Histology & Its Methods of Study PDF

 C H A P T E R Fixation 1 PREPARATION OF TISSUES FOR STUDY Histology & Its Methods of Study...

 C H A P T E R Fixation 1 PREPARATION OF TISSUES FOR STUDY Histology & Its Methods of Study 1 1 AUTORADIOGRAPHY 9 CELL & TISSUE CULTURE 10 Embedding & Sectioning 3 ENZYME HISTOCHEMISTRY 10 Staining 3 LIGHT MICROSCOPY 4 VISUALIZING SPECIFIC MOLECULES 10 Bright-Field Microscopy 4 Immunohistochemistry 11 Fluorescence Microscopy 5 Hybridization Techniques 12 Phase-Contrast Microscopy 5 INTERPRETATION OF STRUCTURES IN TISSUE Confocal Microscopy 5 SECTIONS 14 Polarizing Microscopy 7 SUMMARY OF KEY POINTS 15 ELECTRON MICROSCOPY 8 ASSESS YOUR KNOWLEDGE 16 Transmission Electron Microscopy 8 Scanning Electron Microscopy 9 H istology is the study of the tissues of the body and how these tissues are arranged to constitute organs. This subject involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ. a better knowledge of tissue biology. Familiarity with the tools and methods of any branch of science is essential for a proper understanding of the subject. This chapter reviews common methods used to study cells and tissues, focusing on micro- scopic approaches. Tissues have two interacting components: cells and extra- cellular matrix (ECM). The ECM consists of many kinds of macromolecules, most of which form complex structures, such as collagen fibrils. The ECM supports the cells and con- ››PREPARATION OF TISSUES tains the fluid transporting nutrients to the cells, and carry- FOR STUDY ing away their wastes and secretory products. Cells produce The most common procedure used in histologic research is the ECM locally and are in turn strongly influenced by matrix the preparation of tissue slices or “sections” that can be exam- molecules. Many matrix components bind to specific cell ined visually with transmitted light. Because most tissues and surface receptors that span the cell membranes and connect organs are too thick for light to pass through, thin translu- to structural components inside the cells, forming a contin- cent sections are cut from them and placed on glass slides for uum in which cells and the ECM function together in a well- microscopic examination of the internal structures. coordinated manner. The ideal microscopic preparation is preserved so that the During development, cells and their associated matrix tissue on the slide has the same structural features it had in the become functionally specialized and give rise to fundamen- body. However, this is often not feasible because the prepara- tal types of tissues with characteristic structural features. tion process can remove cellular lipid, with slight distortions Organs are formed by an orderly combination of these tissues, of cell structure. The basic steps used in tissue preparation for and their precise arrangement allows the functioning of each light microscopy are shown in Figure 1–1. organ and of the organism as a whole. The small size of cells and matrix components makes his- tology dependent on the use of microscopes and molecular Fixation methods of study. Advances in biochemistry, molecular biol- To preserve tissue structure and prevent degradation by ogy, physiology, immunology, and pathology are essential for enzymes released from the cells or microorganisms, pieces of 1 01_Mescher_ch01_p001-016.indd 1 18/03/21 11:23 PM  2 CHAPTER 1 Histology & Its Methods of Study FIGURE 1–1 Sectioning fixed and embedded tissue. 52°- 60°C (a) Fixation Dehydration Clearing Infiltration Embedding Drive wheel Block holder Paraffin block Tissue Steel knife b Most tissues studied histologically are prepared as shown, with Similar steps are used in preparing tissue for transmission elec- this sequence of steps (a): tron microscopy (TEM), except special fixatives and dehydrating solutions are used with smaller tissue samples and embedding Fixation: Small pieces of tissue are placed in solutions of involves epoxy resins which become harder than paraffin to allow chemicals that cross-link proteins and inactivate degradative very thin sectioning. enzymes, which preserve cell and tissue structure. Dehydration: The tissue is transferred through a series of (b) A microtome is used for sectioning paraffin-embedded tissues increasingly concentrated alcohol solutions, ending in 100%, for light microscopy. The trimmed tissue specimen is mounted which removes all water. in the paraffin block holder, and each turn of the drive wheel by Clearing: Alcohol is removed in organic solvents in which the histologist advances the holder a controlled distance, gener- both alcohol and paraffin are miscible. ally from 1 to 10 μm. After each forward move, the tissue block Infiltration: The tissue is then placed in melted paraffin until it passes over the steel knife edge and a section is cut at a thickness becomes completely infiltrated with this substance. equal to the distance the block advanced. The paraffin sections Embedding: The paraffin-infiltrated tissue is placed in a small are placed on glass slides and allowed to adhere, deparaffinized, mold with melted paraffin and allowed to harden. and stained for light microscope study. For TEM, sections less than Trimming: The resulting paraffin block is trimmed to expose 1 μm thick are prepared from resin-embedded cells using an ultra- the tissue for sectioning (slicing) on a microtome. microtome with a glass or diamond knife. organs are placed as soon as possible after removal from the microscopy, react with the amine groups (NH2) of proteins, body in solutions of stabilizing or cross-linking compounds preventing their degradation by common proteases. Glutaral- called fixatives. Because a fixative must fully diffuse through dehyde also cross-links adjacent proteins, reinforcing cell and the tissues to preserve all cells, tissues are usually cut into ECM structures. small fragments before fixation to facilitate penetration. To Electron microscopy provides much greater magni- improve cell preservation in large organs, fixatives are often fication and resolution of very small cellular structures, introduced via blood vessels, with vascular perfusion allowing and fixation must be done very carefully to preserve addi- fixation rapidly throughout the tissues. tional “ultrastructural” detail. Typically in such studies, One widely used fixative for light microscopy is forma- glutaraldehyde-treated tissue is then immersed in buffered lin, a buffered isotonic solution of 37% formaldehyde. Both osmium tetroxide, which preserves (and stains) cellular lipids this compound and glutaraldehyde, a fixative used for electron as well as proteins. 01_Mescher_ch01_p001-016.indd 2 18/03/21 11:23 PM  2 CHAPTER 1 Histology & Its Methods of Study FIGURE 1–1 Sectioning fixed and embedded tissue. 