Detection Methods Using High-Affinity Interactions Between Molecules

A molecule present in a tissue section may be identified by using compounds that specifically interact with the molecule. The compounds that will interact with the molecule must be tagged with a label that can be detected under the light or electron microscope (Figure 1–20). The most commonly used labels are fluorescent compounds (which can be seen with a fluorescence or laser microscope), radioactive atoms (which can be detected with autoradiography), molecules of peroxidase (which can be detected after demonstration of the enzyme with hydrogen peroxide and 3,3'-diaminoazobenzidine) or other enzymes (which can be detected with their respective substrates), and metal (usually gold) particles that can be observed with light and electron microscopy. These methods are mainly used for detecting sugars, proteins, and nucleic acids.

Figure 1-20

Compounds that have affinity toward another molecule can be tagged with a label and used to identify that molecule. (1) Molecule A has a high and specific affinity toward a portion of molecule B. (2) When A and B are mixed, A binds to the portion of B it recognizes. (3) Molecule A may be tagged with a label that can be visualized with a light or electron microscope. The label can be a fluorescent compound, an enzyme such as peroxidase, a gold particle, or a radioactive atom. (4) If molecule B is present in a cell or extracellular matrix that is incubated with labeled molecule A, molecule B can be detected.

Phalloidin, protein A, lectins, and antibodies are examples of compounds that interact specifically with other molecules.

Phalloidin, which is extracted from a mushroom (Amanita phalloides), interacts strongly with actin and is usually labeled with fluorescent dyes to demonstrate actin filaments.

Protein A is a protein obtained from Staphylococcus aureus that binds to the Fc region of immunoglobulin (antibody) molecules. When protein A is tagged with a label, immunoglobulins can be detected.

Lectins are proteins or glycoproteins that are derived mainly from plant seeds and that bind with high affinity and specificity to carbohydrates. Different lectins bind to specific sequences of sugar molecules. They may bind to glycoproteins, proteoglycans, and glycolipids and are widely used to characterize membrane molecules containing defined sequences of sugars.

Immunocytochemistry

A highly specific interaction between molecules is that between an antigen and its antibody. For this reason, methods using labeled antibodies have proved most useful in identifying and localizing specific proteins and glycoproteins.

The body has cells that are able to distinguish its own molecules (self) from foreign ones. When exposed to foreign molecules—called antigens—the body may respond by producing proteins—antibodies—that react specifically and bind to the antigen, thus helping to eliminate the foreign substance. Antibodies are proteins of a large family, the immunoglobulin family.

In immunocytochemistry, a tissue section (or cells in culture) that may contain a certain protein is incubated in a solution containing an antibody to this protein. The antibody binds specifically to the protein, whose location can then be seen with either the light or electron microscope, depending on the type of compound used to label the antibody.

One of the most important requirements for immunocytochemistry is the availability of an antibody against the protein that is to be detected. This means that the protein must have been previously purified and isolated so that antibodies can be produced. Some methods for protein isolation can be seen in Figures 1–21 and 1–22.

Figure 1-21

Ultracentrifugation (A) and chromatography (B): methods of protein isolation. A: A mixture of proteins obtained from homogenized cells or tissues is submitted to centrifugation at high speed for several hours. The proteins separate into several bands, depending on the size and density of the protein molecules. The ultracentrifugation medium is drained and collected in several fractions that contain different proteins, which can be analyzed further. B: A solution containing a mixture of proteins obtained from homogenized cells or tissues is added to a column filled with beads that have different chemical properties. For instance, the beads may have different electrostatic charges (attracting proteins according to their charge) or different sizes of pores (acting as sieves for different-sized molecules). As the proteins migrate through the column, their movement is slowed according to their interaction with the particles. When the effluent is recovered, the different groups of proteins may be collected separately.

Figure 1-22

Gel electrophoresis: a method of protein isolation. A: Isolation of proteins. (1) Mixtures of proteins are obtained from homogenized cells or tissues. They are usually treated with a strong detergent (sodium dodecyl sulfate) and with mercaptoethanol to unfold and separate the protein subunits. (2) The samples are put on top of a slab of polyacrylamide gel, which is submitted to an electrical field. The proteins migrate along the gel according to their size and shape. (3) A mixture of proteins of known molecular mass is added to the gel as a reference to identify the molecular mass of the other proteins. B: Detection and identification of the proteins. (1) Staining. All proteins will stain the same color. The color intensity is proportional to the protein concentration. (2) Autoradiography. Radioactive proteins can be detected by autoradiography. An x-ray film is apposed to the gel for a certain time and then developed. Radioactive proteins will appear as dark bands in the film. (3) Immunoblotting. The proteins can be transferred from the gel to a nitrocellulose membrane. The membrane is incubated with a labeled antibody made against proteins that may be present in the sample.

Polyclonal and Monoclonal Antibodies

Let us suppose that our objective is to produce antibodies against protein x of a certain animal species (eg, a rat or a human). If protein x is already isolated, it is injected into an animal of another species (eg, a rabbit or a goat). If the protein is sufficiently different for this animal to recognize it as foreign—that is, as an antigen—the animal will produce antibodies against the protein (eg, rabbit antibody against rat x or goat antibody against human x). These antibodies are collected from the animal's plasma and used for immunocytochemistry.

Several groups (clones) of lymphocytes of the animal that was injected with protein x may recognize different parts of protein x and each group produces an antibody against each part. These antibodies constitute a mixture of polyclonal antibodies.

