Histology and Staining...reducing the effects of autolysis, diffusion of cell contents, and...

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16Histology and Staining

CONTENTS16.1 Introduction16.2 Fixation

Types of Fixative • Post-Fixation • Fixation Artifacts

16.3 Tissue ProcessingDehydration and Clearing • Embedding • Tissue Shrinkage

16.4 Cutting Microtomes • Microtome Blades • Cutting Metal/Tissue Composites

16.5 StainingImmunohistochemistry • In Situ Hybridization • Autoradiography

16.6 Mounting16.7 Microscopy

Resolution • Illumination • Fluorescence Microscopy • Confocal Microscopy • Electron Microscopy

References

16.1 Introduction

Histology is defined as “the science of organic tissue” although it is more commonly regarded as “thatbranch of biology or anatomy concerned with the minute structure of the tissues of plants or animals”(Shorter Oxford Dictionary, 1973). Although its origins have been ascribed to Aristotle, who distin-guished between tissues and organs, histology as a modern science began with the invention of thecompound microscope in about 1600 by Iansen and or Gallileo, and developed with the evolution ofmicroscopy. Nevertheless, many important observations using a single magnifying lens continued to bereported until early in the 18th century, notably by van Leeuwenhoek (1791). Perhaps the first mentionof tissue being composed of separate cells was by Robert Hooke (1665). For a detailed outline andchronology of the history of microscopy and histology see for instance (Kaiser, 1985).

Although it is possible to visualize microscopic structures in living tissue, the amount of informationobtainable is limited by the inability of visible light to penetrate most organisms beyond a depth of a millimeteror so. Consequently, two of the three main aims of practical histology are the preservation of dead tissue andthe cutting of it into slices thin enough to be transparent. Under these conditions, it is often difficult todistinguish different parts of the specimen because they will generally have closely similar refractive indices.Therefore, the third aim is the staining of the specimen to make its components and structure distinguishable.

The acquisition from a living tissue or organ of a magnified image in which the composite materialscan be identified and from which a pathologist can make a diagnosis or scientist can derive structuraland functional information usually follows a well-defined sequence of steps, summarized in Figure 16.1.The aim of this chapter is to expand briefly on the major steps of the sequence.

S.E. GreenwaldUniversity of London

A.G. BrownUniversity of London

© 2004 by CRC Press LLC


Ideally, tissue would be examined in three dimensions. This obviously requires transparency which,for optical microscopy, limits specimen thickness to approximately 600 µm. To study opaque materialor more deeply buried structures, the tissue must be sectioned and stained to produce the necessarycontrast. Using tomographic software, digital images from contiguous or serial sections can be combinedand a three-dimensional image reconstructed. In practice, however, three-dimensional reconstruction isnot commonly performed because processing the large number of sections required to produce a high-resolution image is extremely time consuming. Furthermore, small but cumulative errors in aligningsuccessive sections often result in distortion of the three-dimensional geometry and render measurementsof length and volume unreliable. During the last 20 years or so, advances in confocal microscopy (seebelow) have greatly reduced these problems, although structures more than 600 µm below the specimensurface cannot currently be visualized.

As an alternative approach used in cytological screening, where the pathologist is primarily interestedin changes in the internal structure of cells, a small sample obtained, for instance, by a needle biopsy is

FIGURE 16.1 Summary of the basic steps in histological processing, from living tissue to a stained section suitable for light microscopy. (Redrawn from Horobin, R.W., Histochemistry: An Explanatory Outline of Histochemistry andBiophysical Staining, Gustav Fischer Verlag, Stuttgart; Butterworth’s, London, 1982. With the permission of the authorand publishers.)



smeared into a thin layer onto a glass slide. After staining, these structures can be seen although infor-mation about the architecture of the tissue from which the cells are derived is lost.

16.2 Fixation

Unless it is to be examined very soon after death, nonliving tissue must be fixed. The aims of fixationand its effects are (Sanderson, 1994):

• To maintain geometry and dimension as close as possible to that of the living state.• To prevent autolysis (destruction by enzymes released by dead or dying cells) and putrefaction

(attack and digestion by microorganisms).• To make tissue receptive to staining by changing its chemical composition. Unfixed tissue has little

affinity for most histological stains. An additional benefit of the alteration in chemical structureis to produce differential changes in the refractive index of the tissue components thus improvingcontrast when examined unstained.

• To harden tissue sufficiently to make it more resistant to damage caused by subsequent processing,without making it difficult to cut.

Fixatives can be divided into a number of groups (see below) on the basis of their chemical structureand mode of action (Baker, 1958b), the most important of which stabilize proteins, usually by promotingchemical cross-linking between their molecules forming a gel or a harder polymer surrounding softercomponents. In this way the morphology of the tissue is stabilized making it easier to cut clean sections.Fixation is normally achieved by immersion in an aqueous solution or, for small fragile specimens, byexposure to vapor. The time taken to fix a tissue depends on the rate at which the fixative diffuses, thetemperature and the nature of the tissue itself (Hopwood, 1990). The following empirical relationshipbetween the distance penetrated by the fixative (d) and time (t) was derived by Medewar (1941) fornoncoagulating fixatives and by Baker (cited in Baker (1958b)) for fixatives that coagulate protein (i.e.,form cross-links).

(16.1)

The value of k (at room temperature) for formaldehyde (Baker, 1958b) is 0.06 mm sec–0.5 from whichit follows that it will take approximately 5 min for this fixative to penetrate 1 mm into a homogenouspiece of soft tissue and nearly 8 h to penetrate 10 mm.

In phase partition fixation the tissue is immersed in an aqueous solution of fixative in equilibriumwith an organic solvent (Nettleton and McAuliffe, 1986) (e.g., 50% glutaraldehyde/water in heptane).It is suitable for delicate tissues and has been used to fix and study mucus on the surface of the tracheathat would otherwise be dissolved by aqueous immersion (Sims et al., 1991).

To ensure rapid fixation of whole organs such as lung, heart, liver, etc., the fixative may be perfusedthrough the vasculature (Rostgaard et al., 1993). For reliable morphometric analysis of blood vessels themean pressure should be close to in vivo values (Berry et al., 1993).

16.2.1 Types of Fixative

16.2.1.1 Aldehydes

This group includes formaldehyde, the most commonly used fixative for light microscopy and glutaral-dehyde, favored for electron microscopy. Formaldehyde is usually used as formol saline, a 4% solution offormaldehyde gas in water containing 0.9% sodium chloride. It reacts with protein by a condensationreaction thereby forming cross-links frequently between lysine residues on the exterior of the proteinchains (see, for example, Baker (1958b) for a review of reaction mechanisms).

Fixation with glutaraldehyde often involving the formation of cross-links between pyridine residues(Hilger and Medan, 1987) greatly reduces the immunological activity of proteins, a process sometimes

d kt= 0 5.


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Biomedical Technology and Devices Handbook


referred to as denaturation, thus rendering them unsuitable for immunohistochemical staining (seebelow). On the other hand it disturbs tissue morphology less severely than formaldehyde and is thereforepreferred for investigations of cellular architecture. Acrolein, also an aldehyde, is often mixed withformaldehyde or glutaraldehyde. It has the advantage of rapid penetration and good preservation ofmorphology (Saito and Keino, 1976).

16.2.1.2 Oxidizing Agents

The most commonly used of this group, which includes potassium dichromate and potassium perman-ganate, is osmium tetroxide. The reactions of oxidizing agents with different types of tissue have beenreviewed by Baker (1958b) and more recently by Kiernan (1990). With proteins, osmium tetroxide isthought to form cross-links. It is soluble in some lipids and is also reduced to the black dioxide by them,which then take on this color and become crumbly, making sections difficult to cut. In spite of thesedrawbacks and its slow rate of penetration (Baker, 1958a), osmium tetroxide “preserves the structure ofthe living cell better than any other primary or mixed fixative” (Baker, 1958b) and for this reason is usedextensively both as a fixative and as a stain in electron microscopy. Dissolved in a nonaqueous fluoro-carbon solvent (FC-72), osmium tetraoxide has been used to demonstrate the ultrastructure of theglycocalyx, the fragile coating of vascular endothelial cells (Sims and Horne, 1994), and used alone inperfused brain tissue for both light and electron microscopy (Branchereau et al., 1995).

16.2.1.3 Alcohols and Acetone

Wine has been used as a preservative since antiquity and, according to Baker (1958b), was first used asa preservative for anatomical purposes in 1663 by Robert Boyle. Alcohols and ketones displace waterfrom protein and dehydrate the tissue as a whole, causing it to harden and shrink. Their main advantageis rapid penetration and, when used at low temperature, preservation of enzymatic and immunologicalactivity (Sato et al., 1986). Ethanol, in particular, is the fixative of choice when transporting specimensby air, as the airlines have little objection to their customers carrying large quantities of this materialwhile remaining suspicious of other toxic chemicals.

16.2.1.4 Other Cross-Linking Agents

This group includes the carbodiimides introduced in 1971 (Kendall et al., 1971) and is seeing increasinguse in light and electron microscopy due to their speed of action and specificity (Tymianski et al., 1997),both as a mixture with glutaraldehyde (Willingham and Yamada, 1979) and alone (Panula et al., 1988).They have recently been shown to be suitable for denaturing heart valve tissue prior to implantation asthey can easily be washed out and leave little or no toxic residues (Girardot and Girardot, 1996).

