Methods of Study

OBJECTIVES

This chapter should help you to:

· Know the mathematic relationships among the units of measure used to analyze histologic specimens .
· Name the instruments and techniques used to prepare and study histologic specimens.
· Know the basic steps in preparing specimens for light and electron microscopy.
· Know the advantages and limitations of histologic instruments and techniques.
· Select the appropriate methods to reveal specific microscopic features of cells and tissues.

I.   GENERAL FEATURES OF HISTOLOGY & ITS METHODS

A. Goals of Histology:   Histology, the study of tissues, is largely a visual science that relies on microscopy to reveal cell, tissue, and organ substructure. Its goals include the following:


1. Understanding tissue structure at levels not visible to the unaided eye, including 3-dimensional relationships among the biochemical constituents.
2. Understanding the relationship between tissue structure and function.
3. Establishing a basis for learning histopathology--the relationship between abnormal tissue structure and functional defects.
4. Providing a basis for treating diseased and injured tissues. In medicine, this is the ultimate goal.


B. Histologic Methods: Histology is classed as a subdiscipline of anatomy ("cutting apart"), because its methods involve dividing tissues and organs into pieces to prepare them for microscopic examination and chemical analyses.

1. Microscopy.   Microscopic analysis is the main goal of these methods. The several types of microscopy fall into 2 main groups, light microscopy (iV*) and electron microscopy (EM) (V).
2. Tissue preparation for microscopy.  The optics of each type of microscope make certain demands on tissue preparation (III). Common preparative procedures include sectioning to produce thin, translucent slices and staining to reveal otherwise transparent substructure.
3. Cell, tissue, and organ culture allow observation of the structural or functional effects of certain treatments without interference from regulatory mechanisms present in intact orga nisms (VI).
4. Cell fractionation involves mechanically breaking cells and then separating their compo nents by centrifugation for electron microscopic or biochemical analysis (VII).

C. Advantages and Limitations of Histologic Methods: Only by understanding the advan tages and limitations of histologic methods can one properly interpret the information they provide. The advantages result from making small and complex structures and processes acces sible to observation. The limitations occur because the methods themselves, especially dividing an organism into pieces, often halt the very life processes we wish to analyze.

D. Tissue Structure and Function: These are so closely related that neither can be fully understood without an appreciation of the other. Structure-function relationships should be the main focus of both your initial study and your review.

II. UNITS OF MEASURE

Measurements of cell and tissue components provide useful comparisons of relative sizes. The metric system is used exclusively. The most commonly used units of measure in histology are the millimeter (mm, 10-3 m), micrometer (um, 10-6 m), and nanometer (nm, 10-9 m).

III. PREPARATION OF TISSUES FOR MICROSCOPIC EXAMINATION

Light and electron microscopy share the basic preparative methods outlined in the Row chart in Fig 1-1 and described in Table 1- i. Each method has its limitations and associated artifacts, which must be borne in mind while interpreting histologic images. Pay particular attention to the similarities and differences in tissue preparation for light and electron microscopy. Most methods revolve around the preparation of thin sections. These can seduce the observer into thinking of fdimensional structures in 2-dimensional terms. To overcome this problem, tissues and organs are secrioned in several planes 6 or prepared as serial sections to allow conceptual (often computer-assisted) 3-dimensional reconstruction. Since even complex mixtures of stains are unable to reveal every tissue component, adjacent sections are sometimes treated with different stains.

IV. LIGHT MICROSCOPY

Light microscopes are used to illumine and magnify specimens for detailed observation.

LightSource: Light microscopes are usually illumined by bulbs that emit white light of varymg intensity. White light includes emissions in a limited range of wavelengths (550 nm is often used as an average). Halogen bulbs with tungsten filaments emit intense white light and are commonly used in compound bright-field microscopes.

Types of Microscope Lenses: The lenses of light microscopes are made of glass. The condenser lens is located between the light source and the specimen. It collects light from the source and projects it as a cone through the specimen. The objective lens consists of one or more lenses (III.C.S) and is located between the specimen and the ocular lens. It enlarges and resolves the specimen's image and projects it toward the ocular lens. Several objectives, each providing a different magnitication, are usually mounted on a rotating turret. The ocular lens is located between the objective and the observer or recording device. It further enlarges the image and projects it onto the observer's retina, a screen, or a photographic emulsion.