It is possible, however, to furnish protein x for lymphocytes maintained in cell culture (actually, lymphocytes fused with tumor cells). The different clones of lymphocytes will produce different antibodies against the several parts of protein x. Each clone can be isolated and cultured separately so that the different antibodies against protein x can be collected separately. Each of these antibodies is a monoclonal antibody. There are several advantages to using a monoclonal antibody rather than a polyclonal antibody: for instance, a monoclonal antibody can be selected to be highly specific and to bind strongly to the protein to be detected. Therefore, there will be less nonspecific binding to other proteins similar to the one being looked for.

In the direct method of immunocytochemistry, the antibody (either monoclonal or polyclonal) must be tagged with an appropriate label. A tissue section is incubated with the antibody for some time so that the antibody interacts with and binds to protein x. The section is then washed to remove the unbound antibody (Figure 1–23). Depending on the label that was used (fluorescent compound, enzyme, gold particles), the section can be observed with a light or electron microscope. If peroxidase or another enzyme was used as a label, the enzyme must be detected before the tissue section is observed in the microscope (see Histochemistry & Cytochemistry). The areas of the tissue section that contain protein x will become fluorescent or will be covered by gold particles or by a dark precipitate if an enzyme was used as a marker.

Figure 1-23

Direct method of immunocytochemistry. (1) Immunoglobulin molecule (Ig). (2) Production of a polyclonal antibody. Protein x from a rat is injected into a rabbit. Several rabbit Igs are produced against protein x. (3) Labeling the antibody. The rabbit Igs are tagged with a label. (4) Immunocytochemical reaction. The rabbit Igs recognize and bind to different parts of protein x.

The indirect method of immunocytochemistry is more sensitive but requires more steps. Let us suppose that our objective is to detect protein x, present in rats. Before proceeding to the immunochemical reaction, two procedures are needed: (1) antibodies (polyclonal or monoclonal) to rat protein x must first be produced in an animal of another species (eg, a rabbit); (2) in a parallel procedure, immunoglobulin from a normal (noninjected) rabbit must be injected into an animal of a third species (eg, a goat). Rabbit immunoglobulins are considered foreign by a goat and are thus capable of inducing the production of an antibody (an antiantibody or antiimmunoglobulin) in that animal.

Indirect immunocytochemical detection is performed by initially incubating a section of a rat tissue believed to contain protein x with rabbit anti-x antibody. After washing, the tissue sections are incubated with labeled goat antibody against rabbit antibodies. The antiantibodies will bind to the rabbit antibody that had previously recognized protein x (Figure 1–24). Protein x can then be detected by using a microscopic technique appropriate for the label used in the secondary antibody. There are other indirect methods that involve the use of other intermediate molecules, such as the biotin-avidin technique.

Figure 1-24

Indirect method of immunocytochemistry. (1) Production of a primary polyclonal antibody. Protein x from a rat is injected into a rabbit. Several rabbit immunoglobulins (Ig) are produced against protein x. (2) Production of secondary antibody. Ig from a nonimmune (normal) rabbit is injected into a goat. Goat Igs against rabbit Ig are produced. The goat Igs are then isolated and tagged with a label. (3) First step of the immunocytochemical reaction. The rabbit Igs recognize and bind to different parts of protein x. (4) Second step of the immunocytochemical reaction. Labeled goat Igs recognize and bind to different parts of rabbit immunoglobulin molecules, therefore labeling protein x.

Medical Application

Immunocytochemistry has contributed significantly to research in cell biology and to the improvement of medical diagnostic procedures. Figures 1–25, 1–26, 1–27, and 1–28 show examples of immunocytochemical detection of molecules. Table 1–1 shows some of the routine applications of immunocytochemical procedures in clinical practice.

Table 1–1. Commonly Used Proteins (Antigens) Important for Immunocytochemical Diagnosis and Treatment of Disease.

Antigens Diagnosis Intermediate filament proteins  Cytokeratins Tumors of epithelial origin Glial fibrillary acid protein Tumors of some glial cells Vimentin Tumors of connective tissue Desmin Tumors of muscle Other proteins  Protein and polypeptide hormones Protein or polypeptide hormone–producing tumors Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract and breast Steroid hormone receptors Breast duct cell tumors Antigens produced by viruses Specific virus infections

Figure 1-25

Photomicrograph of a mouse decidual cell grown in vitro. The protein desmin, which forms intermediate filaments, was detected with an indirect immunofluorescence technique. A mesh of fluorescent intermediate filaments occupies most of the cytoplasm. The nucleus (N) is stained blue. High magnification. (Courtesy of FG Costa.)

 

Figure 1-26

Photomicrograph of a section of small intestine in which an antibody against the enzyme lysozyme was applied to demonstrate lysosomes in macrophages and Paneth cells. The brown color, indicating the presence of lysozyme, results from the reaction done to show peroxidase, which was linked to the secondary antibody. Nuclei were counterstained with hematoxylin. Medium magnification.

Figure 1-27

Carcinoembryonic antigen is a protein present in several malignant tumors mainly of the breast and intestines. This photomicrograph is an immunocytochemical demonstration of carcinoembryonic antigen in a section of large intestine adenocarcinoma. The antibody was labeled with peroxidase and the brown precipitate indicates tumor cells. The counterstain was hematoxylin. Medium magnification.

Figure 1–28

Electron micrograph showing a section of a pancreatic acinar cell that was incubated with antiamylase antibody and stained by protein A coupled with gold particles. Protein A has high affinity toward antibody molecules. The gold particles appear as very small black dots over the secretory granules. (Courtesy of M Bendayan.)

Hybridization Techniques

The central challenge in modern cell biology is to understand the workings of the cell in molecular detail. This goal requires techniques that permit analysis of the molecules involved in the process of information flow from DNA to protein. Many techniques are based on hybridization. Hybridization is the binding between two single strands of nucleic acids (DNA with DNA, RNA with RNA, or RNA with DNA) that recognize each other if the strands are complementary. The greater the similarities of the sequences, the more readily complementary strands form "hybrid" double-stranded molecules. Hybridization thus allows the specific identification of sequences of DNA or RNA.