52°- 60°C (a) Fixation Dehydration Clearing Infiltration Embedding Drive wheel Block holder Paraffin block Tissue Steel knife b Most tissues studied histologically are prepared as shown, with Similar steps are used in preparing tissue for transmission elec- this sequence of steps (a): tron microscopy (TEM), except special fixatives and dehydrating solutions are used with smaller tissue samples and embedding Fixation: Small pieces of tissue are placed in solutions of involves epoxy resins which become harder than paraffin to allow chemicals that cross-link proteins and inactivate degradative very thin sectioning. enzymes, which preserve cell and tissue structure. Dehydration: The tissue is transferred through a series of (b) A microtome is used for sectioning paraffin-embedded tissues increasingly concentrated alcohol solutions, ending in 100%, for light microscopy. The trimmed tissue specimen is mounted which removes all water. in the paraffin block holder, and each turn of the drive wheel by Clearing: Alcohol is removed in organic solvents in which the histologist advances the holder a controlled distance, gener- both alcohol and paraffin are miscible. ally from 1 to 10 μm. After each forward move, the tissue block Infiltration: The tissue is then placed in melted paraffin until it passes over the steel knife edge and a section is cut at a thickness becomes completely infiltrated with this substance. equal to the distance the block advanced. The paraffin sections Embedding: The paraffin-infiltrated tissue is placed in a small are placed on glass slides and allowed to adhere, deparaffinized, mold with melted paraffin and allowed to harden. and stained for light microscope study. For TEM, sections less than Trimming: The resulting paraffin block is trimmed to expose 1 μm thick are prepared from resin-embedded cells using an ultra- the tissue for sectioning (slicing) on a microtome. microtome with a glass or diamond knife. organs are placed as soon as possible after removal from the microscopy, react with the amine groups (NH2) of proteins, body in solutions of stabilizing or cross-linking compounds preventing their degradation by common proteases. Glutaral- called fixatives. Because a fixative must fully diffuse through dehyde also cross-links adjacent proteins, reinforcing cell and the tissues to preserve all cells, tissues are usually cut into ECM structures. small fragments before fixation to facilitate penetration. To Electron microscopy provides much greater magni- improve cell preservation in large organs, fixatives are often fication and resolution of very small cellular structures, introduced via blood vessels, with vascular perfusion allowing and fixation must be done very carefully to preserve addi- fixation rapidly throughout the tissues. tional “ultrastructural” detail. Typically in such studies, One widely used fixative for light microscopy is forma- glutaraldehyde-treated tissue is then immersed in buffered lin, a buffered isotonic solution of 37% formaldehyde. Both osmium tetroxide, which preserves (and stains) cellular lipids this compound and glutaraldehyde, a fixative used for electron as well as proteins. 01_Mescher_ch01_p001-016.indd 2 18/03/21 11:23 PM  Preparation of Tissues for Study 3 Embedding & Sectioning Staining C H A P T E R To permit thin sectioning, fixed tissues are infiltrated and Most cells and extracellular material are completely color- embedded in a material that imparts a firm consistency. less, and to be studied microscopically tissue sections must Embedding materials include paraffin, used routinely for light be stained (dyed). Methods of staining have been devised that microscopy, and plastic resins, which are adapted for both make various tissue components not only conspicuous but also light and electron microscopy. distinguishable from one another. Dyes stain material more or Before infiltration with such media, the fixed tissue must less selectively, often behaving like acidic or basic compounds undergo dehydration by having its water extracted gradually and forming electrostatic (salt) linkages with ionizable radicals 1 by transfers through a series of increasing ethanol solutions, of macromolecules in tissues. Cell components, such as nucleic Histology & Its Methods of Study Preparation of Tissues for Study ending in 100% ethanol. The ethanol is then replaced by an acids with a net negative charge (anionic), have an affinity for organic solvent miscible with both alcohol and the embedding basic dyes and are termed basophilic; cationic components, medium, a step referred to as clearing because infiltration with such as proteins with many ionized amino groups, stain more the reagents used here gives the tissue a translucent appearance. readily with acidic dyes and are termed acidophilic. The fully cleared tissue is then placed in melted paraffin Examples of basic dyes include toluidine blue, alcian blue, in an oven at 52°C-60°C, which evaporates the clearing solvent and methylene blue. Hematoxylin behaves like a basic dye, and promotes infiltration of the tissue with paraffin, and then staining basophilic tissue components. The main tissue com- embedded by allowing it to harden in a small container of ponents that ionize and react with basic dyes do so because of paraffin at room temperature. Tissues to be embedded with acids in their composition (DNA, RNA, and glycosaminogly- plastic resin are also dehydrated in ethanol and then infiltrated cans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain with plastic solvents that harden when cross-linking polymer- the acidophilic components of tissues such as mitochondria, izers are added. Plastic embedding avoids the higher tempera- secretory granules, and collagen. tures needed with paraffin, which helps avoid tissue distortion. Of all staining methods, the simple combination of The hardened block with tissue and surrounding embed- hematoxylin and eosin (H&E) is used most commonly. ding medium is trimmed and placed for sectioning in an Hematoxylin stains DNA in the cell nucleus, RNA-rich por- instrument called a microtome (Figure 1–1). Paraffin sections tions of the cytoplasm, and the matrix of cartilage, produc- are typically cut at 3-10 μm thickness for light microscopy, but ing a dark blue or purple color. In contrast, eosin stains other electron microscopy requires sections less than 1 μm thick. cytoplasmic structures and collagen pink (Figure 1–2a). Here One micrometer (1 μm) equals 1/1000 of a millimeter (mm) eosin is considered a counterstain, which is usually a single or 10−6 m. Other spatial units commonly used in microscopy dye applied separately to distinguish additional features of a are the nanometer (1 nm = 0.001 μm = 10−6 mm = 10−9 m) and tissue. More complex procedures, such as