Optical Properties of Lenses:
Magnification increases the apparent size of the specimen and makes it appear closer. It is a property of both objective and ocular lenses. The total magnification value is obtained by multiplying the power of the objective by that of the ocular lens.

Resolution determines the clarity and richness of detail of a microscopic image. It measures how close 2 objects can be and still appear separate; the smaller the value, the greater the resolution. The resolution of the human eye is 200 um; of a light microscope, 0.2 um; of an electron microscope, 0.002 um. Increased magnification is useless without improved resolution. Resolution (R) is thus independent of magnification and is calculated from the numerical aperture (NA) of the objective and the wavelength (h) of illumination:

                                                 R = 0.61h / NA

Numerical aperture is related to the width of the lens aperture. The greater the NA. the greater the resolving power.

Refractive index measures the comparative velocity of light in different media, Owing to the change in refractive index at air-glass interfaces, the air between the lens and coverslip bends some of the light projected through the specimen. At high magnifications, the accompanying loss of resolution reduces image quality. Using immersion oil (which has the same refractive index as glass) between the coverslip and a special oil immersion objective lens avoids the change in refractive index and thereby improves resolution.

Lens-related artifacts. Modern objectives comprise a series of glass lenses. The first (frontal) is spheric or hemispheric and magnifies the image. The others correct for aberra tions or artifacts of lens curvature. Spheric lenses bring light of shorter wavelength into focus closer to the retina than light of longer wavelength, resulting in multiple blurred images. This chromatic aberration can be avoided by using achromatic or apochromatic lenses. Optical properties of the center of a spheric lens differ from those at the periphery. Apochromatic lenses correct for this spheric aberration. Spheric lenses prevent simultaneous focusing on the entire field; either the center or the periphery is out of focus. Planar lenses correct this cnrvatnre of field and provide hat-held focus, The best objective lenses are thus planar apochromatic lenses with a high numerical aperture.

Types of Light Microscope:

Compound bright-field microscopes are the most common tool of histology and histo pathology. They are called compound (versus simple) because they use a series of lenses; bright-field, because the entire field is illuminated by an ordinary condenser. Specimens must be translucent and stained to provide contrast.  

Dark-field microscopes use a special condenser to provide contrast in unstained material, allowing living specimens to be visualized. A disklike shield excludes the center of the light shaft formed by the condenser, so that the specimen is illuminated only from the sides. Only objects that deflect light into the objective lens are visible; these appear bright on a dark background.

Phase contrast microscopes use a special lens system to transform invisible differences in phase (light speed) retardation, caused by the different refractive indices of specimen components, into visible differences in light intensity. Because fixation and staining are not re quired, living and other unstained specimens can be visualized. These have become basic tools for tissue culture. Specimens must be thin and translucent. High resolution is difficult to obtain.

Polarizing microscopes allow selective visualization of birefringent (anisotropic) structures-repetitive or crystalline structures such as collagen fibers or myofibrils. Stain ing is not required. Light from the light source passes through a polarizing filter, the condenser projects the polarized light onto the specimen, and birefringent structures in the specimen rotate the polarized light. The objective lens projects the image through a second polarizing filter, which is oriented so that only light waves oscillating in planes different from that leaving the tirst polarizing filter can enter the ocular lens and be seen. Birefringent structures appear as bright, often colored objects on a dark background.

Fluorescence microscopes allow localization of substances labeled with fluorescing com pounds (fluorochromes. eg, fluorescein or rhodamine). When stimulated by light of the proper wavelength, fluorochromes emit light of a longer wavelength. These microscopes have a special light source and filters. An ultraviolet light source is commonly used, and the emitted light is in the visible spectrum. An excitation filter between the light source and the specimen filters out all wavelengths except that needed to stimulate the fluorochrome. A barrier filter between the objective and ocular lenses protects the eyes from ultraviolet rays and projects only the emitted light.