16.2.4.5 Heat

Heat alone will cause many proteins to coagulate. Microwaves usually in conjunction with fixatives allowrapid heating although the degree of fixation tends to be nonuniform with the center of the specimen lesswell fixed (Leong and Duncis, 1986). For large specimens such as whole organs, preliminary fixation byimmersion in 0.9% saline and irradiation to a temperature of 68 to 74∞C is recommended. The process iscompleted by irradiating 2- to 3-mm pieces to a temperature pf 50 to 68∞C for about 2 min (Leong, 1994).In conjunction with formaldehyde or glutaraldehyde, fixation times as little as 10 sec can be achieved thusreducing the effects of autolysis, diffusion of cell contents, and retaining ultrastructural detail (Leong et al.,1985; Login and Dvorak, 1985). Among other applications in the histology laboratory, microwave irradiationhas been used to preserve immunological activity (Leong et al., 1988), improve the quality of frozen sections,and enhance the staining of ultrathin sections for electron microscopy (EM). Technical details are discussedat some length by Leong (1994) and general reviews may be found in Leong (1988, 1993).

16.2.1.6 Unknown Mechanism

Although fixatives have been in use for many decades, the mode of action of several standard fixativesis not well understood. These include mercuric chloride which penetrates tissue rapidly and coagulatesproteins by reacting with many different amino acid residues (Hopwood, 1990), and picric acid which,


Histology and Staining

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in conjunction with other fixatives such as ethanol or formalin, also coagulates protein while leaving thetissue soft (Leong, 1994).

16.2.2 Post-Fixation

It has been found that staining intensity can be improved by following a period of primary fixation informalin, for example, by a few hours of additional treatment with mercuric chloride (Leong, 1994).Sections are said to be easier to cut and to flatten more readily when post-fixed (Hopwood, 1990). Stainingof cell membranes in samples fixed in glutaraldehyde for electron microscopy is improved by post-fixationwith osmium tetroxide. Blocks destined for both light and electron microscopic investigation can bepost-fixed with 10% formalin following standard glutaraldehyde treatment, which has the additionaladvantage of retaining intracellular structure (Tandler, 1990).

16.2.3 Fixation Artifacts

Tissue volume may change during fixation although the effects are small compared to shrinkage duringembedding. They are discussed in the next section. Specimens that have been stored in formalin forextended periods under acidic conditions become suffused with fine brown crystals of formalin pigment,which is thought to be formed by the reaction of formalin with hematin derived from the hemoglobinof ruptured red blood cells (Baker, 1958b). Its formation is inhibited by the addition of 2% phenol toformalin. If it has formed, it may be removed by brief treatment with a saturated solution of picric acidin ethanol followed by washing in water (Drury and Wallington, 1980).

Although fixation normally stiffens the tissue as well as removing water, highly mobile inorganic ions aswell as large molecules such as hemoglobin (Reale and Luciano, 1970) may diffuse through the specimentoward its periphery as it fixes, giving a spurious view of their localization and distribution. In metabolicstudies of living tissue, the uptake of radioactively labeled amino acids or sugars may be studied postmortemby assessing the distribution of the label in the tissue. However, glutaraldehyde binds strongly to many ofthese substances and therefore their distribution in fixed tissue assessed microscopically by autoradiography(see below) may be altered. Finally, some tissue components, for example, lipids and mucopolysaccharides,do not react with commonly used fixatives and may be lost from the tissue during subsequent processing.

16.3 Tissue Processing

16.3.1 Dehydration and Clearing

Following fixation, tissue must be processed to render it suitable for embedding and subsequent sectioning.Paraffin wax, the most common embedding medium, is hydrophobic, whereas most fixatives are aqueous.Therefore, the material must first be dehydrated and then infiltrated with a solvent that is miscible withthe embedding medium. Typically, the specimen is soaked initially in an aqueous solution of an alcoholand transferred at intervals through successively more concentrated solutions until no more waterremains. The processing continues with the replacement of the alcohol by a transfer agent or transitionmedium (most commonly xylene or toluene) which is both miscible with alcohol and a solvent forparaffin wax, the embedding agent. Finally, the xylene is replaced by molten wax resulting in completeinfiltration of the tissue. Typically, the entire process under automatic control takes around 16 h (Gordon,1990), although small specimens, e.g., needle biopsies, may be processed in 3 h. Dehydration with ethanoland treatment with xylene usually cause hardening, which may be reduced by using n-butanol; and lossof lipids, which is minimized by rapid treatment.

16.3.2 Embedding

Ideally, the embedding medium should be of approximately equal density and resilience to the tissue toallow cutting of sections with minimal damage. The most commonly used medium is paraffin wax


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because it is easy to cut, cheap, and widely available. Sections thinner than 2 µm, which are needed toexamine details of intracellular structure, require a tougher medium such as a plastic resin (see below),from which sections as thin as 0.2 µm can be cut. However, this requires harder and sharper knives madeof tungsten carbide or glass (Bennett et al., 1976).

The tissue permeated with embedding medium is placed in a mold, orientated for subsequent sec-tioning (Hilger and Medan, 1987; Arnolds, 1978). The mold is filled with the medium and allowed tocool, usually under automatic control.

Other embedding media with lower melting points, such as polyethylene glycol or polyester waxes,can be used when excessive heat may reduce immunological activity of proteins that are to be stained byantibodies (see below). For hard and tough specimens or those that contain tissues with differing degreesof hardness, cellulose nitrate has traditionally been used. However, this material is chemically hazardousand is not suitable for sections less than 10-µm thick.

During the last 50 years plastic resins have been developed for use as embedding media driven initiallyby the development of the electron microscope and the consequent need for a material which couldproduce sections no thicker than 80 nm and able to withstand temperatures to 200∞C (Nunn, 1970).Plastic resins are tougher than paraffin wax and therefore make it possible to cut thinner sections andharder tissues with less damage to the section. They also cause less shrinkage during processing thanwaxes. The three main types are briefly compared in Table 16.1.

16.3.3 Tissue Shrinkage

Most fixatives cause tissue to shrink, largely due to their dehydrating effect, although acidic fixativesincluding formalin lead, at least initially, to swelling driven by osmotic pressure (Baker, 1958a). However,as fixation and dehydration proceed, shrinkage inevitably occurs, different tissues being affected todifferent extents. The early literature (reviewed by Baker [1958b]) reports quite variable results on theeffects of dehydration on different tissues. More recent reviews (see, for example, Fox et al. [1985])andmore modern studies agree that fixation and dehydration account for a reduction in volume of between3 and 6% for whole organs (Iwadare et al., 1984) and up to 30% for individual cells (Ross, 1953). Removalof fixative by treatment with transition medium prior to embedding causes a further reduction of around10% (with respect to the original volume). Finally, embedding in wax results in an overall reduction involume of no less than 35%. Resin shrinks less than wax during the embedding process and, whensectioned and dried, may stretch. Overall, tissues in resin sections are nearer to their living dimensions

TABLE 16.1 Summary of the Properties of Embedding Resins

Material Advantages Drawbacks

Epoxy (Spurr, 1969) Tough, thin sections possibleCan be softened with plasticizers for easier

sectioningShrinkage ª3%

Curing temperature 60∞C, may cause tissue damage

Hydrophobic, therefore compatible with limited range of stains

Acrylic (Murray, 1988; Litwin, 1985)

Low viscosity, easier processingMonomer water miscible; polymer water

permeable; dehydration not necessary, compatible with many stains

Shrinkage ª15%

Polyester Low viscosity, easier processingSome can be polymerized at low temp with

UV light, therefore suitable for immunological staining (Altman et al., 1984)

Controllable hardness to match tissue; more water compatible than epoxy, less than acrylic

Tolerant of electron beam; suitable for electron microscopy

Small specimens only due to limited penetration of UV for curing; non-UV-curable have curing temperature



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than those embedded in wax, although factors such as temperature and section thickness affect the overalldegree of volume change (Hanstede and Gerrits, 1983). In a study of arterial sections, Dobrin (1996)has shown that formalin fixation followed by dehydration and embedding in paraffin wax leads to areduction in cross-sectional area of 19%. Fixation in McDowell’s solution (recipe given in Dobrin [1996])followed by embedding in glycol methacrylate (which avoids the need to further dehydrate the tissue)resulted in an overall increase in cross-sectional area as little as 4%. This procedure appears to be optimalat least for vascular histomorphometry. Clearly, when measuring absolute dimensions of cells or othertissue components careful assessment of shrinkage is essential.