In Situ Hybridization

When applied directly to cells and tissue sections, smears, or chromosomes of squashed mitotic cells, the technique is called in situ hybridization. This technique is ideal for determining if a cell has a specific sequence of DNA (such as a gene or part of a gene), for identifying the cells in which a specific gene is being transcribed, or for determining the localization of a gene in a specific chromosome. The DNA inside the cell must be initially denatured by heat or by denaturing agents so that both strands of the DNA separate. They are then ready to be hybridized with a segment of single-stranded DNA or RNA that is complementary to the sequence to be detected. This sequence is called a probe. The probe may be obtained by cloning, by polymerase chain reaction (PCR) amplification of the target sequence, or by synthesis if the desired sequence is short. The probe must be tagged with a label, usually a radioactive isotope (which can be localized by autoradiography) or a modified nucleotide (digoxygenin), which can be identified by immunocytochemistry.

In in situ hybridization, the tissue section, cultured cells, smears, or chromosomes of squashed mitotic cells must first be heated to separate the double strands of their DNA. A solution containing the probe is then placed over the specimen for a period of time necessary for hybridization. After washing off the excess probe, the localization of the bound probe is revealed through its label (Figure 1–29).

Figure 1-29

Tissue section of a benign epithelial tumor (condyloma) submitted to in situ hybridization. The brown areas are places where DNA of human papillomavirus type 2 is present. The counterstain was hematoxylin. Medium magnification. (Courtesy of JE Levi.)

Hybridization can also be performed with purified DNA or RNA in solid supports. Mixtures of DNA or RNA are separated by electrophoresis in an agarose gel or a polyacrylamide gel. After electrophoresis, the fragments of nucleic acids are transferred to a nylon or nitrocellulose sheet by solvent drag: a buffer flows through the gel and membrane by capillarity, carrying the nucleic acid molecules that bind strongly to the nylon or nitrocellulose sheet, where the nucleic acids can be further analyzed. This technique of DNA identification is called Southern blotting. When electrophoresis of RNA is performed, the technique is called Northern blotting.

Hybridization techniques are highly specific and are routinely used in research, clinical diagnosis, and forensic medicine.

References

Alberts B et al: Molecular Biology of the Cell, 3rd ed. Garland, 1994.

Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, 1990.

Cuello ACC: Immunocytochemistry. Wiley, 1983.

Darnell J, Lodish H, Baltimore D: Molecular Cell Biology, 2nd ed. Scientific American Books, 1990.

Hayat MA: Stains and Cytochemical Methods. Plenum, 1993.

James J: Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff, 1976.

Junqueira LCU et al: Differential staining of collagen types I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 1978;41:267. [PMID: 82432]

Meek GA: Practical Electron Microscopy for Biologists. Wiley, 1976.

Pease AGE: Histochemistry: Theoretical and Applied, 4th ed. Churchill Livingstone, 1980.

Rochow TG, Tucker PA: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Plenum, 1994.

Rogers AW: Techniques of Autoradiography, 3rd ed. Elsevier, 1979.

Rubbi CP: Light Microscopy. Essential Data. Wiley, 1994.

Spencer M: Fundamentals of Light Microscopy. Cambridge University Press, 1982.

Stoward PJ, Polak JM (editors): Histochemistry: The Widening Horizons of Its Applications in Biological Sciences. Wiley, 1981.

 

Histochemistry & Cytochemistry

The terms histochemistry and cytochemistry are used to indicate methods for localizing substances in tissue sections. Several procedures are used to obtain this type of information, most of them based on specific chemical reactions or on high-affinity interactions between macromolecules. These methods usually produce insoluble colored or electron-dense compounds that enable the localization of specific substances by means of light or electron microscopy.

Ions

Several ions (eg, calcium, iron, phosphate) have been localized in tissues with these methods, using chemical reactions that produce a dark insoluble product (Figure 1–16).

Figure 1-16

Photomicrograph of a bone section treated with a histochemical technique to demonstrate calcium ions. The dark precipitate indicates the presence of calcium phosphate in calcified bone and cartilage. Noncalcified cartilage tissue (stained in pink) is in the upper portion of the figure. Medium magnification. (Courtesy of PA Abrahamsohn.)

Nucleic Acids

DNA can be identified and quantified in cell nuclei using the Feulgen reaction, which produces a red color in DNA. DNA and RNA can also be analyzed by staining cells or tissue sections with a basic stain. 

Proteins

Although there are general methods to detect proteins in tissue sections, the histochemical methods usually do not permit identification of specific proteins in cells and tissues. Immunocytochemistry, presented later in this chapter, can do so.

Several histochemical methods, however, can be used to reveal, more or less specifically, a large group of proteins, the enzymes. These methods usually make use of the capacity of the enzymes to react with specific chemical bonds. Most histoenzymatic methods work in the following way: (1) tissue sections are immersed in a solution that contains the substrate of the enzyme intended for study; (2) the enzyme is allowed to act on its substrate; (3) at this stage or at a later stage in the method, the section is put in contact with a marker compound; (4) this compound reacts with a molecule that results from the degradation or transformation of the substrate; (5) the final reaction product, which must be insoluble and visible by light or electron microscopy, precipitates over the sites that contain the enzyme. When examining such a section in the microscope, it is possible to see the cells (or organelles) covered with a colored or electron-dense material.

Examples of enzymes that can be detected include the following:

Phosphatases are enzymes widely found in the body. They split the bond between a phosphate group and an alcohol residue of phosphorylated molecules. The colored insoluble reaction product of phosphatases is usually lead phosphate or lead sulfide. Alkaline phosphatases, which have their maximum activity at an alkaline pH, can be detected (Figure 1–17). Acid phosphatases are frequently used to demonstrate lysosomes, cytoplasmic organelles that contain acid phosphatase (Figure 1–18).