Interference microscopes combine optical features of both the phase contrast and polarizing microscopes to provide contrast in unstained material. Relying on differences in refractive index (IV.C.4), they can measure the phase retardation induced by components of the specimen. Unlike standard phase contrast microscopes, they can compare the refracted light with an unimpeded reference beam and provide an electronic readout of the data. Because refractive index and phase retardation are proportionate to mass, these instruments can be used to calculate the mass of cellular components. Modified interference optics, pioneered by Nomarski, are used in differential interference contrast (DIC) microscopes.

V.  ELECTRON MICROSCOPY

A. General Principles: The equation for resolution is the same as for light microscopy (IV.C.2). An electron beam (wavelength ~ 0.005 nm) is used instead of visible light (wavelength ~ 397- 723 nm), giving electron microscopes much greater resolving power and allowing magnification up to 200 times that of light microscopes. Glass lenses are not transparent to wavelengths under 400 nm, but the negatively charged electron beam can be deffected and focused by elec tromagnets as it travels through a vacuum.

B. Major Components cmd Operation of Electron Microscopes: Together, the cathode and anode are analogous to the light source of a light microscope. The cathode is a metallic filament mi that emits a spray of electrons when intensely heated in a vacuum by an electric current. The anode is a positively charged metal plate with a small hole at its center The potential difference Fl between the cathode and anode (60-100 kV) accelerates electrons toward the anode; some of them pass through the hole to form the electron beam. The condenser electromagnet induces ': t an electromagnetic field that deflects the electron beam and focuses a cone of the beam on the specimen. The specimen is typically an ultrathin tissue section stained with electron-absorbing or -scattering substances to provide contrast. The image formed is actually the shadow of the contrast material. The objective electromagnet deflects the portion of the electron beam that has passed through the specimen, to form and magnify the image. The one or 2 projector electromagnets are analogous to the light microscope's ocular lenses. They further enlarge the image produced by the objective electromagnet and project it onto a fluorescent screen or photographic emulsion. The fluorescent screen is a plate coated with material that fluoresces as ij electrons strike it. Electrons deflected or absorbed by the specimen do not reach the screen, ~i whereas those that pass through the specimen do. The result is a transmission image formed by shadows of the electron-dense components of the specimen.

C. Limitations of Electron Microscopy: Because the electron beam must travel in a high vacuum, living tissue cannot be used. Tissue sections must be very thin, or they will absorb or deflect the entire beam. The electron beam may damage or otherwise alter specimen structure. The image cannot be visualized directly, but must be used to create a fluorescent or photographic image.

D. Types of Electron Microscope:


1. The transmission electron microscope (TEM) permits visualization of the internal ultra structure of cells and tissues as well as minute structures within cells or in intercellular spaces (limit of resolution ~ 0.2 nm). It operates as described above (V.B). Specimens are prepared as described in Table 1-I and Fig 1-I.
2. The scanning electron microscope (SEM) permits visualization of surface ultrastructure (limit of resolution ~ 2 nm). After the specimen is coated with a thin layer of heavy metal (Table I-I), a narrow electron beam is directed across its surface in a point-by-point se quence, generating 2 major signals. Secondary electrons are released from the specimen surface, collected on detectors, and converted electronically into an image that is displayed on a cathode ray tube. This image provides a 3-dimensional representation of the specimen surface. X-rays are generated when the electron beam strikes atoms heavier than sodium. Analysis of the x-ray signal can supply information about the concentration and distribution of certain elements in the specimen.

VI. CELL, TISSUE, and ORGAN CULTURE

These methods are used to study the function of living cells and tissues without the interference of the organism's normal homeostatic mechanisms. They permit easier control and manipulation of the cell or tissue environment. Cells and tissue isolated and grown in culture are referred to as in vitro ("in glass") and those in the intact organism as in vivo ("in the living"). It should be remembered that cells may react differently to a particular treatment in vitro and in vivo.

A. Culture Medium: The medium in which cells and tissues are grown is intended to substitute for the plasma that normally bathes them in vive. it consists of a buffered isotonic saline solution to which is added an array of nutrients (amino acids, vitamins, hormones, carbohydrates) of rigidly controlled composition. Recent advances in knowledge of cell and tissue growth require ments have decreased the use of serum and tissue extracts of less well-defined composition to supplement the medium. Antibacterial and antifungal agents are often added to the medium.