16.4 Cutting

16.4.1 Microtomes

The purpose of the microtome is to cut sections of known and uniform thickness through tissue, whichis surrounded by and infused with embedding medium, in the form of a cuboidal or cylindrical block.At its most basic, the microtome consists of a chuck to hold the tissue block near to a blade that is passedover the specimen and that removes thin slices in the manner of a carpenter’s plane. A later developmentallows the specimen to advance toward the blade, as each slice is removed, by rotating a lead screwattached to the chuck (Figure 16.2a). This type of rocking blade device was introduced in 1881 (Kaiser,1985). In its modern form, the base sledge microtome (Figure 16.2b), the chuck and lead screw assembly,in a heavy casting moving on runners within a massive frame, is passed repeatedly under a fixed blade.Each pass of the specimen causes the lead screw to advance by a preset amount. The inertia of the rockingmechanism, which is usually driven by hand, minimizes chattering between the blade and the block, andconsequent tearing of the specimen. The base sledge microtome is largely confined to the cutting of waxembedded sections. As the sections are cut, they are pushed onto the upper surface of the blade wherethey collect as a delicate ribbon of crinkled sections. From time to time these are carefully picked up andfloated onto water held at some 10∞C below the melting point of the wax. The softened wax is stretchedby surface tension and the flattened section is transferred to a glass slide by passing the slide underneaththe section, to which it adheres by surface tension, and lifting it out of the water (Figure 16.3). Thin resinsections suitable for electron microscopy may be flattened by exposure to xylene or chloroform vaporand picked up onto a fine metal grid rather than a glass slide (Nunn, 1970). Slides with sections containingelastin such as blood vessels, skin and lung tissue, which retain some resilience even after prolongedfixation and which tend to curl at the edges, are placed on a hot plate that softens the wax further andallows greater stretching. These processes require considerable manual dexterity and training. The tricksof the trade may be found in practical texts such as Bancroft and Stevens (1990) and Sanderson (1994).

In the rotary microtome, the chuck and embedded specimen block are moved against a blade in areciprocating motion by a crank connected to a motor or manually driven flywheel. These widely useddevices can cut sections varying in thickness from 0.5 to 25 µm in wax and resins and lend themselveswell to automated section cutting (Vincent, 1991).

Sections once picked up onto the slides are placed in an incubator at just below the melting point ofthe wax for about 30 min. This dries the slide and increases the adhesion between section and glass.Finally, they are soaked in xylene and, if they are to be stained with an aqueous dye, are rehydrated withaqueous ethanol and water. (If alcoholic dyes are to be used, the rehydration stage is omitted.)

Fixed or unfixed tissue can be hardened by freezing and cut in a freezing microtome designed or modifiedto maintain the specimen at a low temperature (–20 to –40∞C). This allows the rapid assessment ofbiopsies obtained during a surgical procedure so that the surgeon’s subsequent decisions may then bebased on the pathologist’s diagnosis. Frozen sections are also used for investigating tissues that are proneto rapid enzymatic degradation (e.g., liver, central nervous system), or that contain diffusible or solublematerials such as lipid.

In the vibrating blade microtome, or vibratome, a thin razor-like blade oscillating at main frequencyis advanced across the specimen glued to a stub, held in a chuck, and immersed in water, saline, or fixative


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to prevent undue heating. The amplitude of the vibration and the speed at which the blade advancesthrough the specimen can be adjusted to suit different types of tissue (Zelander and Kirkeby, 1978; Salleeand Russell, 1993). The major advantage of these devices is that they will cut cleanly through unfixedand unembedded tissue at room temperature. Their major drawback is that unprocessed specimens lessthan 20-µm thick cannot be cut because they tend to disintegrate.

16.4.2 Microtome Blades

The profile and sharpness of the blade is of critical importance in maintaining a clean cut and uniformsection thickness. In paraffin sections under optimal conditions, the thickness of adjacent sections canvary by 10% in a standard 5-µm section and by as much as 50% in 1-µm sections. In tests on typicalsections of nominal thickness 5 µm, although the mean measured thickness was 4.8 µm (SD 0.14 µm),individual sections varied between 3 and 7 µm. In thinner sections (nominal thickness 1 µm), the variation

FIGURE 16.2 (Upper panel) “Cambridge Rocker” microtome. This instrument, built in the late 19th century, was in routine use until the 1930s. The spring-loaded handle (A) is pulled toward the operator causing arm (B) to liftthe specimen embedded in a wax block (E) above the edge of the blade (F). At the same time a pawl engages in thetoothed wheel causing and the attached threaded rod to rotate lifting arm (D) and advancing the specimen towardthe blade by a set amount which determines the section thickness. On releasing the handle, the specimen is drawndown over the blade, thus producing a thin slice that rides up onto the blade. (Lower panel) Base sledge microtome.



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was as much as 50% (Merriam, 1957; Helander, 1983). Thickness variation in resin sections that areharder and more homogenous than wax is, however, considerably smaller (Helander, 1983). Nevertheless,thickness variation must be carefully assessed, especially in studies involving three-dimensional recon-struction from serial sections (Bibb et al., 1993).

Microtome blades are typically made of high carbon steel and commonly have a plane wedge-shapedprofile with a raked edge, although other profiles are used for particularly hard or soft materials (Ellis,1994). Sharpening is a highly skilled procedure, details of which may be found in standard practicalhistology texts (Ellis, 1994; Sanderson, 1994). For harder materials such as teeth and bone, a cutting edgeof sintered tungsten carbide is bonded to a steel body.

For very thin sections of hard material, glass knives made by fracturing a glass block under controlledconditions (Reid and Beesley, 1991) are suitable. They can be used in a base sledge, rotating blade, orspecialized power-driven microtomes (for example, the Ultramicrotome, Dupont Biomedical ProductsDivision, Wilmington, Delaware) designed to cut sections from hard resin blocks as thin as 10 nm,suitable for electron microscopy. In this device, section thickness is controlled by advancing the blockby a stepper motor under microprocessor control. Diamond knives may be used in place of glass withthe advantage of increased sharpness and durability. The drawbacks are high cost and a cutting widthlimited to 4 mm.

16.4.3 Cutting Metal/Tissue Composites

When studying the pathology of the interaction between living tissue and metal implants such as jointprostheses or intravascular stents, it is frequently necessary to cut sections containing soft tissue inproximity to metals such as stainless steel or titanium (Figure 16.4). A standard technique developed todeal with these problems involves the following steps (Donath, 1985):

• Embed fixed specimen in resin to form a cylindrical block.• Cut a disk (say, 5 mm in thickness) from the cylindrical block using a diamond-coated band saw.• Glue disk to plastic base plate, typically with epoxy resin.• Remove cutting marks by grinding one face of the disk flat using graded grinding paste and polish.

FIGURE 16.3 Sections cut from a wax block floating on water and flattened by surface tension. A single slice hasbeen lifted onto a glass slide.


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• Glue plastic slide to polished face of the disk.• Cut the sandwich formed by the base plate, the disk, and the plastic slide with the band saw so

that the distance between the plastic slide and the cut edge is slightly greater than the desiredsection thickness.

• Grind the cut face until the desired section thickness is achieved and polish.• Glue cover slip over the exposed polished face of the disk.

This elaborate and time-consuming process minimizes the risk of crushing the soft tissue or tearingthe metal out of the section while preserving its geometry and producing a section of known thicknessand good optical quality. Sections as thin as 25 µm may be reliably produced.

16.5 Staining

A biological stain has been defined as “a dye for making biological objects more clearly visible thanthey would be unstained” (Lillie, 1969). Initially, all dyes were of natural origin, obtained by extractionfrom plants such as crocus (saffron) (Leeuwenhoek, 1791) cited in Baker (1958b), the tree haemotoxyloncampechianum (hemotoxylin) (Waldeyer, 1863) and the cochineal beetle (carmine) (Goppert andCohn, 1849), the latter being the first systematically to study dyed tissues with the microscope although,as mentioned above, von Leeuwenhoek certainly employed dyes. The early history of histologicalstaining has been briefly reviewed by Conn (1969) and Baker (1958b) and in more detail by, forinstance, Lewis (1942).

The term staining is generally used to include any method of coloring tissue. This may be achieved byusing a dye containing a chemical group (or groups) that binds with reactive sites in the tissue. Thisprocess is sometimes referred to as dyeing (Kiernan, 1990). Alternatively, the tissue may be allowed toabsorb a solution of a coloring agent that remains in the tissue when the solvent evaporates in the mannerof a coffee stain on a tablecloth.

Baker (1958b) defines dyes as “aromatic, salt-like, crystalline solids, that dissolve in aqueous solutionsin the form of coloured ions which can attach themselves chemically to tissue components. When theattachment takes place they do not lose or change colour.” This raises two questions: What makes ionscolored? And how do they attach to tissues?

FIGURE 16.4 (See color insert following page 14-10.) Section of the femoral head and adjoining stainless steelimplant. Note the “clean” interface between the tissue and the implant. Elliptical areas contain osteoclasts andosteoblasts, cells involved in the growth and remodeling of bone. (With thanks to Professor P. Revell.)



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The majority of dyes used in histological staining are organic molecules containing conjugateddouble bonds consisting of electronic orbitals delocalized over several atoms (a chromogen) and polarresidues which enable them to form ionic bonds with polar molecules in the tissue (auxochromes).The energy required to raise electrons in the delocalized orbitals to an excited state often correspondsto frequencies in the visible part of the EM spectrum, and most organic compounds with conjugatedbonds (for instance, quinoids in which two of the hydrogen atoms on the benzene ring are replacedby an oxygen) are therefore colored.

There are many types of chemical bonds formed between dye and tissue. For instance, acidic dyes arecommonly used to stain basic components in cellular cytoplasm and collagen in connective tissue; whereasbasic dyes are more suitable for nucleic acids in the cell nucleus and other acidic moieties such asphospholipids or mucins. Neutral or amphoteric dyes such as hematoxylin require the presence of amordant, usually a metal ion, which has the effect of making the amphoteric dye basic, thus strengtheningthe bond between dye and tissue. Mordants in general are able to bond chemically both with the tissueand the dye and are often used to ensure that the dye does not leach out during subsequent treatmentsuch as using a second dye to stain another tissue component.