Figure 1-17

Photomicrograph of a rat kidney section treated by the Gomori method to demonstrate the enzyme alkaline phosphatase. The sites where this enzyme is present are covered by a black precipitate (arrows). Medium magnification.

Figure 1-18

Detection of acid phosphatase. Electron micrograph of a rat kidney cell showing three lysosomes (ly) close to the nucleus (N). The dark material on the lysosomes is lead phosphate that precipitated on places where acid phosphatase was present. (Courtesy of E Katchburian.)

Dehydrogenases remove hydrogen from one substrate and transfer it to another. There are many dehydrogenases in the body, and they play an important role in several metabolic processes. Dehydrogenases are detected histochemically by incubating nonfixed tissue sections in a substrate solution containing a molecule that receives hydrogen and precipitates as an insoluble colored compound. By this method, succinate dehydrogenase—a key enzyme in the citric acid (Krebs) cycle—can be localized in mitochondria.

Peroxidase, which is present in several types of cells, is an enzyme that promotes the oxidation of certain substrates with the transfer of hydrogen ions to hydrogen peroxide, forming molecules of water.

In this method, sections of adequately fixed tissue are incubated in a solution containing hydrogen peroxide and 3,3'-diaminoazobenzidine. The latter compound is oxidized in the presence of peroxidase, resulting in an insoluble, brown, electron-dense precipitate that permits the localization of peroxidase activity by light and electron microscopy. Peroxidase activity in blood cells, which is important in the diagnosis of leukemias, can be detected by this method.

Because peroxidase is extremely active and produces an appreciable amount of insoluble precipitate in a short time, it has also been used for an important practical application: tagging other compounds. Molecules of peroxidase can be purified, isolated, and coupled with another molecule. Later in this chapter, applications of tagging molecules with peroxidase are presented.

Polysaccharides & Oligosaccharides

Polysaccharides in the body occur either in a free state or combined with proteins and lipids. In the combined state, they constitute an extremely complex heterogeneous group. Polysaccharides can be demonstrated by the periodic acid–Schiff (PAS) reaction, which is based on the transformation into aldehyde of 1,2-glycol groups present in the sugar molecules. These aldehydes are then revealed by Schiff's reagent, which produces a purple or magenta color in areas of the section with an accumulation of polysaccharides.

A ubiquitous free polysaccharide in the body is glycogen, which can be demonstrated by the PAS reaction in liver, striated muscle, and other tissues where it accumulates.

Glycoproteins are protein molecules associated with small, branched chains of sugars (oligosaccharides). The protein chain predominates in weight and volume over the oligosaccharide chain. Because both glycogen and neutral glycoproteins are PAS positive, the specificity of the PAS reaction can be improved by comparing the staining of regular sections with that of sections pretreated with an enzyme that breaks glycogen (eg, amylase present in the saliva). Structures that stain intensely with the PAS reaction but do not stain after treatment with amylase contain glycogen. Figure 1–19 shows examples of structures stained by the PAS reaction.

Figure 1-19

Photomicrograph of an intestinal villus stained by PAS. Staining is intense in the cell surface brush border (arrows) and in the secretory product of goblet cells (G) because of their high content of polysaccharides. The counterstain was hematoxylin. High magnification.

Glycosaminoglycans are strongly anionic, unbranched long-chain polysaccharides containing aminated monosaccharides (amino sugars). The complex molecules formed by the attachment of glycosaminoglycan chains to a protein core constitute the proteoglycans. Some of the significant constituents of connective tissue matrices are proteoglycans (see Chapter 5: Connective Tissue and Chapter 7: Cartilage). Unlike the glycoproteins, the carbohydrate chains in proteoglycans constitute the major component of the molecule. Glycosaminoglycans and acidic glycoproteins are strongly anionic because of their high content of carboxyl and sulfate groups. For this reason, they react strongly with the alcian blue dye.

Lipids

Lipids are best revealed with dyes that are soluble in lipids. Frozen sections are immersed in alcohol solutions saturated with the dye. Sudan IV and Sudan black are the most commonly used dyes. The dye dissolves in the cellular lipid droplets, which become stained in red or black. Additional methods used for the localization of cholesterol and its esters, phospholipids, and glycolipids are useful in diagnosing metabolic diseases in which there are intracellular accumulations of different kinds of lipids.

Medical Application

Several histochemical procedures are frequently used in laboratory diagnosis of diseases that result in the storage of iron, glycogen, glycosaminoglycans, and other substances. Examples are Perls' reaction for iron (eg, hemochromatosis, hemosiderosis), the PAS-amylase reaction for glycogen (glycogenosis), alcian blue staining for glycosaminoglycans (mucopolysaccharidosis), and lipid staining (sphingolipidosis).

References

Alberts B et al: Molecular Biology of the Cell, 3rd ed. Garland, 1994.

Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, 1990.

Cuello ACC: Immunocytochemistry. Wiley, 1983.

Darnell J, Lodish H, Baltimore D: Molecular Cell Biology, 2nd ed. Scientific American Books, 1990.

Hayat MA: Stains and Cytochemical Methods. Plenum, 1993.

James J: Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff, 1976.

Junqueira LCU et al: Differential staining of collagen types I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 1978;41:267. [PMID: 82432]

Meek GA: Practical Electron Microscopy for Biologists. Wiley, 1976.

Pease AGE: Histochemistry: Theoretical and Applied, 4th ed. Churchill Livingstone, 1980.

Rochow TG, Tucker PA: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Plenum, 1994.

Rogers AW: Techniques of Autoradiography, 3rd ed. Elsevier, 1979.

