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12/17/12 ZEISS Microscopy Online Campus | Microscopy Basics | Enhancing Contrast in Transmitted Light

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Introduction

In transmitted light microscopy, the specimen quality does not always lenditself to easy observation and image recording with excellent contrast insimple brightfield imaging mode. Investigations dealing with inherently low-contrast specimens, such as unstained bacteria, thin tissue slices, andadherent live cells, rely on specialized contrast-enhancing techniques toassist with imaging these virtually transparent samples. In the course ofexamining unstained specimens, poor light absorption by the specimenresults in extremely small variations in the intensity distribution differencebetween the specimen and the background. When the background is bright,the human eye requires local intensity fluctuations of at least 10 to 20percent to be able to recognize specimen details. Unfortunately, this level of modulation isseldom seen with transparent specimens, which are usually rendered almost invisible against abackground of similar intensity. The term transmitted light, when used in optical microscopy,refers to any imaging modality where light is passed from the illumination source on the oppositeside of the specimen to the objective (thus, illumination is transmitted through the specimen).The contrast-enhancing techniques described in this section represent a variety of methods insample preparation as well as optical tricks that generate intensity changes which are useful forobservation and imaging.

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Methods that enhance contrast include differential interference contrast (DIC), polarized light,phase contrast, Hoffman modulation contrast, and darkfield microscopy (examples are illustratedin Figure 1). Several of these techniques are limited by light originating in regions removed fromthe focal plane when imaging thicker plant and animal tissues, while polarized light requiresbirefringence (usually not present to a significant degree in animal cells) to generate contrast.

Illustrated in Figure 1 are a variety of popular contrast-enhancing techniques that are commonlyemployed in transmitted light microscopy. The thin tissue section in Figure 1(a) reveals a humanbasal cell carcinoma stained with eosin and hematoxylin to generate color contrast in brightfieldimaging mode. Figure 1(b) shows living HeLa cells in a plastic tissue culture vessel imaged withphase contrast. A fixed culture of Indian Muntjac cells mounted in an aqueous medium arepresented in differential interference contrast image in Figure 1(c). A fresh tissue section ofmouse heart muscle bathed in aqueous saline solution is displayed in the Figure 1(d) panel,where contrast is generated using Hoffman modulation (or ZEISS VAREL) contrast, an obliqueillumination technique. The brilliant bright-on-dark contrast observed with darkfield illumination isshown in Figure 1(e) using an Obelia hydroid specimen. Finally, rabbit skeletal muscle fibers(Figure 1(f)) are among the biological specimens that are birefringent and demonstrate contrastin polarized light.

Brightfield Illumination

One of the primary and favorite techniques used in all forms of optical microscopy for the pastthree centuries, brightfield illumination relies upon changes in light absorption, refractive index,or color for generating contrast. As light passes through the specimen, regions that alter thedirection, speed, and/or spectrum of the wavefronts generate optical disparities (contrast) whenthe rays are gathered and focused by the objective. Resolution in a brightfield system dependson both the objective and condenser numerical apertures, and an immersion medium is requiredon both sides of the specimen (for numerical aperture combinations exceeding a value of 2.3).Digital cameras provide the wide dynamic range and spatial resolution required to capture theinformation present in a brightfield image. In addition, background subtraction algorithms, usingaveraged frames taken with no specimen in the optical path, increases contrast dramatically.

Simple brightfield imaging, with the microscope properly adjusted for Köhler illumination,provides a limited degree of information about the cell outline, nuclear position, and the locationof larger vesicles in unstained specimens. Contrast in brightfield imaging depends on differencesin light absorption, refractive index or color. Optical disparities (contrast) are developed as lightpasses through the specimen altering the direction, speed or spectral characteristics of theimaging wavefront. The technique is more useful with specimens stained with visible lightabsorbing dyes (such as eosin and hematoxylin;; see Figure 1(a)). However, the general lack ofcontrast in brightfield mode when examining unstained specimens renders this techniquerelatively useless for serious investigations of living cell structure.