Perhaps the most frequently used coloring process in histology combines the dyes hematoxylin which,ideally, stains cell nuclei blue and eosin, the “counterstain,” which is taken up by the cytoplasm, renderingit red or pink (Figure 16.5).

Polychrome stains result when three or more dyes are applied sequentially to the same section to givea multicolored preparation. The classic trichrome technique described by Masson (Masson, 1929) givesstriking results, wherein the connective tissue protein collagen stains blue, while muscle cells are red(Figure 16.6), which also shows the results of the MSB stain. In Johansen’s quadruple stain for planttissue, for example, parasitic fungi stain green, cytoplasm stains orange, cellulose appears as a yellowishgreen, and lignin takes on a red color (Johansen, 1939). Polychrome stains have been reviewed by Cullinget al. (1985).

The rubber-like protein elastin, which is unusually hydrophobic, is effectively stained by water-solubledyes such as Orcein or acid fuchsin which contain hydrophobic groups surrounded by molecular clustersof water stabilized by hydrogen bonding. A similar process takes place when the dye congo red is usedto demonstrate the presence of amyloid, a protein formed in the brain and other organs as a result of

FIGURE 16.5 (See color insert following page 14-10.) Section through the lining of the small intestine stained with hematoxylin and eosin.


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degenerative disease. When the hydrophobic groups in the dye and on the protein come together, thewater clusters are destabilized and the entropy of the system is increased. This process has been termedhydrophobic bonding. It and other types of chemical bonding between dye and tissue are summarizedin Horobin (1988, 1990).

In wax-embedded material, the wax is dissolved away after mounting the section on the slide and thetissue can then be exposed to a wide variety of aqueous or nonaqueous dye solutions. Resin-embeddedmaterial normally cannot be removed in this way, thus the variety of dyes is limited to those that arecompatible with the resin used. Nevertheless, the number of resin-compatible dyes continues to increaseand several polychrome varieties have been developed (see, for example, Johansen [1939] and Scala etal. [1993]). Acrylic resins on the other hand, being water soluble do not suffer from this incompatibilitywith aqueous dyes.

Other methods of coloring tissue include:

FIGURE 16.6 (See color insert following page 14-10.) (Upper panel) Transverse section of the tongue treated with Masson’s trichrome stain, showing muscle in red, connective tissue in blue/green, and cell nuclei in purple. (Lowerpanel) Section of placenta stained with MSB. With this stain, fibrin is red, other connective tissue is blue, and redblood cells are yellow.



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• The staining of lipid by nonpolar dyes such as Sudan Red dissolved in nonaqueous solvents(e.g., iso-propanol). The degree of uptake depends on the relative solubility of the dye in solventand tissue.

• Metallic impregnation used in electron microscopy to increase electron density.• The Gram stain (see, for example, Lillie [1965]) which exploits the difference between types of

bacteria in the solubility of trapped dye molecules. Gram-positive bacteria retain the dye aftertreatment with iodine and appear blue, whereas Gram-negative organisms appear colorless andmay then be visualized by treatment with a counterstain, usually neutral red, the background.The presence or absence of this stain together with the shape of the bacterium provides thepathologist with a simple method of classifying microorganisms found in infectious diseasesand infected wounds.

• Vital staining in which a dye is taken up by living cells — for example, acridine orange stainsintracellular DNA and connective tissue green, and intracellular RNA, red/orange. The dyes canbe introduced via the vasculature, injected directly into muscle or, for freshly obtained biopsies,by immersion. Such stains are of particular value to the pathologist to demonstrate cell functionin biopsies (Aschoff et al., 1982; Foskett and Grinstein, 1990).

16.5.1 Immunohistochemistry

A major problem with most staining techniques involving dyes is that staining intensity and even colorare variable and interpretation depends on the knowledge and skill of the observer. The introduction ofimmunocytochemistry in the 1950s (Coons and Kaplan, 1950; Coons et al., 1955) largely overcame this,while at the same time introducing a different set of problems. In essence immunocytochemistry relieson the highly specific reaction between an applied antibody and the tissue constituent to which it binds(antigen). In order to visualize the point of reaction, the antibody must carry a molecule (label) thatenables a colored end product to be formed. Originally, these labels were enzymes, which were thendemonstrated by standard histochemical staining techniques already in use for the visualization ofenzymes in tissue. Of those originally tried out, peroxidase and alkaline phosphatase have survived thetest of time and are currently the most favored in routine use. In addition to enzymes, which remain themost commonly used labels for light microscopy, fluorescent dyes, colloidal metals or radioactive isotopesare used (Polak and Van Noorden, 1984).

Initially, a single labeled primary antibody was used in what has come to be known as the directtechnique. This method had the advantage that it was quick to carry out, and nonspecific reactions wereminimized as only the single antibody was used. However, it suffered from the fact that each antigenicsite had only one colored molecule attached to it giving little signal amplification. Through a number ofintermediate methods, the avidin-biotin complex (ABC) techniques most favored today were developed.These rely on the fact that avidin has four binding sites available for reaction with biotin with which ithas a high affinity. Biotin can also be coupled to an antibody as well as an enzyme label such as peroxidase.

The first step in carrying out the ABC technique is to apply an unlabeled primary antibody to theantigen of interest. Step two is to apply a secondary antibody labeled with biotin that reacts with theprimary. For example, if the first antibody was raised in a rabbit (rabbit anti-antigen), the secondantibody could be biotinylated mouse anti-rabbit. The reason for using a secondary antibody ratherthan directly labeling the primary is that the general technique can then be applied to any antibody,no matter which animal it was raised in, simply by changing the secondary antibody species. The thirdstep is to apply a preformed complex of avidin and peroxidase-labeled biotin. This is prepared suchthat not all of the biotin binding sites on the avidin are occupied. These free sites are able to join tothe biotin of the secondary antibody and also to other avidin-biotin complex molecules. In this waythe number of peroxidase molecules available for color formation is greatly increased providing therequired signal amplification.

One of the major problems with the demonstration of antigenic sites is that they must be well preservedby the fixation process, but also be available for demonstration. This contradiction can be overcome by


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a number of poorly understood treatments grouped under the heading of antigen unmasking. Initially,enzymatic treatment was employed using enzymes such as trypsin and pronase. These are applied at thestart of the technique under conditions that free the antigenic sites without destroying the structure ofthe remaining tissue. Enzymatic treatment has largely been replaced by heat-mediated antigen retrievaltechniques where the sections are heated in the presence of heavy metal salts or, more often, buffers suchas citrate at pH 6.0. Heating is carried out using microwaves or by boiling in a pressure cooker orautoclave. This would seem to be a very harsh treatment but it would appear that antigens survive betterusing these treatments than when unmasked by enzymes.

Great care must be taken when employing unmasking techniques as many antigens may be destroyedand larger proteins may be broken down to reveal molecules displaying antigenic sites leading to falsenegative and positive results, respectively. It is therefore very important when introducing a new antibodyin to the laboratory that adequate tests are carried out using tissues known to contain the antigen forinvestigation and other tissues in which the antibody is lacking.

16.5.2 In Situ Hybridization

A further development of the principles of immunocytochemistry has been the introduction of in situhybridization (ISH) for the demonstration of DNA and RNA. This technique relies upon the hybridizationof labeled single-stranded fragments of nucleic acid (probes) to complementary strands of nucleic acidlocated within the tissue sample. This has the distinct advantage over other methods of nucleic aciddemonstration in that it is the only technique that allows their localization to specific cells within tissues.As with immunocytochemistry, the major problem with this technique is that the nucleic acids are maskedin a complex matrix of other tissue elements which have been cross-linked by the process of fixation. Inaddition, DNA is already masked by being double stranded, and one of these strands must be removedbefore hybridization can occur. As with immunocytochemistry, careful fixation preserves more of thenucleic acid of interest and gives better morphology, but also decreases the accessibility of the probe tothe tissue. Similarly, mild protease treatment is employed, commonly proteinase K, and again the extentof this treatment must be determined in a series of trial runs.

Probe selection is an important step in carrying out successful hybridization and a number of differentprobes are available to suite particular circumstances. Double-stranded DNA probes are available thatcontain both complementary strands that have been labeled. As there is no way of controlling which ofthe two strands will be removed from the tissue by the pretreatment, double-stranded probes have theadvantage that either will do. However, they suffer from the fact that the two strands of the probe willreanneal with themselves in solution thereby reducing their sensitivity. Single-stranded DNA and RNAprobes are available, both providing greater sensitivity. However, it is the introduction of oligonucleotideprobes that has allowed in situ hybridization to mature into a technique available for use in routinelaboratories. These are readily synthesized short lengths (normally 20 to 30 bases) of nucleotide that havethe label incorporated during the production process. Their synthesis allows the production of “designer”probes of known base sequence that can be used against specific regions on nucleic acid present in thetissue sample. They also have the advantage that their small size allows them easily to penetrate the spaceswithin the fixed tissue. However, because of their short length the choice of base sequence must be carefullycontrolled to avoid mismatches occurring with other similar regions on nucleic acid of the target tissue.