Rubbi CP: Light Microscopy. Essential Data. Wiley, 1994.

Spencer M: Fundamentals of Light Microscopy. Cambridge University Press, 1982.

Stoward PJ, Polak JM (editors): Histochemistry: The Widening Horizons of Its Applications in Biological Sciences. Wiley, 1981.

Cell Fractionation

Organelles and other components of cells and tissues can be isolated by cell fractionation. This is the physical process by which centrifugal force is used to separate organelles and cellular components as a function of their sedimentation coefficients. The sedimentation coefficient of a particle depends on its size, form, and density and on the viscosity of the medium (Figure 1–14). The organelles obtained with these techniques can be analyzed for purity in the electron microscope (Figure 1–15), and their chemical composition and functions can be studied in vitro.

Figure 1–14

Cell fractionation allows the isolation of cell constituents by differential centrifugation. The drawings at the right show the cellular organelles at the bottom of each tube after centrifugation. Centrifugal force is expressed by g, which is equivalent to the force of gravity. (1) A fragment of tissue is minced with razor blades or scissors and dissociated with a homogenizer or by ultrasound. (2) The dissociated tissue is left standing for about 20 min. Clumps of cells and fibers of extracellular matrix precipitate to the bottom. (3) The supernatant is centrifuged at 1000 g for 20 min. Nuclei precipitate. (4) The supernatant is centrifuged at 10,000 g for 20 min. Mitochondria and lysosomes precipitate. (5) The supernatant is centrifuged at 105,000 g for 120 min. Microsomes precipitate. (6) If the supernatant is first treated with sodium deoxycholate and then centrifuged at 105,000 g for 120 min, the microsomes dissociate and precipitate separately as endoplasmic reticulum membranes and ribosomes. (Redrawn and reproduced, with permission, from Bloom W, Fawcett DW: A Textbook of Histology, 9th ed. Saunders, 1968.)

Figure 1–15

Electron micrographs of three cell fractions isolated by density gradient centrifugation. A: Mitochondrial fraction, contaminated with microsomes. B: Microsomal fraction. C: Lysosomal fraction. High magnifications. (Courtesy of P Baudhuin.)

References Alberts B et al: Molecular Biology of the Cell, 3rd ed. Garland, 1994. Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, 1990. Cuello ACC: Immunocytochemistry. Wiley, 1983. Darnell J, Lodish H, Baltimore D: Molecular Cell Biology, 2nd ed. Scientific American Books, 1990. Hayat MA: Stains and Cytochemical Methods. Plenum, 1993. James J: Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff, 1976. Junqueira LCU et al: Differential staining of collagen types I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 1978;41:267. [PMID: 82432] Meek GA: Practical Electron Microscopy for Biologists. Wiley, 1976. Pease AGE: Histochemistry: Theoretical and Applied, 4th ed. Churchill Livingstone, 1980. Rochow TG, Tucker PA: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Plenum, 1994. Rogers AW: Techniques of Autoradiography, 3rd ed. Elsevier, 1979. Rubbi CP: Light Microscopy. Essential Data. Wiley, 1994. Spencer M: Fundamentals of Light Microscopy. Cambridge University Press, 1982. Stoward PJ, Polak JM (editors): Histochemistry: The Widening Horizons of Its Applications in Biological Sciences. Wiley, 1981.

Cell & Tissue Culture

Live cells and tissues can be maintained and studied outside the body. In a complex organism, tissues and organs are formed by several kinds of cells. These cells are bathed in blood plasma, which contains hundreds of different molecules. Cell and tissue culture has been very helpful in isolating the effect of a single molecule on one type of cell or tissue. It also allows the direct observation of the behavior of living cells under a microscope. Several experiments that cannot be performed in the living animal can be reproduced in vitro.

The cells and tissues are grown in complex solutions of known composition (salts, amino acids, vitamins) to which serum components are frequently added. In preparing cultures from a tissue or organ, cells must be initially dispersed either mechanically or by treating the tissue with enzymes. Once isolated, the cells can be cultivated in a suspension or spread out on a Petri dish or glass slide, to which they adhere, usually as a single layer of cells (Figure 1–3). Cultures of cells that are isolated in this way are called primary cell cultures. Many cell types were once isolated in this way from normal or pathological tissue and have been maintained in vitro ever since because they have been immortalized and now constitute a permanent cell line. Most cells obtained from normal tissues have a finite, genetically programmed life span. Certain changes, however (mainly related to oncogenes; see Chapter 3: The Cell Nucleus), can promote cell immortality, a process called transformation, which may be a first step in transforming a normal cell into a cancer cell. Because of transformation and other improvements in culture technology, most cell types can now be maintained in the laboratory indefinitely. All procedures with living cells and tissues must be performed in a sterile area, using sterile solutions and equipment.

Medical Application

Cell culture has been widely used for the study of the metabolism of normal and cancerous cells and for the development of new drugs. This technique is also useful in the study of parasites that grow only within cells, such as viruses, mycoplasma, and some protozoa (Figure 1–13). In cytogenetic research, determination of human karyotypes (the number and morphology of an individual's chromosomes) is accomplished by the short-term cultivation of blood lymphocytes or of skin fibroblasts. By examining cells during mitotic division in tissue cultures, it is possible to detect anomalies in the number and morphology of the chromosomes that have been shown to be related and are diagnostic of numerous diseases collectively called genetic disorders. In addition, cell culture is central to contemporary techniques of molecular biology and recombinant DNA technology.

Figure 1–13

Photomicrograph of chicken fibroblasts that were grown in tissue culture and infected by the protozoan Trypanosoma cruzi. Although the borders of the cells are not readily visible, their nuclei (N) can be easily seen. Many trypanosomes are present within each cell (arrows). High magnification. (Courtesy of S Yoneda.)