trichrome stains (eg, angstrom (1 Å = 0.1 nm or 10−4 μm). The sections are placed Masson trichrome), allow greater distinctions among various on glass slides and stained for light microscopy or on metal extracellular tissue components. grids for electron-microscopic staining and examination. The periodic acid–Schiff (PAS) reaction utilizes the hexose rings of polysaccharides and other carbohydrate-rich tissue structures and stains such macromolecules distinctly › ›› MEDICAL APPLICATION purple or magenta. Figure 1–2b shows an example of cells with Biopsies are tissue samples removed during surgery or routine carbohydrate-rich areas well-stained by the PAS reaction. The medical procedures. In the operating room, biopsies are fixed DNA of cell nuclei can be specifically stained using a modifi- in vials of formalin for processing and microscopic analysis in cation of the PAS procedure called the Feulgen reaction. a pathology laboratory. If results of such analyses are required Basophilic or PAS-positive material can be further identi- before the medical procedure is completed, for example to fied by enzyme digestion, pretreatment of a tissue section with know whether a growth is malignant before the patient is an enzyme that specifically digests one substrate. For example, closed, a much more rapid processing method is used. The pretreatment with ribonuclease will greatly reduce cytoplas- biopsy is rapidly frozen in liquid nitrogen, preserving cell mic basophilia with little overall effect on the nucleus, indicat- structures and at the same time making the tissue hard and ing the importance of RNA for the cytoplasmic staining. ready for sectioning. A microtome called a cryostat in a cabi- Lipid-rich structures of cells are revealed by avoiding the net at subfreezing temperature is used to section the block processing steps that remove lipids, such as treatment with with tissue, and the frozen sections are placed on slides for heat and organic solvents, and staining with lipid-soluble rapid staining and microscopic examination by a pathologist. dyes such as Sudan black, which can be useful in diagnosis Freezing of tissues is also effective in histochemical stud- of metabolic diseases that involve intracellular accumulations ies of very sensitive enzymes or small molecules because of cholesterol, phospholipids, or glycolipids. Less common freezing, unlike fixation, does not inactivate most enzymes. methods of staining can employ metal impregnation tech- Finally, because clearing solvents often dissolve cell lipids in niques, typically using solutions of silver salts to visualize fixed tissues, frozen sections are also useful when structures certain ECM fibers and specific cellular elements in nervous containing lipids are to be studied histologically. tissue. The Appendix lists important staining procedures used for most of the light micrographs in this book. 01_Mescher_ch01_p001-016.indd 3 18/03/21 11:23 PM  4 CHAPTER 1 Histology & Its Methods of Study FIGURE 1–2 Hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining. G G G L L G G G a b Micrographs of epithelium lining the small intestine, (a) stained lumen, where projecting microvilli have a prominent layer of with H&E, and (b) stained with the PAS reaction for glycoproteins. glycoproteins at the lumen (L) and in the mucin-rich secretory With H&E, basophilic cell nuclei are stained purple, while cyto- granules of goblet cells. Cell surface glycoproteins and mucin are plasm stains pink. Cell regions with abundant oligosaccharides PAS-positive because of their high content of oligosaccharides on glycoproteins, such as the ends of the cells at the lumen (L) and polysaccharides, respectively. The PAS-stained tissue was or the scattered mucus-secreting goblet cells (G), are poorly counterstained with hematoxylin to show the cell nuclei. stained. With PAS, however, cell staining is most intense at the (a. X400; b. X300) Slide preparation, from tissue fixation to observation (or ocular lens) further magnifying this image and projecting with a light microscope, may take from 12 hours to 2½ days, it onto the viewer’s retina or a charge-coupled device (CCD) depending on the size of the tissue, the embedding medium, highly sensitive to low light levels with a camera and a monitor. and the method of staining. The final step before microscopic The total magnification is obtained by multiplying the magni- observation is mounting a protective glass coverslip on the fying power of the objective and ocular lenses. slide with clear adhesive. The critical factor in obtaining a crisp, detailed image with a light microscope is its resolving power, defined as the smallest distance between two structures at which they can be ››LIGHT MICROSCOPY seen as separate objects. The maximal resolving power of the light microscope is approximately 0.2 μm, which can permit Conventional bright-field microscopy and more specialized clear images magnified 1000-1500 times. Objects smaller or applications like fluorescence, phase-contrast, confocal, and thinner than 0.2 μm (such as a single ribosome or cytoplasmic polarizing microscopy are all based on the interaction of light microfilament) cannot be distinguished with this instrument. with tissue components and are used to reveal and study tissue Likewise, two structures such as mitochondria will be seen as features. only one object if they are separated by less than 0.2 μm. The microscope’s resolving power determines the quality of the Bright-Field Microscopy image, its clarity and richness of detail, and depends mainly on With the bright-field microscope, stained tissue is examined the quality of its objective lens. Magnification is of value only with ordinary light passing through the preparation. As shown when accompanied by high resolution. Objective lenses pro- in Figure 1–3, the microscope includes an optical system and viding higher magnification are designed to also have higher mechanisms to move and focus the specimen. The optical resolving power. The eyepiece lens only enlarges the image components are the condenser focusing light on the object obtained by the objective and does not improve resolution. to be studied; the objective lens enlarging and projecting the Virtual microscopy, typically used for the study of image of the object toward the observer; and the eyepiece bright-field microscopic preparations, involves the conversion 01_Mescher_ch01_p001-016.indd 4 18/03/21 11:24 PM  Light Microscopy 5 FIGURE 1–3 Components and light path of a Fluorescence Microscopy C H A P T E R bright-field microscope. When certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength— Eyepiece Interpupillar adjustment a phenomenon called fluorescence. In fluorescence Binocular tubes Head microscopy, tissue sections