An important factor that has made in situ hybridization such a useful technique is that the specificityof reaction can be very accurately controlled by varying the conditions under which hybridization iscarried out. Thus length and concentration of the probe, pH, temperature and buffer composition areall factors that affect the sensitivity and specificity of the reaction. The variation of these factors determinesthe “stringency” under which the reaction occurs. Reactions carried out at high stringency (high tem-perature, low salt, high formamide buffer concentrations) ensure that only reactions with high homologyare stable. Under low stringency conditions (low temperature, high salt, low formamide) some degreeof stable mismatching will occur giving nonspecific reactions. One might ask why high stringencyconditions are not employed all of the time. The answer is that, to a large extent, they are incompatible



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with the aims of keeping tissue sections on the slide and maintaining adequate tissue morphology because,for sufficient hybridization to occur, prolonged periods under harsh conditions are required. It is thereforeleft to the user to decide the level of mismatching that can be tolerated and in most cases moderate conditionsover 4 to 6 h are used, with mismatches being removed by subsequent washing in buffer of high stringency.

Visualization of the label is carried out using the same techniques employed for immunocytochemistry.Today probes are mainly labeled with bioton or digoxigenin. The former can be demonstrated by anABC technique while the latter requires an antidigoxigenin antibody followed by ABC.

16.5.3 Autoradiography

Autoradiography, a method of locating small quantities of radioactivity in biological material, can traceits origins to the observation in 1896 by Henri Bequerel that uranium and its compounds are able to foga nearby photographic plate (for an account of its early history see Rogers, [1973]). A radioactively labeledmarker chosen for its ability to bind with the tissue or cell under investigation is administered to anexperimental animal, a tissue culture or a cell culture system and is taken up by the target cells. Thetissue is then fixed, embedded and sectioned after which the section is placed in close contact with aspecially prepared photographic emulsion, consisting of a suspension of silver bromide (AgBr) in gelatinebonded to the surface of the section. Radioactivity from the section is absorbed by the emulsion,producing free bromine ions which, in turn, yield free electrons. These are trapped by defects in the AgBrcrystal lattice where they react with silver ions to produce atomic silver. This process is analogous to theexposure to light of a conventional photographic film. The free silver atoms act as nuclei for the formationof more atomic silver and during the development process, grains of silver are formed that are largeenough to be detected microscopically. After development, the emulsion is treated with a fixative thatdissolves the unreacted AgBr, rendering the emulsion transparent.

In principle it is possible to measure the amount of radioactivity in the section by counting the silvergrains although changes in pressure, temperature or the effects of additional chemical reactions can allchange their size as well as the number formed. To ensure the accuracy and repeatability of quantitativework, great care must be taken to standardize the experimental conditions. Ideally, an internal standardof known activity is processed in tandem with the test tissue or, where possible, actually incorporatedwith the tissue itself (Flitney, 1991).

When used in conjunction with conventional staining for light or electron microscopy, sites within atissue or cell where a particular metabolic or synthetic process occurs can be related to microscopicstructure and calibration is not normally necessary.

16.5.3.1 Isotopes Used

Of the isotopes in common use, which include [14C], [35S], and [125I], tritium [3H] is the most widelyused for three reasons. First, hydrogen is found in most molecules of biological interest. Second, [3H] isa ß emitter of low energy and therefore low penetrating power, giving sharper and higher resolutionimages. Third, it has a half-life of approximately 12 years, so the radioactive intensity does not changeappreciably during the course of a typical experiment.

16.5.3.2 Emulsions

There are two general methods of applying emulsion to slides. Stripping (Doniach and Pelc, 1950) involvesthe following steps:

• The section, cut in the normal way, is mounted in the normal way on a slide precoated withgelatine and allowed to dry.

• A piece of emulsion is cut from the glass plate on which it is supplied and floated onto water,where it is left to absorb water for a few minutes.

• The slide and section are dipped into the water and withdrawn so that the emulsion is lifted outand covers the section.

• After drying, the emulsion is bonded to the slide and remains in close contact with the radioactive tissue.


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Dipping, introduced in 1955 (Joftes and Warren, 1955), involves dipping the slide and attached spec-imen into liquid emulsion, withdrawing it, allowing the excess liquid to drain off and drying the prep-aration by evaporation. The advantages of dipping over stripping (Flitney, 1991) are:

• Closer contact between section and emulsion• Better control of AgBr crystal size and therefore resolution• Easier to make thin emulsion layers and hence better staining• Speed and ease of preparation lending itself to partial automation

The main drawback is the difficulty in producing an emulsion layer of uniform thickness, limiting theaccuracy and repeatability of quantitative work.

For electron microscopy resin, sections are mounted on ultra-clean microscope slides, pretreated witha very thin layer of emulsion (Salpeter and Bachmann, 1964) and stored during exposure. Due to thethinness of the emulsion exposure times approximately 10 times longer than comparable preparationsfor light microscopy are necessary. The autoradiographs are developed and fixed on the slide and stained.Finally, they are transferred to electron microscope grids and coated with a 5-nm layer of carbon tominimize chemical reaction between tissue and emulsion.

16.5.3.3 Resolution

Resolution has been defined empirically as the distance in the plane of the section from the radioactivesource at which the grain density is half its value directly above the source (Doniach and Pelc, 1950)or the radius of a circle centered on the source which contains half the grains produced by the source(Bachmann and Salpeter, 1965) or, similarly, as the distance from a linear source of a strip parallel tothe source which contains half the grains associated with the source (Salpeter et al., 1969). Factorsaffecting resolution include the size of the AgBr crystals and the thickness of the emulsion as well asthe energy of the radioactive particles that determines the distance they penetrate into the emulsion.[3H], which emits ß particles with energies up to 18 keV, has, under optimal conditions, a resolutionof 0.5 µm; whereas a higher energy emitter such as [14C] or [35S] will have a resolution of 2 to 5 µm(Flitney, 1991).

16.5.3.4 Sensitivity

Sensitivity depends on emulsion thickness and efficiency, the fraction of ß particles captured by theemulsion and giving rise to detectable grains (typically 60 to 80% of those traveling toward theemulsion). As the thickness of the emulsion increases, the probability of a particle hitting an AgBrmolecule, and thus ultimately producing a silver grain, increases. In general, increased sensitivityimplies decreased resolution so, in practice, a compromise must be sought for individual experiments.Under ideal conditions approximately 10–9 µCi can be detected. This is equivalent to around 1 disin-tegration of a [3H] atom per day. At this rate however, exposure times of several weeks would berequired, assuming that approximately 100 disintegrations are needed to produce a grain visible underthe light microscope (Rogers, 1973).

16.5.3.5 Tissue Fixation

The aims of fixation, preservation of tissue structure and no interference with subsequent staining aresimilar to those of conventional histology with the additional need of maintaining the resolution andsensitivity of the emulsion. Formalin and glutaraldehyde desensitize the emulsion while fixatives that donot, such as methanol, tend to harden the tissue and disrupt its morphology (Flitney, 1991).

16.5.3.6 Staining

If staining is carried out before applying the photographic emulsion, care must be taken to compensatefor loss of staining intensity during development and photographic fixation; whereas if the staining isperformed after the autoradiography stains must be chosen so as not to affect the stability of the silvergrains. Suitable stains for each alternative are listed in Flitney (1991).


Histology and Staining 16-17


The techniques of immunohistochemistry and in situ hybridization may be combined with autorad-iography to map the distribution of a particular antigen (Beckman et al., 1983), rates of cellular prolif-eration (Lacy, 1991) or the phase of DNA replication (Lockwood, 1980). However, as the range of availableantibodies increases and their specificity improves, purely immunological techniques are finding favorover the combined approach, a tendency that is encouraged by the technical complexity of autoradiog-raphy as well as radiation safety issues.

16.6 Mounting

Once the stained tissue has been picked up onto a slide, it must be dehydrated and cleared in much thesame way as the aqueous fixative was removed before embedding. Thus, water remaining from the stainingprocess is removed by treatment with successively more concentrated solution of a water-miscible organicsolvent such as ethanol or acetone, taking care to do this rapidly to avoid leaching out those stains thatare soluble in these agents. Finally, the ethanol or acetone is removed by treatment with a transitionagent such as xylene after which the section on the slide is ready for the application of the mountingagent, followed by a protective glass coverslip. Most resins used for embedding are only sparingly solublein noncorrosive solvents in which case clearing is not attempted and the mounting agent is applied tothe intact section.

The purpose of the mounting agent is to seal the space between the slide and the coverslip, keepingout air and moisture, and to “fine-tune” the optical properties of the entire preparation. With stainedtissue, spherical aberration is minimized if the refractive index of the mounting agent is close to that ofglass. For unstained tissue, on the other hand, contrast is enhanced by choosing a mounting agent witha refractive index different to that of glass. The most common mounting agents for stained tissue areCanada Balsam and DPX, an artificial resin introduced in 1941 (Kirkpatrick and Lendrum, 1941) withrefractive indices (RIs) of 1.52, close to that of glass. More recently, methacrylate mounting resins havebeen introduced that polymerize on exposure to light, do not undergo shrinkage and do not cause fadingof the stain with time (Silverman, 1986).