References Alberts B et al: Molecular Biology of the Cell, 3rd ed. Garland, 1994. Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, 1990. Cuello ACC: Immunocytochemistry. Wiley, 1983. Darnell J, Lodish H, Baltimore D: Molecular Cell Biology, 2nd ed. Scientific American Books, 1990. Hayat MA: Stains and Cytochemical Methods. Plenum, 1993. James J: Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff, 1976. Junqueira LCU et al: Differential staining of collagen types I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 1978;41:267. [PMID: 82432] Meek GA: Practical Electron Microscopy for Biologists. Wiley, 1976. Pease AGE: Histochemistry: Theoretical and Applied, 4th ed. Churchill Livingstone, 1980. Rochow TG, Tucker PA: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Plenum, 1994. Rogers AW: Techniques of Autoradiography, 3rd ed. Elsevier, 1979. Rubbi CP: Light Microscopy. Essential Data. Wiley, 1994. Spencer M: Fundamentals of Light Microscopy. Cambridge University Press, 1982. Stoward PJ, Polak JM (editors): Histochemistry: The Widening Horizons of Its Applications in Biological Sciences. Wiley, 1981.

 

Autoradiography of Tissue Sections

Autoradiography is the study of biological events in tissue sections using radioactivity. Autoradiography permits the localization of radioactive substances in tissues by means of emitted radiation effects on photographic emulsions. Silver bromide crystals present in the emulsion act as microdetectors of radioactivity in the same way that they respond to light in common photography. The first step of autoradiography is to deliver a radioactive compound to the cells. A variety of molecules, including radioactive amino acids, radioactive nucleotides, and radioactive sugars, can be used, depending on the purpose of the study. These molecules are called precursors, because they may be used by the cells to synthesize larger molecules, such as proteins, nucleic acids, or polysaccharides and glycoproteins. The tissue sections are prepared and are covered with photographic emulsion. The slides are kept in light-proof boxes; after an adequate exposure time they are developed photographically and examined. When the silver bromide crystals present in the photographic emulsion are hit by radiation they are transformed into small black granules of metallic silver, thus revealing the existence of radioactivity in the tissue. The structures that contain radioactive molecules become covered by these granules. This procedure can be used in both light and electron microscopy (Figure 1–11).

Figure 1–11

Autoradiographs from the submandibular gland of a mouse injected with [³H] fucose 8 h before being killed. A: With a light microscope it is possible to observe black silver grains indicating radioactive regions in the cells. Most radioactivity is in the granules of the cells of the granular ducts of the gland. High magnification. B: The same tissue prepared for electron microscope autoradiography. The silver grains in this enlargement appear as coiled structures localized mainly over the granules (G) and in the gland lumen (L). High magnification. (Courtesy of TG Lima and A Haddad.)

Much information becomes available by localizing radioactivity in tissue components. Thus, if a radioactive amino acid is used, it is possible to know which cells in a tissue produce more protein and which cells produce less, because the number of silver granules formed over the cells is proportional to the intensity of protein synthesis. If a radioactive precursor of DNA (such as radioactive thymidine) is used, it is possible to know which cells in a tissue (and how many) are preparing to divide. Dynamic events may also be analyzed. For instance, to determine where in the cell a protein is produced, if it is secreted, and which path it follows in the cell before being secreted, several animals are injected with a radioactive amino acid and are killed at different times after the injection. Autoradiographs of the sections, taken at various times throughout the experiment, will show the migration of the radioactive proteins. To determine where new cells are produced in an organ and where they migrate, several animals are injected with radioactive thymidine and are killed at different times after the injection. Autoradiographs of the sections will show where the cells divide and where (or if) they migrate (Figure 1–12).

Figure 1–12

Autoradiographs of tissue sections from a mouse that was injected with [³H] thymidine 1 h before the organs were collected. Because the autoradiographs were exposed for a very long time, the radioactive nuclei became heavily labeled and appear covered by clouds of dark granules. A: Many cells were dividing at the base of the intestinal glands (arrowheads), but no cells were dividing along the villi (arrows). Low magnification. B: A section of a lymph node shows that cell division occurs mostly at the germinal centers of this structure (arrow). Low magnification. (Courtesy of TMT Zorn, M Soto-Suazo, CMR Pellegrini, and WE Stumpf.)

References

Alberts B et al: Molecular Biology of the Cell, 3rd ed. Garland, 1994.

Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, 1990.

Cuello ACC: Immunocytochemistry. Wiley, 1983.

Darnell J, Lodish H, Baltimore D: Molecular Cell Biology, 2nd ed. Scientific American Books, 1990.

Hayat MA: Stains and Cytochemical Methods. Plenum, 1993.

James J: Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff, 1976.

Junqueira LCU et al: Differential staining of collagen types I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 1978;41:267. [PMID: 82432]

Meek GA: Practical Electron Microscopy for Biologists. Wiley, 1976.

Pease AGE: Histochemistry: Theoretical and Applied, 4th ed. Churchill Livingstone, 1980.

Rochow TG, Tucker PA: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Plenum, 1994.

Rogers AW: Techniques of Autoradiography, 3rd ed. Elsevier, 1979.

Rubbi CP: Light Microscopy. Essential Data. Wiley, 1994.

Spencer M: Fundamentals of Light Microscopy. Cambridge University Press, 1982.

Stoward PJ, Polak JM (editors): Histochemistry: The Widening Horizons of Its Applications in Biological Sciences. Wiley, 1981.

Electron Microscopy

Transmission and scanning electron microscopes are based on the interaction between electrons and tissue components.