are usually irradiated with ultra- violet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear bright on Stand a dark background. For fluorescent microscopy, the instru- 1 Measuring ment has a source of UV or other light and filters that select Histology & Its Methods of Study Light Microscopy graticule Beamsplitter rays of different wavelengths emitted by the substances to be Revolving nosepiece visualized. Specimen Objective Fluorescent compounds with affinity for specific cell holder macromolecules may be used as fluorescent stains. Acridine Mechanical stage orange, which binds both DNA and RNA, is an example. On/off switch When observed in the fluorescence microscope, these nucleic Condenser Illumination intensity acids emit slightly different fluorescence, allowing them to be Field lens control localized separately in cells (Figure 1–4a). Other compounds, Field such as DAPI and Hoechst stain, specifically bind DNA and diaphragm Collector are used to stain cell nuclei, emitting a characteristic blue fluo- lens rescence under UV. Another important application of fluores- cence microscopy is achieved by coupling compounds such as X-Y Base translation fluorescein to molecules that will specifically bind to certain Tungsten mechanism cellular components and thus allow the identification of these halogen lamp structures under the microscope (Figure 1–4b). Antibodies labeled with fluorescent compounds are extremely important Photograph of a bright-field light microscope showing its in immunohistologic staining. (See the section Visualizing mechanical components and the pathway of light from the Specific Molecules.) substage lamp to the eye of the observer. The optical system has three sets of lenses: Phase-Contrast Microscopy The condenser collects and focuses a cone of light that illuminates the tissue slide on the stage. Unstained cells and tissue sections, which are usually trans- Objective lenses enlarge and project the illuminated parent and colorless, can be studied with these modified image of the object toward the eyepiece. Interchangeable light microscopes. Cellular detail is normally difficult to see objectives with different magnifications routinely used in in unstained tissues because all parts of the specimen have histology include X4 for observing a large area (field) of the tissue at low magnification; X10 for medium magnification roughly similar optical densities. Phase-contrast micros- of a smaller field; and X40 for high magnification of more copy, however, uses a lens system that produces visible images detailed areas. from transparent objects and, importantly, can be used with The two eyepieces or oculars magnify this image another living, cultured cells (Figure 1–5). X10 and project it to the viewer, yielding a total magnifica- Phase-contrast microscopy is based on the principle that tion of X40, X100, or X400. light changes its speed when passing through cellular and 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. of a stained tissue preparation to high-resolution digital Because they allow the examination of cells without fixation or images and permits study of tissues using a computer or other staining, phase-contrast microscopes are prominent tools in digital device, without an actual stained slide or a microscope. all cell culture laboratories. A modification of phase-contrast In this technique, regions of a glass-mounted specimen are microscopy is differential interference contrast micros- captured digitally in a grid-like pattern at multiple magnifica- copy with Nomarski optics, which produces an image of liv- tions using a specialized slide-scanning microscope and saved ing cells with a more apparent three-dimensional (3D) aspect as thousands of consecutive image files. Software then con- (Figure 1–5c). verts 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 Confocal Microscopy in cost and ease of use, virtual microscopy is rapidly replacing With a regular bright-field microscope, the beam of light light microscopes and collections of glass slides in histology is relatively large and fills the specimen. Stray (excess) light laboratories for students. reduces contrast within the image and compromises the 01_Mescher_ch01_p001-016.indd 5 18/03/21 11:24 PM  6 CHAPTER 1 Histology & Its Methods of Study FIGURE 1–4 Appearance of cells with fluorescent microscopy. N N R a b Components of cells are often stained with compounds visible by filaments show nuclei with blue fluorescence and actin filaments fluorescence microscopy. stained green. Important information such as the greater den- (a) Acridine orange binds nucleic acids and causes DNA in cell sity of microfilaments at the cell periphery is readily apparent. nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to (Both ×500) appear orange in these cells of a kidney tubule. (Figure 1–4b, used with permission from Drs Claire E. Walczak and Rania Rizk, Indiana University School of Medicine, (b) Cultured cells stained with DAPI (4′,6-diamino-2-phenylindole) Bloomington.) that binds DNA and with fluorescein phalloidin that binds actin FIGURE 1–5 Unstained cells’ appearance in three types of light microscopy. a b c Living neural crest cells growing in culture appear differently in-phase light differently and produce an image of these features with various techniques of light microscopy. Here the same field in all the cells. of unstained cells, including two differentiating pigment cells, is (c) Differential interference contrast microscopy: Cellular details shown using three different methods (all ×200): are highlighted in a different manner using Nomarski optics. (a) Bright-field microscopy: Without fixation and staining, only Phase-contrast microscopy, with or without differential interfer- the two pigment cells can be seen. ence, is widely used to observe live cells grown in tissue culture. (b) Phase-contrast microscopy: Cell boundaries, nuclei, and (Used with permission from Dr Sherry Rogers, Department of Cell cytoplasmic structures with different refractive indices affect Biology and Physiology, University of New Mexico, Albuquerque, NM.) 01_Mescher_ch01_p001-016.indd 6 18/03/21 11:24 PM  Light Microscopy 7 such optical sections at a series of focal planes through the FIGURE 1–6 Principle of confocal microscopy. specimen allows them to be digitally reconstructed into a C H A P T E R 3D image. Laser Polarizing Microscopy Polarizing microscopy allows the recognition of stained or unstained structures made of highly organized subunits. When normal light passes through a polarizing filter, it exits 1 Scanner vibrating in only one direction. If a second filter is placed in Histology & Its Methods of Study Light Microscopy the microscope above the first one, with its main axis per- Detector pendicular