Aqueous mounting agents include Apathy’s medium (RI 1.52), Farrant’s medium (RI 1.42), andglycerol jelly (RI 1.4–1.47). Details of their composition and preparation are given in Sanderson (1994).

Figure 16.7 (left-hand side) shows that the presence of the coverslip between the section and theobjective lens of the microscope can cause additional refraction at the glass/air interface. The resulting

FIGURE 16.7 Ray diagrams for dry and oil immersion objectives showing how the oil (of refractive index similarto that of the glass coverslip) increases the “half-angle of acceptance” (a) and hence the resolving power of the lens.


16-18 Biomedical Technology and Devices Handbook


spherical aberration is only a significant problem for lenses with a numerical aperture greater than 0.5(see below). These lenses are normally designed for use with coverslips of a standard thickness (0.17mm). The effect of deviations from these standard conditions has been investigated by Rawlins (1992)and Pluta (1988). Objective lenses fitted with an adjustable correction collar in which the spacing betweenthe component lenses is adjustable may also be used to compensate for variations in coverslip thicknessor depth of the mounting medium (White, 1974).

16.7 Microscopy

16.7.1 Resolution

The most obvious function of microscopy is to produce a magnified image of a specimen. Of equalimportance is the ability to resolve two closely spaced objects. Resolution or minimum resolved distanceis “the least separation between two points at which they may be distinguished as separate” (Bradburyand Bracegirdle, 1998). The healthy human eye with a near point of 250 mm can resolve two objects 70µm apart. To achieve higher resolution, a lens or lenses are required to reduce the near point. Theresolution (r) of a lens that is restricted by interference between the light diffracted by two closelyseparated points on the object is expressed by:

(16.2)

where l is the wavelength of the illumination and NA, the numerical aperture of the lens is given by theexpression:

(16.3)

where n is the refractive index of the medium between the object and the lens, and a is the half angleof acceptance of the lens (Figure 16.7). Thus, resolving power may be increased by using shorter wave-length illumination or by increasing the numerical aperture of the lens. Modern high power objectivelenses designed for use in air have an acceptance angle approaching 90∞ and numerical apertures up to0.95. In practice, the diffraction of light passing through the glass coverslip over the specimen into theair above reduces the acceptance angle. This effect can be reduced and the resolution increased by usingan immersion lens (Figure 16.7, right-hand side) in which the space between the cover slip and the lensis filled with a medium such as oil whose refractive index matches that of the coverslip (ª1.5).

16.7.2 Illumination

Most histological specimens viewed through the light microscope are illuminated by transmitted light, someof which is absorbed by dyes chosen to stain particular structures, which then appear as dark areas againsta light background. When viewing living tissues or other materials that cannot be stained and that have arefractive index similar to that of the medium in which they are suspended or embedded, the structure isdifficult to visualize (Figure 16.8a). To avoid this problem dark ground or dark field illumination must beused. In this mode the light source is prevented from entering the microscope objective lens directly by asuitably shaped mask. Thus, only light that has been reflected or refracted by parts of the specimen can passthrough the objective and hence reach the eyepiece. In this way the specimen appears as a light image againsta dark background (Figure 16.8b). The main drawback of this technique is loss of fine detail and poor contrast.

Interference microscopy overcomes this problem by splitting the light beam into two different pathlengths arranged so that the observed field in the absence of a specimen consists of closely spacedinterference fringes. The presence of the specimen in the beam path will cause slight shifts in the phaseof one or both beams, and the interference pattern is correspondingly changed.

rNA

=l

2

NA n= sin a




Phase contrast microscopes achieve a similar effect by passing an annular light beam through thespecimen which is then shifted in phase by a similarly shaped phase plate in the objective lens system.Light that has been refracted by the specimen does not pass through the phase plate and when combinedwith the phase-shifted beam produces an interference pattern which corresponds to the shape andstructure of the specimen (Figure 16.8c).

In polarized light microscopy, the birefringent properties of materials such as protein, bone and lipidare exploited to enhance contrast with or without staining. A plain polarizing filter is positioned betweenthe light source and the specimen, and a similar filter is placed between the objective and eyepiece lenses.One of the filters is rotated so that its plane of polarization is nearly at right angles to the other, givinga dark background in the field of view. The birefringent components of the specimen, having rotatedthe plane of polarization, will therefore appear lighter than the background (Figure 16.9). Both phase

FIGURE 16.8 A brain section (10-mm-thick cryostat section) is shown visualized with four different types ofillumination. The section contains neuronal cell bodies and myelinated fiber tracts, and the neurons have beenmarked with two different specialized histochemical stains. One is an immunofluorescence marker that identifies aneuropeptide and the other is a radiolabeled oligonucleotide probe that identifies a receptor mRNA. The probe isrevealed using the technique of autoradiography (see text). Under normal bright field illumination (a), the tissue isdifficult to see and the silver grains are just visible as faint dots. Dark field illumination (b) reveals the silver grainsas bright white dots against a dark background (it also reveals the myelinated fiber tracts), and phase contrastillumination (c) reveals the tissue. Epifluorescence (d) illumination reveals the immunofluorescence marker. (Withthanks to Professor J.V. Priestley.)




contrast and polarized light techniques allow visualization of unstained and or living tissue, although atthe expense of high contrast images.

16.7.3 Fluorescence Microscopy

Fluorescence is defined as the absorption of electromagnetic radiation of a specific wavelength (excitation)and its reemission at a longer wavelength. When a specimen containing naturally fluorescent material(e.g., vitamin A, chlorophyll, collagen), or one stained with a fluorescent compound having a specificaffinity for a component of interest (i.e., a fluorochrome), is illuminated by visible or ultraviolet (UV)light of the appropriate wavelength, the fluorescence, which is generally much less intense than the excitinglight, is normally masked by the background illumination. Common fluorochromes such as rhodamineor fluoroscein are excited by UV light and emit in the visible part of the EM spectrum. The shorterwavelength of UV light improves the resolving power of the system (Equation 16.2). In early fluorescencemicroscopes the specimen was illuminated by transmission in the conventional manner and the excitinglight as well as background illumination was attenuated by suitable bandpass filters. In modern fluores-cence microscopes the specimen is normally visualized by incident light or epi-illumination (Figure16.8d). In such instruments the light is directed via a filter and a dichroic mirror (a mirror that reflectslight of some wavelengths and transmits at others) through the objective and the fluorescence returnsvia a second filter through the mirror and into the eyepiece. By careful choice of filter and mirror, it ispossible to visualize low intensity fluorescence at a wavelength close to that of the exciting light.

In the technique of immunofluorescence originally reported in 1941 (Coons et al., 1941; Weller andCoons, 1954), a fluorescent stain is combined with an antibody having an affinity for an antigen in thespecimen. The advantages of immunological specificity, fluorescent sensitivity and short wavelengthresolving power are thus combined.

16.7.4 Confocal Microscopy

A major drawback of optical microscopy, especially at high magnification, is the low depth of focus andthe difficulty of visualizing objects below the surface of the section. For instance, at an overall magnificationsufficient to examine a cell nucleus, say, 300¥, objects more than a few microns from the focal point will

FIGURE 16.9 (See color insert following page 14-10.) Transverse section of a porcine carotid artery viewed underpolarized light. The birefringent collagen fibers appear bright against a dark background.




not be clearly resolved. In 1961 Minsky (1961) patented a device in which a pinhole is placed between theobjective and the eyepiece, and the object is illuminated by a point source produced by a second pinholein the light path. The pinholes allow light originating from object lying within or close to the focal planeto pass through, while blocking light from points remote from this plane. An image is then synthesizedby moving the specimen in a raster pattern or by tandem scanning of the pinholes. The first successfuluse of such a system was reported by Egger and Petran (1967) who were able to examine unstained neuraltissue; since then, neuroscientists have remained the most frequent and enthusiastic users of confocalmicroscopy (Fine et al., 1988). Commercial development was encouraged by the availability of compactand relatively low cost lasers that provided a source of light sufficiently intense and compact to overcomethe low image brightness inherent in any pinhole device. In modern instruments, which usually incorporateepi-illumination suitable for fluorescence techniques, the laser beam is scanned over the object (Carlssonet al., 1985) and the final image is captured by a high definition video camera interfaced to a PC equippedwith copious video memory. By moving the pinhole along the optical axis of the microscope, the positionof the focal plane can be changed and a three-dimensional tomographic image can be constructed. Integralsoftware allows these images to be reconstructed, displayed from different viewpoints and animated. Witha combination of fluorescence induced by high intensity excitation and high sensitivity CCD cameras,objects as much as 600 µm below the surface of a specimen can be visualized.

Recently it has been reported that by using a modified form of confocal fluorescence microscopy,structures smaller than the resolution limit imposed by Equation 16.3 may be resolved (Dyba and Hell,2002), although this claim has been questioned (Stelzer, 2002). Nevertheless, there is little doubt thattechnical improvements will continue to extend the scope and utility of confocal microscopy for theforeseeable future.

