. The lipid composition of each half of the bilayer is different. For example, in red blood cells (erythrocytes), phosphatidylcholine and sphingomyelin are more abundant in the outer half of the membrane, w hereas phosphatidylserine and phosphatidylethanolamine are more concentrated in the inner half. Some of the lipids, know n as glycolipids, possess oligosaccharide chains that extend outw ard from the surface of the cell membrane and thus contribute to lipid asymmetry (Figures 2–3A and 2–4). Figure 2–3. A: The fluid mosaic model of membrane structure. The membrane consists of a phospholipid double layer with proteins inserted in it (integral proteins) or bound to the cytoplasmic surface (peripheral proteins). Integral membrane proteins are firmly embedded in the lipid layers. Some of these proteins completely span the bilayer and are called transmembrane proteins, whereas others are embedded in either the outer or inner leaflet of the lipid bilayer. The dotted line in the integral membrane protein is the region where hydrophobic amino acids interact with the hydrophobic portions of the membrane. Many of the proteins and lipids have externally exposed oligosaccharide chains. B: Membrane cleavage occurs when a cell is frozen and fractured (cryofracture). Most of the membrane particles (1) are proteins or aggregates of proteins that remain attached to the half of the membrane adjacent to the cytoplasm (P, or protoplasmic, face of the membrane). Fewer particles are found attached to the outer half of the membrane (E, or extracellular, face). For every protein particle that bulges on one surface, a corresponding depression (2) appears in the opposite surface. Membrane splitting occurs along the line of weakness formed by the fatty acid tails of membrane phospholipids, since only weak hydrophobic interactions bind the halves of the membrane along this line. (Modified and reproduced, with permission, from Krstíc RV: Ultrastructure of the Mammalian Cell. Springer- Ve

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