to the first filter, no light passes through. If, how- ever, tissue structures containing oriented macromolecules are located between the two polarizing filters, their repeti- tive structure rotates the axis of the light emerging from the polarizer and they appear as bright structures against a dark background (Figure 1–7). The ability to rotate the direction Plate with of vibration of polarized light is called birefringence and is pinhole Beam splitter FIGURE 1–7 Tissue appearance with bright-field and polarizing microscopy. Lens Other out-of-focus Focal plane Specimen planes Although a very small spot of light originating from one plane of the section crosses the pinhole and reaches the detector, rays originating from other planes are blocked by the blind. Thus, only one very thin plane of the specimen is focused at a a time. The diagram shows the practical arrangement of a confo- cal 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 speci- men 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 micros- copy (Figure 1–6) avoids these problems and achieves high resolution and sharp focus by using (1) a small point of high-intensity light, often from a laser and (2) a plate with a pinhole aperture in front of the image detector. The point b light source, the focal point of the lens, and the detector’s pin- point aperture are all optically conjugated or aligned to each Polarizing light microscopy produces an image only of material other in the focal plane (confocal), and unfocused light does having repetitive, periodic macromolecular structure; features not pass through the pinhole. This greatly improves resolu- without such structure are not seen. Pieces of thin, unsec- tion of the object in focus and allows the localization of speci- tioned mesentery were stained with red picrosirius, orcein, and men components with much greater precision than with the hematoxylin, placed on slides and observed by bright-field (a) bright-field microscope. and polarizing (b) microscopy. Confocal microscopes include a computer-driven mirror (a) With bright-field microscopy, collagen fibers appear red, with thin elastic fibers and cell nuclei darker. (X40) system (the beam splitter) to move the point of illumination across the specimen automatically and rapidly. Digital images (b) With polarizing microscopy, only the collagen fibers are visible and exhibit intense yellow or orange birefringence. captured at many individual spots in a very thin plane of focus (a: X40; b: X100) are used to produce an “optical section” of that plane. Creating 01_Mescher_ch01_p001-016.indd 7 18/03/21 11:24 PM  8 CHAPTER 1 Histology & Its Methods of Study a feature of crystalline substances or substances containing The wavelength in an electron beam is much shorter than that highly oriented molecules, such as cellulose, collagen, micro- of light, allowing a 1000-fold increase in resolution. tubules, and actin filaments. The utility of all light microscopic methods is greatly Transmission Electron Microscopy extended through the use of digital cameras. Many features The transmission electron microscope (TEM) is an imag- of digitized histologic images can be analyzed quantitatively ing system that permits resolution around 3 nm. This high using appropriate software. Such images can also be enhanced resolution allows isolated particles magnified as much as to allow objects not directly visible through the eyepieces to be 400,000 times to be viewed in detail. Very thin (40-90 nm), examined on a monitor. resin-embedded tissue sections are typically studied by TEM at magnifications up to approximately 120,000 times. ››ELECTRON MICROSCOPY Figure 1–8a indicates the components of a TEM and the basic principles of its operation: a beam of electrons focused Transmission and scanning electron microscopes are based on using electromagnetic “lenses” passes through the tissue sec- the interaction of tissue components with beams of electrons. tion to produce an image with black, white, and intermediate FIGURE 1–8 Electron microscopes. Electron gun Electron gun Cathode Cathode 3 mm Anode Anode Copper grid Condensor lens with three sections Lens Specimen Column Objective lens holder Lens Column Scanner Intermediate lens Electron detector TEM image Lens SEM image Projector lens Image on viewing screen Specimen Electron detector with CCD camera (a) Transmission electron microscope (b) Scanning electron microscope Electron microscopes are large instruments generally housed in a In a TEM image, areas of the specimen through which elec- specialized EM facility. trons passed appear bright (electron lucent), while denser areas (a) Schematic view of the major components of a transmission elec- or those that bind heavy metal ions during specimen preparation tron microscope (TEM), which is configured rather like an upside- absorb or deflect electrons and appear darker (electron dense). down light microscope. With the microscope column in a vacuum, a Such images are therefore always black, white, and shades of gray. metallic (usually tungsten) filament (cathode) at the top emits elec- (b) The scanning electron microscope (SEM) has many similarities trons that travel to an anode with an accelerating voltage between to a TEM. However, here the focused electron beam does not pass 60 and 120 kV. Electrons passing through a hole in the anode form a through the specimen, but rather is moved sequentially (scanned) beam that is focused electromagnetically by circular electric coils from point to point across its surface similar to the way an electron in a manner analogous to the effect of optical lenses on light. beam is scanned across a television tube or screen. For SEM, speci- The first lens is a condenser focusing the beam on the section. mens are coated with metal atoms with which the electron beam Some electrons interact with atoms in the section, being absorbed interacts, producing reflected electrons and newly emitted secondary or scattered to different extents, while others are simply transmit- electrons. All of these are captured by a detector and transmitted to ted through the specimen with no interaction. Electrons reaching amplifiers and processed to produce a black-and-white image on the the objective lens form an image that is then magnified and finally monitor. The SEM shows only surface views of the coated specimen projected on a fluorescent screen or a charge-coupled device but with a striking 3D, shadowed quality. The inside of organs or cells (CCD) monitor and camera. can be analyzed after sectioning to expose their internal surfaces. 