16.7.5 Electron Microscopy

By the end of the 19th century, the resolving power of the light microscope was approaching its theoreticallimit, i.e., half the wavelength of blue light or approximately 0.2 µm (Equation 16.2). In 1931 (Kaiser,1985) the introduction of the electron beam as a source of “illumination” led to the development oftransmission electron microscopes (TEMs) with a resolution approaching 0.1 nm, with which it is possibleto resolve the structure of intracellular organelles and even that of large molecules. In the TEM, electron-dense regions of the specimen scatter the electron beam, whereas electrons passing through the specimenare focused onto a fluorescent screen or photographic plate. The electron-dense regions thus appear darkagainst a light background. The degree of scatter depends on the energy of the electron beam, the atomicnumber of the scattering atoms and the thickness of the specimen. In the scanning electron microscope(Oatley, 1972), which has a lower resolution (5 to 10 nm) but a greater depth of focus, the specimen isscanned by the beam in a raster pattern and information is obtained primarily from scattered electronsand those produced by secondary emission. Thus it is possible to study surface details of a solid specimenwhatever its thickness and to construct a pseudo-three-dimensional image of the surface. In addition tothe scattered and secondary emission electrons, the primary beam gives rise to x-rays, the spectra ofwhich are characteristic of the atoms from which they are emitted. By combining this information withthe scattered electron signal, the spatial distribution of specific elements in the specimen may be mapped.

Due to their expense, electron microscopes are not widely used in diagnostic histopathology, althoughthey are increasingly being called upon to provide specialist diagnostic information.

References

Shorter Oxford Dictionary, 3rd ed., Oxford University Press, Oxford, 1973.Altman, L.G., Schneider, B.G., and Papermaster, D.S., Rapid embedding of tissues in Lowicryl K4M for

immunoelectron microscopy, J. Histochem. Cytochem., 32, 1217–1223, 1984.Arnolds, W.J., Oriented embedding of small objects in agar-paraffin, with reference marks for serial

section reconstruction, Stain Technol., 53, 287–288, 1978.




Aschoff, A., Fritz, N., and Ilert, M., Axonal transport of fluorescent compounds in the brain and spinalcord of cat and rat, in Axonal Transport in Physiology and Pathology, Weiss, D.G. and Gorio, A.,Eds., Springer, Berlin, 1982, p. 177.

Bachmann, L. and Salpeter, E.E., Autoradiography with the electron microscope. A quantitative investi-gation, Lab. Invest., 14, 1041–1051, 1965.

Baker, J.R., Experiments on fixation, Unpublished observations, 1958a.Baker, J.R., Principles of Biological Microtechnique, 1st ed., Methuen & Co. Ltd., London, 1958b.Bancroft, J.D. and Stevens, A., Theory and Practice of Histological Techniques, Churchill Livingstone,

Edinburgh, 1990.Beckman, W.C.J., Stumpf, W.E., and Sar, M., Localisation of steroid hormones and their receptors. A

comparison of autoradiographic and immunocytochemical techniques, in Techniques in Imunocy-tochemistry, Bullock, G.R. and Petrusz, P., Eds., 1983, Academic Press, London.

Bennett, H.S., Wyrick, A.D., Lee, S.W., and McNeil, J.H., Science and art in preparing tissues embeddedin plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simplestains, Stain Technol., 51, 71–97, 1976.

Berry, C.L., Sosa-Melgarejo, J.A., and Greenwald, S.E., The relationship between wall tension, lamellarthickness and intercellular junctions in the fetal and adult aorta: its relevance to the pathology ofdissecting aneurysm, J. Pathol., 169, 15–20, 1993.

Bibb, C.A., Pullinger, A.G., and Baldioceda, F., Serial variation in histological character of articular softtissue in young human adult temporomandibular joint condyles, Arch. Oral Biol., 38, 343–352,1993.

Bradbury, S. and Bracegirdle, B., Introduction to Light Microsocopy, Royal Microscopical Society Hand-books, Vol. 42, BIOS Scientific Publishers, Oxford, 1998.

Branchereau, P., Van Bockstaele, E.J., Chan, J., and Pickel, V.M., Ultrastructural characterization ofneurons recorded intracellularly in vivo and injected with lucifer yellow: advantages of immu-nogold-silver vs. immunoperoxidase labeling, Microsc. Res. Tech., 30, 427–436, 1995.

Carlsson, K., Danielsson, P.E., Lenz, R., Liljeborg, A., Majlof, L., and Aslund, N., Three-dimensionalmicroscopy using a confocal laser scanning microscope, Opt. Lett., 10, 53–55, 1985.

Coons, A.H. and Kaplan, M.H., Localization of antigen in tissue cells, J. Exp. Med., 91, 1–13, 1950.Coons, A.H., Creech, H.J., and Jones, R.N., Immunological properties of an antibody containing fluo-

rescent groups, Proc. Soc. Exp. Biol. Med., 47, 200, 1941.Coons, A.H., Leduce, E.H., and Connolly, J.M., Studies on antibody production. I. A method for the

histochemical demonstration of specific antibody and its application to a study of the hyperim-mune rabbit, J. Exp. Med., 102, 49–60, 1955.

Culling, C.F.A., Allison, R.T., and Barr, W.T., Cellular Pathology Technique, 4th ed., London, Butterworths,London, 1985.

Dobrin, P.B., Effect of histologic preparation on the cross-sectional area of arterial rings, J. Surg. Res.,61, 413–415, 1996.

Donath, K., The diagnostic value of the new method for the study of undecalcified bones and teeth withattached soft tissue (Sage-Schliff (sawing and grinding) technique), Pathol. Res. Pract., 179,631–633, 1985.

Doniach, I. and Pelc, S.R., Autoradiographic technique, Br. J. Radiogr., 23, 184–192, 1950.Drury, R.A.B. and Wallington, E.A., Carleton’s Histological Technique, 5th ed., Oxford University Press,

Oxford, 1980.Dyba, M. and Hell, S.W., Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33

nm axial resolution, Phys. Rev. Lett., 88, 163901-1–163901-4, 2002.Egger, M.D. and Petran, M., New reflected-light microscope for viewing unstained brain and ganglion

cells, Science, 157, 305–307, 1967.Ellis, R.C., The microtome: function and design, in Laboratory Histopathology A complete reference,

Woods, A.E. and Ellis, R.C., Eds., Churchill Livingstone, New York, 1994, pp. 4.4.1–4.4.23.




Fine, A., Amos, W.B., Durbin, R.M., and McNaughton, P.A., Confocal microscopy: applications inneurobiology, Trends Neurosci., 11, 346–351, 1988.

Flitney, E., Autoradiography, in Theory and Practice of Histological Techniques, Bancroft, J.D. and Stevens,A., Ed., Churchill Livingstone, Edinburgh, 1991, pp. 645–665.

Foskett, J.K. and Grinstein, S., Non Invasive Techniques in Molecular Biology, Wiley-Liss, New York, 1990.Fox, C.H., Johnson, F.B., Whiting, J., and Roller, P.P., Formaldehyde fixation, J. Histochem. Cytochem.,

33, 845–853, 1985.Girardot, J.M. and Girardot, M.N., Amide cross-linking: an alternative to glutaraldehyde fixation, J. Heart

Valve Dis., 5, 518–525, 1996.Goppert, H.R. and Cohn, F., Uber die Rotation des Zellinhaltes von Nitella fiexilis, Bot. Ztg., 7, 665–719,

1849.Gordon, K.C., Tissue processing, in The Theory and Practice of Histological Techniques, Bancroft, J.D. and

Stevens, A., Eds., Churchill Livingstone, Edinburgh, 1990, pp. 44–59.Hanstede, J.G. and Gerrits, P.O., The effects of embedding in water-soluble plastics on the final dimensions

of liver sections, J. Microsc., 131, 79–86, 1983.Hardy, P.M., Nicholls, A.C., and Rydon, H., The nature of the cross linking of proteins with glutaralde-

hyde, J. Chem. Soc., 1, 958–962, 1976.Helander, K.G., Thickness variations within individual paraffin and glycol methacrylate sections, J.

Microsc., 132, 223–227, 1983. Hilger, H.H. and Medan, D., A simple method for exact alignment of small paraffin embedded specimens

to the cutting plane, Stain Technol., 62, 282–283, 1987.Hooke, R., Micrographia (facsimile edition), Royal Society London (1665), Dover, New York, 1961.Hopwood, D., Fixation and fixatives, in Theory and Practice of Histological Techniques, Bancroft, J.D. and

Stevens, A., Eds., Churchill Livingstone, Edinburgh, 1990, pp. 21–42.Horobin, R.W., Understanding Histochemistry: Evaluation and Design of Biological Stains, Ellis Horwood,

Chichester, 1988.Horobin, R.W., An overview of the theory of staining, in Theory and Practice of Histological Techniques,

Bancroft, J.D. and Stevens, A., Eds., Churchill Livingstone, Edinburgh, 1990, pp. 93–105.Iwadare, T., Mori, H., Ishiguro, K., and Takeishi, M., Dimensional changes of tissues in the course of

processing, J. Microsc., 136, 323–327, 1984.Joftes, D.L. and Warren, S., Simplified liquid emulsion radioautography, J. Biol. Photogr. Assoc., 23,

145–151, 1955.Johansen, D.A., A quadruple stain combination for plant tissues, Stain Technol., 14, 125–128, 1939. Kaiser, H.E., Functional comparative histology, Gegenbaurs Morph Jahrb Lepzig, 131, 815–862, 1985.Kendall, P.A., Polak, J.M., and Pearse, A.G., Carbodiimide fixation for immunohistochemistry: observa-

tions on the fixation of polypeptide hormones, Experientia, 27, 1104–1106, 1971.Kiernan, J.A., Histological and Histochemical Methods: Theory and Practice, 2nd ed., Pergamon Press,

Oxford, 1990.Kirkpatrick, J. and Lendrum, A.C., Further observations on the use of synthetic resin as a substitute for

Canada balsam; precipitation of paraffin wax in medium and an improved plasticizer, J. Pathol.Bacteriol., 53, 441–443, 1941.