Transmission Electron Microscopy

The transmission electron microscope is an imaging system that theoretically permits very high resolution (0.1 nm) (Figure 1–8). In practice, however, the resolution obtained by most good instruments is around 3 nm. This high resolution allows magnifications of up to 400,000 times to be viewed with detail. Unfortunately, this level of magnification applies only to isolated molecules or particles. Very thin tissue sections can be observed with detail at magnifications of up to about 120,000 times.

Figure 1–8

Photograph of the JEM-1230 transmission electron microscope. (Courtesy of JEOL USA, Inc., Peabody, MA.)

The transmission electron microscope functions on the principle that a beam of electrons can be deflected by electromagnetic fields in a manner similar to light deflection in glass lenses. In the electron microscope, electrons are released by heating a very thin metallic (usually tungsten) filament (the cathode) in a vacuum. The electrons released are then submitted to a voltage difference of 60–120 kV between the cathode and the anode, which is a metallic plate with a hole in its center (Figure 1–9). Electrons are thus attracted to the anode and accelerated to high speeds. They pass through the central opening in the anode, forming a constant stream (or beam) of electrons that penetrates the tube of the microscope. The beam passes inside electric coils and is deflected in a way roughly analogous to what occurs in optical lenses, because electrons change their path when submitted to electromagnetic fields. For this reason, the electric coils of electron microscopes are called electromagnetic lenses.

Figure 1–9

 

Schematic view of a transmission electron microscope with its lenses and the pathway of the electrons. CCD, charged coupled device.

The configuration of the electron microscope is very similar to that of the optical microscope, although the optics of the electron microscope are usually placed upside down (Figure 1–9). The first lens is a condenser that focuses the beam of electrons on the section. Some electrons interact with atoms of the section and continue their course, whereas others simply cross the specimen without interacting. Most electrons reach the objective lens, which forms a magnified image that is then projected through other magnifying lenses. Because the human eye is not sensitive to electrons, the image is finally projected on a fluorescent screen or is registered by photographic plates or a charged coupled device camera. Because most of the image in the transmission electron microscope is produced by the balance between the electrons that hit the fluorescent screen (or the photographic plate) and the electrons that are retained in the tube of the microscope, the resulting image is always in black and white. Dark areas of an electron micrograph are usually called electron dense, whereas light areas are called electron lucent.

To provide a good interaction between the specimen and the electrons, electron microscopy requires very thin sections (40–90 nm); therefore, embedding is performed with a resin that becomes very hard. The blocks thus obtained are so hard that glass or diamond knives are usually necessary to section them. The extremely thin sections are collected on small metal grids and transferred to the interior of the microscope to be analyzed.

Freezing techniques allow the examination of tissues by electron microscopy without the need for fixation and embedding. There are fewer artifacts than with conventional tissue preparation, although the technique is usually arduous. Frozen tissues may be sectioned and submitted to cytochemistry or immunocytochemistry or may be fractured (cryofracture, freeze fracture) to reveal details of the internal structure of the membranes.

Scanning Electron Microscopy

Scanning electron microscopy permits pseudo-three-dimensional views of the surfaces of cells, tissues, and organs. This electron microscope produces a very narrow electron beam that is moved sequentially (scanned) from point to point across the specimen. Unlike the electrons in the transmission electron microscope, those in the scanning electron microscope do not pass through the specimen (Figure 1–10). The electron beam interacts with a very thin metal coating previously applied to the specimen and produces reflected or emitted electrons. These electrons are captured by a detector that transmits them to amplifiers and other devices so that in the end the signal is projected into a cathode ray tube (a monitor), resulting in a black-and-white image. The resulting photographs are easily understood, since they present a view that appears to be illuminated from above, just as our ordinary macroscopic world is filled with highlights and shadows caused by illumination from above. The scanning electron microscope shows only surface views. The inside of organs can be analyzed by freezing the organs and fracturing them to expose their internal surfaces. Examples of scanning electron microscopy can be seen in Figures 12–3 and 12–4.

Figure 1–10

Schematic view of a scanning electron microscope.

References Alberts B et al: Molecular Biology of the Cell, 3rd ed. Garland, 1994. Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, 1990. Cuello ACC: Immunocytochemistry. Wiley, 1983. Darnell J, Lodish H, Baltimore D: Molecular Cell Biology, 2nd ed. Scientific American Books, 1990. Hayat MA: Stains and Cytochemical Methods. Plenum, 1993. James J: Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff, 1976. Junqueira LCU et al: Differential staining of collagen types I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 1978;41:267. [PMID: 82432] Meek GA: Practical Electron Microscopy for Biologists. Wiley, 1976. Pease AGE: Histochemistry: Theoretical and Applied, 4th ed. Churchill Livingstone, 1980. Rochow TG, Tucker PA: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Plenum, 1994. Rogers AW: Techniques of Autoradiography, 3rd ed. Elsevier, 1979. Rubbi CP: Light Microscopy. Essential Data. Wiley, 1994. Spencer M: Fundamentals of Light Microscopy. Cambridge University Press, 1982. Stoward PJ, Polak JM (editors): Histochemistry: The Widening Horizons of Its Applications in Biological Sciences. Wiley, 1981.

Phase-Contrast Microscopy & Differential Interference Microscopy

Some optical arrangements allow the observation of unstained cells and tissue sections. Unstained biological specimens are usually transparent and difficult to view in detail, since all parts of the specimen have almost the same optical density. Phase-contrast microscopy, however, uses a lens system that produces visible images from transparent objects (Figure 1–3).

Figure 1–3

Cultured neural crest cells seen with different optical techniques. The cells are unstained, and the same cells appear in all photographs. Two pigmented cells are used for orientation in each image. A: Conventional light microscopy. B: Phase-contrast microscopy. C: Nomarski differential interference microscopy. High magnification. (Courtesy of S Rogers.)

Phase-contrast microscopy is based on the principle that light changes 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 relative to each other, which makes this kind of microscopy a powerful tool with which to observe living cells. Another way to observe unstained cells or tissue sections is Nomarski differential interference microscopy, which produces an apparently three-dimensional image (Figure 1–3).

Polarizing Microscopy

Polarizing microscopy allows structures made of highly organized molecules to be recognized. When normal light passes through a polarizing filter (such as a Polaroid), it exits vibrating in only one direction. If a second filter is placed in the microscope above the first one, with its main axis perpendicular to the first filter, no light passes through. If, however, tissue structures containing oriented molecules (such as cellulose, collagen, microtubules, and microfilaments) are located between the two polarizing filters, their repetitive, oriented molecular structure rotates the axis of the light emerging from the polarizer. Consequently, they appear as bright structures against a dark background (Figure 1–4). The ability to rotate the direction of vibration of polarized light is called birefringence and is a feature of crystalline substances or substances containing highly oriented molecules.

Figure 1–4

Polarized light microscopy. A small piece of rat mesentery was stained with the picrosirius method, which stains collagen fibers. The mesentery was then placed on the slide and observed by transparency. Under polarized light, collagen fibers exhibit intense birefringence and appear brilliant or yellow. Medium magnification.

Confocal Microscopy

The depth of focus in the regular light microscope is relatively long, especially when small magnification objectives are used. This means that a rather wide extent of the specimen is seen in focus simultaneously, causing superimposition of the image of a three-dimensional object. With confocal microscopy, on the other hand, only a very thin plane of the specimen is seen in focus at one time. There are two principles on which this is based: (1) the specimen is illuminated by a very small beam of light (whereas in the common light microscope, a large beam of light floods the specimen) and (2) the image collected from the specimen must pass through a small pinhole. The result is that only the image originating from the focused plane reaches the detector whereas the images in front of and behind this plane are blocked (Figure 1–5). The harmful glare of the out-of-focus objects is lost, and the definition of the focused object becomes better and allows the localization of any specimen component with much greater precision than in the common light microscope.

Figure 1–5

 

Principle of confocal microscopy. While a very small spot of light originating from one plane of the section crosses the pinhole and reaches the detector, rays originating from other planes are blocked by the plate. Thus, only one very thin plane of the specimen is focused at a time.

For practical reasons, the following arrangement is used in most confocal microscopes (Figure 1–6): (1) the illumination is provided by a laser source; (2) because it is a very small point, it must be moved over the specimen (scanned) to allow the observation of a larger area of the specimen; (3) the component of the specimen that is of interest must be labeled with a fluorescent molecule (meaning that a routine section cannot be studied); (4) the light that is reflected by the specimen is used to form an image; (5) because the reflected light is captured by a detector, the signal can be electronically enhanced to be seen in a monitor.

Figure 1–6

Practical arrangement of a confocal microscope. Light from a laser source hits the specimen and is reflected. A beam splitter directs the reflected light to a pinhole and a detector. Light from components of the specimen that are above or below the focused plane are blocked by the plate. The laser scans the specimen so that a larger area of the specimen can be observed.

Because only a very thin focal plane (also called an optical section) is focused at a time, it is possible to reunite several focused planes of one specimen and reconstruct them into a three-dimensional image. To accomplish the reconstruction and many of its other features, the confocal microscope depends on heavy computing capacity.

Fluorescence Microscopy When certain substances are irradiated by light of a certain wavelength, they emit light with a longer wavelength. This phenomenon is called fluorescence. In fluorescence microscopy, tissue sections are irradiated with either ultraviolet (UV) light or laser, and the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant or colored on a dark background. Fluorescent compounds that have an affinity for cell macromolecules may be used as fluorescent stains. Acridine orange, which can combine with DNA and RNA, is an example. When observed in the fluorescence microscope, the DNA–acridine orange complex emits a yellowish-green light, and the RNA–acridine orange complex emits a reddish-orange light. It is thus possible to identify and localize nucleic acids in the cells (Figure 1–7). Another important application of fluorescence microscopy is achieved by coupling fluorescent substances (such as fluorescein isothiocyanate, FITC) to molecules that will specifically bind to components of the tissues and will thus allow the identification of these components under the microscope (see Detection Methods Using High-Affinity Interactions between Molecules). Figure 1–7 Photomicrograph of kidney cells in culture, stained with acridine orange. Under a fluorescence microscope, DNA (within the nuclei) emits yellow light, and the RNA-rich cytoplasm appears reddish or orange. (Courtesy of A Geraldes and JMV Costa.)

References Alberts B et al: Molecular Biology of the Cell, 3rd ed. Garland, 1994. Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, 1990. Cuello ACC: Immunocytochemistry. Wiley, 1983. Darnell J, Lodish H, Baltimore D: Molecular Cell Biology, 2nd ed. Scientific American Books, 1990. Hayat MA: Stains and Cytochemical Methods. Plenum, 1993. James J: Light Microscopic Techniques in Biology and Medicine. Martinus Nijhoff, 1976. Junqueira LCU et al: Differential staining of collagen types I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn 1978;41:267. [PMID: 82432] Meek GA: Practical Electron Microscopy for Biologists. Wiley, 1976. Pease AGE: Histochemistry: Theoretical and Applied, 4th ed. Churchill Livingstone, 1980. Rochow TG, Tucker PA: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Plenum, 1994. Rogers AW: Techniques of Autoradiography, 3rd ed. Elsevier, 1979. Rubbi CP: Light Microscopy. Essential Data. Wiley, 1994. Spencer M: Fundamentals of Light Microscopy. Cambridge University Press, 1982. Stoward PJ, Polak JM (editors): Histochemistry: The Widening Horizons of Its Applications in Biological Sciences. Wiley, 1981.