01_Mescher_ch01_p001-016.indd 8 18/03/21 11:24 PM  Autoradiography 9 shades of gray regions. These regions of an electron micro- thin layer of heavy metal (often gold) that reflects electrons graph correspond to tissue areas through which electrons in a beam scanning the specimen. The reflected electrons are C H A P T E R passed readily (appearing brighter or electron-lucent) and captured by a detector, producing signals that are processed areas where electrons were absorbed or deflected (appearing to produce a black-and-white image. SEM images are usually darker or more electron-dense). To improve contrast and reso- easy to interpret because they present a three-dimensional lution in TEM, compounds with heavy metal ions are often view that appears to be illuminated in the same way that large added to the fixative or dehydrating solutions used for tissue objects are seen with highlights and shadows caused by light. preparation. These include osmium tetroxide, lead citrate, 1 and uranyl compounds, which bind cellular macromolecules, ››AUTORADIOGRAPHY Histology & Its Methods of Study Autoradiography increasing their electron density and visibility. Cryofracture and freeze etching are techniques that Microscopic autoradiography is a method of localizing newly allow TEM study of cells without fixation or embedding and synthesized macromolecules in cells or tissue sections. Radio- have been particularly useful in the study of membrane struc- actively labeled metabolites (nucleotides, amino acids, sugars) ture. In these methods, very small tissue specimens are rap- provided to living cells or experimental animals are incorpo- idly frozen in liquid nitrogen and then cut or fractured with a rated into specific macromolecules (DNA, RNA, protein, glyco- knife. A replica of the frozen exposed surface is produced in a proteins, and polysaccharides). After tissue fixation, processing, vacuum by applying thin coats of vaporized platinum or other and sectioning only the new, labeled macromolecules continue metal atoms. After removal of the organic material, the replica to emit weak and localized radiation, unincorporated isotope of the cut surface can be examined by TEM. With membranes, having been washed out during the processing steps. Slides or the random fracture planes often split the lipid bilayers, expos- ing protein components whose size, shape, and distribution TEM grids with radiolabeled cells or tissue sections are coated are difficult to study by other methods. in a darkroom with photographic emulsion in which silver bro- mide crystals act as microdetectors of the radiation in the same way that they respond to light in photographic film. After an Scanning Electron Microscopy adequate exposure time in lightproof boxes, the slides are devel- Scanning electron microscopy (SEM) provides a high- oped photographically. Silver bromide crystals reduced by the resolution view of the surfaces of cells, tissues, and organs. Like radiation produce small black grains of metallic silver, which the TEM, this microscope produces and focuses a very narrow under either the light microscope or TEM indicate the locations beam of electrons, but in this instrument, the beam does not of radiolabeled macromolecules in the tissue (Figure 1–9). pass through the specimen (Figure 1–8b). Instead, the surface Much histological information becomes available by of the specimen is first dried and spray-coated with a very autoradiography. If a radioactive precursor of DNA (such FIGURE 1–9 Microscopic autoradiography. L G G a b Autoradiographs are tissue preparations in which particles called (a) Black grains of silver from the light-sensitive material coating silver grains indicate the cells or regions of cells in which specific the specimen are visible over cell regions with secretory granules macromolecules were synthesized just prior to fixation. Shown and the duct indicating glycoprotein locations. (X1500) here are autoradiographs from the salivary gland of a mouse (b) The same tissue prepared for TEM autoradiography shows sil- injected with 3H-fucose 8 hours before tissue fixation. Fucose was ver grains with a coiled or amorphous appearance again localized incorporated into oligosaccharides, and the free 3H-fucose was mainly over the granules (G) and in the gland lumen (L). (X7500) removed during fixation and sectioning of the gland. Autoradio- (Figure 1–9b, used with permission from Drs Ticiano G. Lima and graphic processing and microscopy reveal locations of newly syn- A. Antonio Haddad, School of Medicine, Ribeirão Preto, Brazil.) thesized glycoproteins containing that sugar. 01_Mescher_ch01_p001-016.indd 9 18/03/21 11:24 PM  10 CHAPTER 1 Histology & Its Methods of Study as tritium-labeled thymidine) is used, it is possible to know which cells in a tissue (and how many) are replicating DNA ››ENZYME HISTOCHEMISTRY and preparing to divide. Dynamic events may also be analyzed. Enzyme histochemistry (or cytochemistry) is a method for For example, if one wishes to know where in the cell protein is localizing cellular structures using a specific enzymatic activ- produced, if it is secreted, and its path in the cell before being ity present in those structures. To preserve the endogenous secreted, several animals are injected with a radioactive amino enzymes, histochemical procedures usually use unfixed or acid and tissues collected at different times after the injections. mildly fixed tissue, which is sectioned on a cryostat to avoid Autoradiography of the tissues from the sequential times will adverse effects of heat and organic solvents on enzymatic indicate the migration of the radioactive proteins. activity. For enzyme histochemistry: (1) tissue sections are immersed in a buffer containing the substrate of the enzyme ››CELL & TISSUE CULTURE to be localized at the appropriate temperature and pH; (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 Live cells and tissues can be maintained and studied outside a product of the enzymatic action on the substrate; and (4) the the body in culture (in vitro). In the organism (in vivo), cells final product from the marker, which must be insoluble and are bathed in fluid derived from blood plasma and containing visible by light or electron microscopy, precipitates over the many different molecules required for survival and growth. site of the enzymes, identifying their location. Cell culture allows the direct observation of cellular behavior Examples of enzymes that can be detected histochemi- under a phase-contrast microscope, and many experiments cally include the following: technically impossible to perform in the intact animal can be accomplished in vitro. Phosphatases, which remove phosphate groups from The cells and tissues are grown in complex solutions of macromolecules (Figure 1–10) known composition (salts, amino acids, vitamins) to which Dehydrogenases, which transfer hydrogen ions from serum or specific growth factors are added. Cells to be cultured