B. CultureTypes:


1. Cell culture. In suspension culture, cells are suspended in culture medium either free or attached to the surface of floating beads. In some cases cells are suspended in semisolid, 3-dimensional matrices composed of extracellular matrix materials or agarose. Plate-cultured cells behave differently at different densities. In plate culture, cells are attached to plastic or glass tissue culture dishes. The dishes may be coated with substances that improve attach ment and cell function: gelatin, collagen, polylysine, serum albumin, or extracellular matrix extracts. They may be cultured in confluent monolayers (entire culture surface covered with cells in contact with one another) or at clonal densities (seeded at low densities to avoid cell cell contact). The latter method allows growth of individual cell colonies, or clones.
2. Tissue and organ culture. Fragments of tissues or organs are removed from the body and grown as intact explants, usually at the air-medium interface. This method is often used to study embryonic differentiation and morphogenesis, away from the complex environment of the embryo.

C. Isolation and Study of Pure Cell Strains: Individual cell types may be isolated and studied in vitro to explore their separate contributions to tissue and organ function. Cell suspensions are commonly obtained from tissues by enzymatic digestion leg, with trypsin, collagenase, or hyaluronidase) of the cellular and intercellular components that hold cells together. Cell types may then be separated on the basis of size and mass through specialized forms of centrifugation (elutriation, density gradient centrifugation). Newer methods use specific antibodies to isolate particular cell types from a heterogeneous population in suspension. Some such methods exploit differential binding of cells by antibodies attached to a culture surface; others use a fluorescence activated cell sorter, which separates cells labeled with fluorescent antibodies from cells lacking the label.

VII. CELL FRACTIONATION

Cell fractionation is used to isolate and collect cellular components in quantity to study their contributions to cell function. This procedure begins with the mechanical homogenization of cells and tissues to break plasma membranes and release the cell components into suspension. The components (individual organelles) are then separated on the basis of size and density by using either of 2 centrifugation methods. In differential centrifugation, components are separated by their characteristic sedimentation rates, using different amounts of centrifugal force for various periods. In density gradient centrifugation, the homogenate is layered on top of a gradient of solute and centrifuged until its components come to rest in portions of the gradient with densities similar to their own.

OBJECTIVES

This chapter should help you to:

· Describe the basic principles of histochemistry.
· Know the substances of biologic interest that can be localized by histochemical techniques.
· Name the classes of histochemical techniques and describe the advantages and limitations of each.
· Choose appropriate techniques to reveal the location of specific substances in cells and tissues.

I.  BASIC PRINCIPLES OF HISTOCHEMISTRY

Histochemistry marries the methods of histology with those of chemistry or biochemistry. The goal is to reveal the chemical and biochemical composition of tissues and cells beyond the acid-base distribution shown by standard staining methods (see Chapter 1) without disrupting the normal distribution of the chemicals. To achieve this goal, the following criteria must be met.

A. Presentation of Normal Chemical Distribution: The substance being analyzed must not diffuse away from its original site. Otherwise, standard chemical procedures would suffice.

B. Presenration of Normal Chemical Composition: The procedure must not block or dena ture the reactive chemical groups being analyzed or change normally unreactive groups into reactive groups.

C. Specificity of the Reaction: The method should be highly specific for the substance or chemical groups being analyzed, to avoid false-positive results.

D. Detectability of the Reaction Product: The reaction product should be colored or electron scattering, so that it can be visualized easily with a light or electron microscope.

E. Insolubility of the Reaction Product: The reaction product should be insoluble, so that it remains in close proximity to the substance it marks.

II. SOME IMPORTANT BIOLOGIC SUBSTANCES & CLASSIC METHODS FOR DETECTING THEM

A. ions: It is difficult to localize most ions accurately because of their small size and tendency to diffuse. However, certain ions are normally immobilized by their association with tissue pro teins. Examples include the iron bound by hemoglobin in red blood cells (see Chapter 12) and the phosphate bound by collagen and other matrix proteins in mineralized bone (see Chapter 8).