Lacy, E.R., Kuwayama, H., Cowart, K.S., King, J.S., Deutz, A.H., and Sistrunk, S., A rapid, accurate,immunohistochemical method to label proliferating cells in the digestive tract. A comparison withtritiated thymidine, Gastroenterology, 100, 259–262, 1991.

Leeuwenhoek, A., Epistolae Physiologicae Super Compluribus Naturae Arcanis, Beman, Delft, 1791.Leong, A.S., Microwave irradiation in histopathology, Pathol Annu., 23, 213–234, 1988.Leong, A.S., Microwave techniques for diagnostic laboratories, Scanning, 15, 88–98, 1993.Leong, A.S., Tissue and section preparation, in Laboratory Histopathology A Complete Reference, Woods,

A.E. and Ellis, R.C., Eds., Churchill Livingstone, New York, 1994, pp. 4.1.1–4.1.26.Leong, A.S., Daymon, M.E., and Milios, J., Microwave irradiation as a form of fixation for light and

electron microscopy, J. Pathol., 146, 313–321, 1985.




Leong, A.S. and Duncis, C.G., A method of rapid fixation of large biopsy specimens using microwaveirradiation, Pathology, 18, 222–225, 1986.

Leong, A.S., Milios, J., and Duncis, C.G., Antigen preservation in microwave-irradiated tissues: a com-parison with formaldehyde fixation, J. Pathol., 156, 275–282, 1988.

Lewis, F.T., The introduction of biological stains: employment of Saffron by Vieussens and van Leeuwen-hoek, Anat. Rec., 83, 229–253, 1942.

Lillie, R.D., Histopathologic Technic and Modern Histochemistry, 2nd ed., McGraw-Hill, New York, 1965.Lillie, R.D., H.J. Conn’s Biological Stains, 8th ed., Williams & Wilkins, Baltimore, 1969.Litwin, J.A., Light microscopic histochemistry on plastic sections, Prog. Histochem.Cytochem., 16, 1–84,

1985.Lockwood, A.H., Immunofluorescence radioautography. Simultaneous visualization of DNA replication

and supramolecular antigens in individual cells, Exp. Cell Res., 128, 383–394, 1980.Login, G.R. and Dvorak, A.M., Microwave energy fixation for electron microscopy, Am. J. Pathol., 120,

230–243, 1985.Masson, P., Some histological methods. Trichrome stainings and their preliminary technique, Bull. Int.

Assoc. Med., 12, 75–90, 1929.Medewar, P.B., The rate of penetration of fixatives, J. R. Microsc. Soc., 61, 46–57, 1941.Merriam, R.W., Determination of section thickness in quantitative microspectrophotometry, Lab. Invest.,

6, 28–43, 1957.Minsky, M., Microscopy Apparatus, U.S. Patent No. 3013467, 1961.Murray, G.I., Is wax on the wane? J. Pathol., 156, 187–188, 1988.Nettleton, G.S. and McAuliffe, W.G., A histological comparison of phase-partition fixation with fixation

in aqueous solutions, J. Histochem. Cytochem., 34, 795–800, 1986.Nunn, R.E., Electron Microscopy: Microtomy, Staining and Specialized Techniques, Butterworths Laboratory

Aids, Baker, F.J., Ed., Butterworths, London, 1970.Nunn, R.E., Electron Microscopy: Preparation of Biological Specimens, Butterworths Laboratory Aids,

Baker, F.J., Ed., Butterworths, London, 1970.Oatley, C.W., The Scanning Electron Microscope, Cambridge University Press, London, 1972.Panula, P., Happola, O., Airaksinen, M.S., Auvinen, S., and Virkamaki, A., Carbodiimide as a tissue fixative

in histamine immunohistochemistry and its application in developmental neurobiology, J. His-tochem. Cytochem., 36, 259–269, 1988.

Pearse, A.G.E., Histochemistry, Theoretical and Applied, 4th ed., Vol. 1, Churchill Livingstone, Edinburgh,1980.

Pluta, M., Advanced Light Microscopy. Principles and Basic Properties, Vol.1, Elsevier, Amsterdam, 1988.Polak, J.M. and Van Noorden, S., An introduction to immunocytochemistry: current techniques and

problems, in Royal Microscopy Society Handbook, Vol. 111, Oxford University Press, Oxford, 1984.Rawlins, D.J., Light Microscopy, Bios Scientific, Oxford, 1992.Reale, E. and Luciano, L., Fixation with aldehydes. Their usefulness for histological and histochemical

studies in light and electron microscopy, Histochemie, 23, 144–170, 1970.Reid, N. and Beesley, J.E., Sectioning and cryosectioning for electron microscopy, in Methods in Electron

Microscopy, Glauert, A.M., Ed., Elsevier, Amsterdam, 1991.Rogers, A.W., Techniques of Autoradiography, Elsevier, London, 1973.Ross, K.F.A., Cell shrinkage caused by fixatives and paraffin wax embedding in ordinary cytological

preparations, Q. J. Microsc., 94, 125–139, 1953.Rostgaard, J., Qvortrup, K., and Poulsen, S.S., Improvements in the technique of vascular perfusion-

fixation employing a fluorocarbon-containing perfusate and a peristaltic pump controlled bypressure feedback, J. Microsc., 172, 137–151, 1993.

Saito, T. and Keino, H., Acrolein as a fixative for enzyme cytochemistry, J. Histochem Cytochem., 24,1258–1269, 1976.

Sallee, C.J. and Russell, D.F., Embedding of neural tissue in agarose or glyoxyl agarose for vibratomesectioning, Biotechnol. Histochem., 68, 360–368, 1993.




Salpeter, E.E. and Bachmann, L., Autoradiography with the electron microscope, J. Cell. Biol., 22, 469–474,1964.

Salpeter, M.M., Bachmann, L., and Salpeter, E.E., Resolution in electron microscope radioautography, J.Cell. Biol., 41, 1–32, 1969.

Sanderson, J.B., Biological Microtechnique, Royal Microscopical Society, Microscopical Handbooks, Vol.28, Bradbury, S., Ed., BIOS Scientific Publ., Oxford, 1994.

Sato, Y., Mukai, K., Watanabe, S., Goto, M., and Shimosato, Y., The AMeX method. A simplified techniqueof tissue processing and paraffin embedding with improved preservation of antigens for immun-ostaining, Am. J. Pathol., 125, 431–435, 1986.

Scala, C., Preda, P., Cennacchi, G., Martinelli, G.N., Manara, G.C., and Pasquinelli, G., A new polychromestain and simultaneous methods of histological, histochemical and immunohistochemical stainingsperformed on semithin sections of Bioacryl-embedded human tissues, Histochem. J., 25, 670–677,1993.

Silverman, M., Light-polymerizing plastics as slide mounting media, Stain Technol., 61, 135–137, 1986.Sims, D.E. and Horne, M.M., Non-aqueous fixative preserves macromolecules on the endothelial cell

surface: an in situ study, Eur. J. Morphol., 32, 59–64, 1994.Sims, D.E., Westfall, J.A., Kiorpes, A.L., and Horne, M.M., Preservation of tracheal mucus by nonaqueous

fixative, Biotechnol. Histochem., 66, 173–180, 1991.Spurr, A.R., A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res.,

26, 31–43, 1969.Stelzer, E.H., Beyond the diffraction limit? Nature, 417, 806–807, 2002.Tandler, B., Improved slides of semithin sections, J. Electron. Microsc. Tech., 14, 285–286, 1990.Tymianski, M., Bernstein, G.M., Abdel-Hamid, K.M., Sattler, R., Velumian, A., Carlen, P.L., Razavi, H.,

and Jones, O.T., A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments, Cell Calcium, 21,175–183, 1997.

Vincent, J.F.V., Automating the microtome, Microsc. Anal., 23, 19–21, 1991.Waldeyer, R., Untersuchungen uber den Ursprung und den Verlauf des Axencylinders bei Wirbellosen

und Wirbelthieren sowie uber dessen Endverhalten in der querdestreiften Muskelfaser, Henle Pfe-iffer Ztg Ration Med., 20, 193–256, 1863.

Weller, T.H. and Coons, A.H., Fluorescent antibody studies with agents of varicella and Herpes Zoster,Proc. Soc. Exp. Biol. Med., 86, 789, 1954.

White, G.W., The correction of tube length to compensate for coverglass thickness variations, Microscopy,32, 411–420, 1974.

Willingham, M.C. and Yamada, S.S., Development of a new primary fixative for electron microscopicimmunocytochemical localization of intracellular antigens in cultured cells, J. Histochem.Cytochem., 27, 947–960, 1979.

Zelander, T. and Kirkeby, S., Vibratome sections of difficult tissues, Stain Technol., 53, 251–255, 1978.


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