one substrate to another, such as many enzymes of the are dispersed mechanically or enzymatically from a tissue or citric acid (Krebs) cycle, allowing histochemical identifi- organ and placed with sterile procedures in a clear dish to cation of such enzymes in mitochondria which they adhere, usually as a single layer (Figure 1–5). Such Peroxidase, which promotes the oxidation of sub- preparations are called primary cell cultures. Some cells can strates with the transfer of hydrogen ions to hydrogen be maintained in vitro for long periods because they become peroxide immortalized and constitute a permanent cell line. Most cells obtained from normal tissues have a finite, genetically pro- grammed life span. However, certain changes (some related to oncogenes; see Chapter 3) can promote cell immortality, a › ›› MEDICAL APPLICATION process called transformation, and are similar to the initial Many enzyme histochemical procedures are used in the changes in a normal cells becoming a cancer cell. Improve- medical laboratory, including Perls’ Prussian blue reaction for ments in culture technology and use of specific growth factors iron (used to diagnose the iron storage diseases, hemochro- now allow most cell types to be maintained in vitro. matosis and hemosiderosis), the PAS-amylase and alcian blue As shown in Chapter 2, incubation of living cells in vitro reactions for polysaccharides (to detect glycogenosis and with a variety of new fluorescent compounds that are seques- mucopolysaccharidosis), and reactions for lipids and sphin- tered and metabolized in specific compartments of the cell pro- golipids (to detect sphingolipidosis). vides a new approach to understanding these compartments, both structurally and physiologically. Other histologic tech- niques applied to cultured cells have been particularly important for understanding the locations and functions of microtubules, microfilaments, and other components of the cytoskeleton. ››VISUALIZING SPECIFIC MOLECULES A specific macromolecule present in a tissue section may also be identified by using tagged compounds or macromolecules › ›› MEDICAL APPLICATION that bind specifically with the molecule of interest. The com- pounds that interact with the molecule must be visible with Cell culture is very widely used to study molecular changes the light or electron microscope, often by being tagged with a that occur in cancer; to analyze infectious viruses, myco- detectible label. The most commonly used labels are fluores- plasma, and some protozoa; and for many routine genetic or cent compounds, radioactive atoms that can be detected with chromosomal analyses. Cervical cancer cells from a patient autoradiography, molecules of peroxidase or other enzymes later identified as Henrietta Lacks, who died from the disease that can be detected with histochemistry, and metal (usually in 1951, were used to establish one of the first cell lines, gold) particles that can be seen with light and electron micros- called HeLa cells, which are still used in research on cellular copy. These methods can be used to detect and localize spe- structure and function throughout the world. cific sugars, proteins, and nucleic acids. 01_Mescher_ch01_p001-016.indd 10 18/03/21 11:24 PM  Visualizing Specific Molecules 11 Examples of molecules that interact specifically with FIGURE 1–10 Enzyme histochemistry. other molecules include the following: C H A P T E R Phalloidin, a compound extracted from mushroom, Amanita phalloides, interacts strongly with the actin protein of microfilaments. L Protein A, purified from Staphylococcus aureus bacte- ria, binds to the Fc region of antibody molecules, and can therefore be used to localize naturally occurring or 1 applied antibodies bound to cell structures. Histology & Its Methods of Study Visualizing Specific Molecules 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 macro- molecules 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 L L to identify and localize many specific proteins, not just those with enzymatic activity that can be demonstrated by histochemistry. aa The body’s immune cells interact with and produce anti- bodies against other macromolecules—called antigens—that are recognized as “foreign,” not a normal part of the organism, and potentially dangerous. Antibodies belong to the immu- noglobulin family of glycoproteins and are secreted by lym- phocytes. These molecules normally bind specifically to their provoking antigens and help eliminate them. Ly Widely applied for both research and diagnostic pur- poses, every immunohistochemical technique requires an antibody against the protein or other antigen that is to be Ly detected. This means that the protein must have been pre- viously 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 a rat), the isolated protein is injected into an ani- mal of another species (eg, a rabbit or a goat). If the protein’s b N 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. (a) Micrograph of cross-sections of kidney tubules treated Different groups (clones) of lymphocytes in the injected histochemically to demonstrate alkaline phosphatases (with animal recognize different parts of protein x and each clone maximum activity at an alkaline pH) showing strong activity of produces an antibody against that part. These antibodies are this enzyme at the apical surfaces of the cells at the lumens (L) of the tubules. (X200) collected from the animal’s plasma and constitute a mixture of polyclonal antibodies, each capable of binding a different (b) TEM image of a kidney cell in which acid phosphatase has been localized histochemically in three lysosomes (Ly) near the region of protein x. nucleus (N). The dark material within these structures is lead It is also possible, however, to inject protein x into a phosphate that precipitated in places with acid phosphatase mouse and a few days later isolate the activated lymphocytes activity. (X25,000) and place them into culture. Growth and activity of these (Figure 1–10b, used with permission from Dr Eduardo cells can be prolonged indefinitely by fusing them with lym- Katchburian, Department of Morphology, Federal University of São Paulo, Brazil.) phocytic tumor cells to produce hybridoma cells. Different hybridoma clones produce different antibodies against the several parts of protein x, and each clone can be isolated and 01_Mescher_ch01_p001-016.indd 11 18/03/21 11:24 PM  12 CHAPTER 1 Histology & Its Methods of Study cultured separately so that the different antibodies against level of antibody binding serving to amplify the visible signal. protein x can be collected separately. Each of these antibodies Moreover, the same preparation of labeled secondary antibody is a monoclonal antibody. An advantage of using a mono- can be used in