1. Iron. Incubating iron-containing tissue in potassium fermcyanide and hydrochloric acid results in precipitation of dark blue ferric ferrocyanide (Perls' reaction). This reaction is used to identify cells involved in hemoglobin metabolism and to diagnose diseases characterized by iron deposits in tissues (hemosiderosis).
2. Phosphate, Tissue phosphates react with silver nitrate to form silver phosphate, which reacts with hydroquinone to form a black precipitate of reduced silver. This reaction is used in studies of calcium phosphate deposition during bone formation.

B. Lipids: Lipids are usually dissolved by organic fixatives or clearing agents, leaving gaps in the tissue, but they are preserved in frozen sections. For light microscopy, lipids are best demonstrated by dyes that are more soluble in lipid than in the dye solvents leg, Sudan IV, Sudan black, and oil red 0). EM specimens are treated with reagents that react with lipids to form insoluble precipitates leg, osmium tetroxide). Such methods are used to show normal lipid distribution and disease-related lipid accumulation leg, fatty change in the liver).

C. Nucleic Acids: The nucleic acids, DNA and RNA, can be localized by specific and non specific methods (see below). DNA is found mainly in nuclei, and its amount is much the same in every cell. RNA is found both in nuclei and in cytoplasm, and its amount varies widely, depending on a cell's functional state.


1. Feulgen's reaction, Hydrochloric acid is used to partially hydrolyze DNA, promoting the formation of free aldehydes. These then react with Schiff's reagent (bleached fuchsin) to form an insoluble magenta precipitate in amounts proportionate to the amount of DNA present.
2. Acridine orange, Complexes of acridine orange and nucleic acids emit a Ruorescence whose intensity is proportionate to the amount of nucleic acid present. The fluorescence is yellow green if the complex contains DNA and red-orange if it contains RNA. Neoplastic and other rapidly growing cells contain more RNA than slower-growing cells and may thus be distin guished from them.
3. Basic dyes. Both DNA and RNA stain nonspecifically with basic dyes. Because of the strong affinity of RNA for such dyes, its distribution in cells and tissues may be studied by subtraction. In this procedure, one of 2 adjacent sections is treated with ribonuclease (RNase) to remove RNA; then both are stained with basic dyes leg, hematoxylin, toluidine blue, methylene blue). Basophilic structures present in the untreated section leg. ribosomes) but absent in the RNase-treated section contain RNA.

D. Proteins and Amino Acids: Older methods of protein identification are nonspecific for t proteins but specific for particular amino acids. Examples: Million reaction for tyrosine, Sakaguchi reaction for arginine, tetrazotized benzidine reaction for tryptophan. Specific classes of enzymes can be detected by the techniques of enzyme histochemistry (III), Specific proteins can now be localized by using immunohistochemistry (IV).

E. Carbohydrates: Complex carbohydrates, ie, polysaccharides and oligosaccharides, can be localized by the histochemical techniques described below. In addition, some carbohydrates are immunogenic owing to their large size or their presence as covalently linked components of glyeoronjugates (proteogiycans, glycoproteins, glycolipids); these can be analyzed by immunohistochemical methods.


1. PAS reaction, The periodic acid-Schiff (PAS) reaction is a common technique for demon strating polysaccharides, particularly glycogen, Periodic acid reacts with the 1,2-glycol groups of sugars to form aldehydes. Schiff's reagent then reacts with the aldehydes to form an insoluble magenta pigment (compare II.C.I). Because the PAS reaction stains many complex carbohydrates, the specific localization of glycogen requires enzymatic subtraction of glycogen from an adjacent section with amylase. This method is used to distinguish among types of glycogen storage diseases.
2. Alcian blue, Alcian blue is a nonspecific basic stain at neutral pH, but it is specific for sulfate groups at pH I. It is used to demonstrate sulfated glycosaminoglycans leg, chondroitin sulfate) that are abundant in the extracellular matrix of cartilage.
3. Ruthenium red, Ruthenium red binds nonspecifically to polyanions and forms an electron- scattering precipitate useful in EM demonstration of polysaccharides.
4. Lectins, Lectins are highly specific sugar-binding proteins found in plants and animals. Fluorescently labeled lectins can show the distribution of specific terminal sugar residues on oligosaccharides, such as those in the glycocalyx of cell membranes. Examples: Concanavalin A binds to mannose; peanut agglutinin and Ricinus cornmunis agglutinin bind to galactose; and wheat germ agglutinin binds to N-acetyl-D-glucosamine.