Phase Contrast

First described by the Dutchman Frits Zernike in 1934, phase contrast earned its discoverer theNobel Prize for physics in 1953 while revolutionizing basic biomedical research on living cells.The technique is ideal for thin unstained specimens (such as culture cells on glass), which areapproximately 5 to 10 micrometers thick above the nucleus, but less than a micrometer thick atthe periphery. Such specimens barely exhibit any light absorption in the visible portion of thespectrum and the human eye cannot detect them in brightfield and darkfield illumination. Phasecontrast (as illustrated in Figure 1(b)) employs an optical mechanism to translate minutevariations in phase into corresponding changes in amplitude, which can be visualized asdifferences in image contrast. The microscope must be equipped with a specialized condensercontaining an annulus or a series of annuli matched to a set of objectives containing phase rings(designed Ph) in the rear focal plane (phase contrast objectives can also be used withfluorescence, but with a slight reduction in transmission). Phase contrast is an excellent methodto increase contrast when viewing or imaging living cells in culture, but typically results in halossurrounding the outlines of edge features. These halos are optical artifacts that often reduce thevisibility of boundary details. The technique is not useful for thick specimens (such as plant andanimal tissue sections) because shifts in phase occur in regions removed from the focal planethat distort image detail. Furthermore, floating debris and other out-of-focus phase objectsinterfere with imaging adherent cells on coverslips.

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Polarized Light

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As presented in Figure 2, very small differences exist between the refractive indices of the cellsand their surrounding aqueous solutions and within the cells between the cytoplasm (Figure2(b)) and the cell nucleus (Figure 2(c)). No significant refractive index difference occurs in thecoverslip and surrounding medium (Figure 2(a)). Phase contrast makes these tiny differencesvisible by the use of optical manipulation as, for example, it translates them into differences inintensity that can be visually observed and recorded. The optical effect employed in this caseconsists of a shift of phase relationships between light wavefronts traveling through differentportions of the specimen. During their journey through cell nuclei, cytoplasm, or water, the lightwaves are shifted (retarded) by small degrees, since these media have slightly different refractiveindices (the higher the refractive index of a medium, the smaller the speed or velocity of light inthe medium). As a result, a light wave that has passed through a cell nucleus lags behind thelight waves that only had to pass through water (compare Figure 2(a) and Figure 2(c)). Theamount of lag is called a phase shift. Before their entry into the specimen, the waves are still inphase (wavefronts beneath the coverslip), but this is no longer the case when they have passedthrough the various materials having different refractive indices. The amount of the phase shiftdepends on what media (refractive index) the waves have passed through on their paths, andhow long the paths were through these media. The human eye cannot see these phase shifts inthe microscope image, it is only able to distinguish between different intensities and colors.Therefore, the phase contrast technique uses optical tricks to translate phase shifts into greyvalues.

In phase contrast microscope configuration, the condenser aperture diaphragm is replaced by aphase stop (the size of which depends upon objective and condenser numerical aperture) thatilluminates the specimen via the condenser optical components in a hollow cone of light fashion,as illustrated in Figure 3(a). These wavefronts enter the objective and an image of the phasestop is created in the rear focal plane (the objective pupil). Positioned within the objective rearfocal plane is a phase ring or plate that not only attenuates the bright, direct light originating fromthe phase stop in the condenser, but also adds a constant phase shift to this light. If the specimencontains sub-structures that have mixed refractive indices, these entities guide the light from thedirect waves into new paths (red dotted line in Figure 3(a)). Wavefronts diffracted by thespecimen (in effect, those containing structural information) will not pass through the phase ringin the objective, meaning that they will not be attenuated or retarded. All of the wavefronts areultimately recombined to form the intermediate image by the tube lens.



The wavefronts that have all been retarded to varying degrees by details in the specimen aresuperimposed in the intermediate image over the shifted and attenuated wavefronts where theyamplify or attenuate each other (depending on the phase), forming the final phase contrast imageobserved in the eyepieces. These interference processes in the intermediate image create brightand dark areas of the various structures having refractive index (and/or thickness) variations inthe specimen. Optimum contrast is created by selecting the correct retardation and attenuationvalues, which is a function of the optical properties of the phase ring in the objective aperture.

A major artifact of phase contrast imaging is the bright halos of light that appear on the borderssurrounding the specimen. Halos occur in phase contrast microscopy because the circularphase-retarding (and neutral density) ring located in the objective phase plate also transmits asmall degree of diffracted light from the specimen (it is not restricted to passing surround wavesalone). The problem is compounded by the fact that the width of the non-diffracted light (zeroth-order) surround wavefront projected onto the phase plate by the condenser annulus is smallerthan the actual width of the phase plate ring. Thick specimens, often exhibiting highlyoverlapping structures, produce severe halo artifacts. Therefore, phase contrast is a method thatis only recommended for very thin specimens where several structures are not physically lyingon top of each other. In a thick specimen, details may be blended into an image that, in the finalanalysis, is no longer legible.

Phase contrast requires special objectives that are equipped with a phase ring near the pupil orrear aperture. They are easy to recognize by the green inscription that denotes the size of thecorresponding condenser phase stop: Ph1, