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  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