F. Catecholamines: The catecholamines, including epinephrine and norepinephrine, fluoresce in the presence of dry formaldehyde vapor at 60-80 "C. This reaction is used in studies of catecholamine distribution in nerve tissue.

III. ENZYME HISTOCHEMISTRY

The techniques of enzyme histochemistry, which relate structure and function, can be used to locate many enzymes, including acid phosphatase, dehydrogenases, and peroxidases. Because fixation and clearing typically inactivate enzymes, frozen sections are commonly used. The sections are incubated in solutions containing substrates for the enzymes of interest and reagents that yield insoluble colored or electron-dense precipitates at the sites of enzyme activity.

A. Acid Phosphatase: In the Gomori method for acid phosphatase, the tissue is incubated with glycerophosphate and lead nitrate. The enzyme liberates phosphate, which combines with lead to produce lead phosphate, a colorless precipitate. The tissue is then immersed in a solution of ammonium sulfide, which reacts with lead phosphate to form lead sulfide, a black precipitate. Owing to their characteristic content of acid phosphatase. lysosomes can be distinguished from other cytoplasmic granules and organelles through the use of enzyme histochemistry.

B. Dehydrogenases: Dehydrogenases can be localized by incubating tissue sections with an appropriate substrate and tetrazole. The enzyme transfers hydrogen ions from the substrate to tetrazole, reducing tetrazole to formazan, a dark precipitate. Specific dehydrogenases can be targeted by choosing specific substntes.

C. Peroxidases: Peroxidases are most often demonstrated by incubating tissue with 3,3' diaminobenzidine (DAB) and hydrogen peroxide. The enzyme transfers hydrogen from DAB to the peroxide, and the oxidized DAB forms an electron-dense dark brown to black precipitate at the site of enzyme activity. This reaction is useful for both light and electron microscopy.

IV. IMMUNOHISTOCHEMISTRY

Immunohistochemistry utilizes labeled antibodies to localize specific cell and tissue antigens and is among the most sensitive and specific histochemical techniques. Because many targeted antigens are proteins whose structure might be altered by fixation and clearing, frozen sections are commonly used. In some cases, water-soluble plastics and waxes can be used for embedding.

A. Raising Antibodies: Repeated injection of antigens (proteins, glycoproteins, proteoglycans, and some polysaccharides) causes the injected animal's B lymphocytes to differentiate into plasma cells and produce antibodies. Members of a lymphocyte clone (descendents of a single lymphocyte) produce a single type of antibody, which binds to a specific antigenic site, or epitope,


1. Polyclonal antibodies. Large complex antigens may have multiple epitopes and elicit sev- eral antibody types. Mixtures of different antibodies to a single antigen (obtained through fractionation of the injected animal's serum) are called polyclonal antibodies and are com- monly raised in rabbits and goats.
2. Monoclonal antibodies, Antibodies specific for a single epitope and produced by a single clone are called monoclonal antibodies and are commonly raised in mice. Lymphocytes from the spleen of an antigen-injected mouse are mixed with myeloma cells (lymphocyte-derived tumor cells) under conditions that cause the lymphocytes and myeloma cells to fuse. Each resulting hybridoma cell has the myeloma's capacity for rapid cell division in culture and the lymphocyte's capacity for unique antibody secretion. An isolated hybridoma gives rise to a large clone that produces large quantities of pure antibody.

B. Labeling Antibodies: Antibodies are not visible with standard microscopy and must be labeled in a manner that does not interfere with their binding specificity. Common labels include tluorochromes leg, ffuorescein, rhodamine), enzymes demonstrable via enzyme histochemical techniques leg, peroxidase, alkaline phosphatase), and electron-scattering compounds for use in electron microscopy leg, ferritin, colloidal gold).

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