studies with many different primary antibodies clonal antibody rather than polyclonal antibodies is that it can (specific for different antigens) as long as all these are made be selected to be highly specific and to bind strongly to the in the same species. There are other indirect methods that protein to be detected, with less nonspecific binding to other involve the use of other intermediate molecules, such as the proteins that are similar to the one of interest. biotin-avidin technique, which are also used to amplify detec- In immunohistochemistry, a tissue section that one believes tion signals. contains the protein of interest is incubated in a solution con- Examples of indirect immunocytochemistry are shown taining antibody (either monoclonal or polyclonal) against this in Figure 1–12, demonstrating the use of this method with protein. The antibody binds specifically to the protein and after cells in culture or after tissue sectioning for both light micros- a rinse to remove unbound antibody the protein’s location in the copy and TEM. Indirect immunohistochemistry has largely tissue or cells can be seen with either the light or electron micro- replaced autoradiography in studies of localized cell prolif- scope by visualizing the antibody bound to its specific antigen. eration; by providing bromodeoxyuridine, a thymidine ana- Antibodies are commonly tagged with fluorescent compounds, log, instead of radiolabeled thymidine, nuclei of growing cells with peroxidase or alkaline phosphatase for histochemical can be recognized by antibodies specific for bromodeoxyuri- detection, or with electron-dense gold particles for TEM. dine incorporated into DNA, and the time-consuming steps of As Figure 1–11 indicates, there are direct and indirect photographic processing are eliminated. methods of immunocytochemistry. The direct method just utilizes a labeled antibody that binds the protein of interest. Indirect immunohistochemistry involves sequential appli- › ›› MEDICAL APPLICATION cation of two antibodies and additional washing steps. The Because cells in some diseases, including many cancer cells, (primary) antibody specifically binding the protein of interest often produce proteins unique to their pathologic condition, is not labeled. The detectible tag is conjugated to a second- immunohistochemistry can be used by pathologists to diag- ary antibody made in an animal species different (“foreign”) nose many diseases, including certain types of tumors and from that which made the primary antibody. For example, pri- some virus-infected cells. Table 1–1 shows some applications mary antibodies made by mouse lymphocytes (such as most of immunocytochemistry routinely used in clinical practice. monoclonal antibodies) are specifically recognized and bound by antibodies made in a rabbit or goat injected with mouse antibody immunoglobulin. Hybridization Techniques The indirect method is used more widely in research and Hybridization usually implies the specific binding between pathologic tests because it is more sensitive, with the extra two single strands of nucleic acid, which occurs under FIGURE 1–11 Immunocytochemistry techniques. Labeled secondary Labeled Unlabeled antibody antibody primary antibody Antigen Antigen Tissue section Glass slide Direct Indirect Immunocytochemistry (or immunohistochemistry) can be direct labeled secondary antibody is obtained that was (1) made in or indirect. Direct immunocytochemistry (left) uses an antibody another species against immunoglobulin proteins (antibodies) made against the tissue protein of interest and tagged directly from the species in which the primary antibodies were made and with a label such as a fluorescent compound or peroxidase. When (2) labeled with a fluorescent compound or peroxidase. When placed with the tissue section on a slide, these labeled antibod- the labeled secondary antibody is applied to the tissue section, it ies bind specifically to the protein (antigen) against which they specifically binds the primary antibodies, indirectly labeling the were produced and can be visualized by the appropriate method. protein of interest on the slide. Because more than one labeled Indirect immunocytochemistry (right) uses first a primary secondary antibody can bind each primary antibody molecule, antibody made against the protein (antigen) of interest and labeling of the protein of interest is amplified by the indirect applied to the tissue section to bind its specific antigen. Then a method. 01_Mescher_ch01_p001-016.indd 12 18/03/21 11:24 PM  Visualizing Specific Molecules 13 FIGURE 1–12 Cells and tissues stained by immunohistochemistry. 1 C H A P T E R Histology & Its Methods of Study Visualizing Specific Molecules c a 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 b 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. Immunocytochemical methods to localize specific proteins can (X5000) be applied to either light microscopic or TEM preparations using a (Figure 1–12c, used with permission from Dr Moise Bendayan, variety of labels. Departments of Pathology and Cell Biology, University of Montreal, Montreal, Canada.) (a) A single cultured uterine cell stained fluorescently to reveal a meshwork of intermediate filaments (green) throughout appropriate conditions if the strands are complementary. The specific identification of sequences in genes or RNA. This can greater the similarities of their nucleotide sequences, the more occur with cellular DNA or RNA when nucleic acid sequences readily the complementary strands form “hybrid” double-strand in solution are applied directly to prepared cells and tissue sec- molecules. Hybridization at stringent conditions allows the tions, a procedure called in situ hybridization (ISH). TABLE 1–1     Examples of specific antigens with diagnostic importance. Antigens Diagnosis Specific cytokeratins Tumors of epithelial origin Protein and polypeptide hormones Certain endocrine 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 01_Mescher_ch01_p001-016.indd 13 18/03/21 11:24 PM  14 CHAPTER 1 Histology & Its Methods of Study 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 › ›› MEDICAL APPLICATION (Figure 1–13), (2) identifying the cells containing specific Warts on the skin of the genitals and elsewhere are due to messenger RNAs (mRNAs) (in which the corresponding infection with the human papillomavirus (HPV) which causes gene is being transcribed), or (3) determining the localiza- the characteristic benign proliferative growth. As shown in tion of a gene in a specific chromosome. DNA and RNA of Figure 1–12, such virus-infected cells can often be demon- the cells must be initially denatured by heat or other agents strated by ISH. Certain cancer cells with unique or elevated to become completely s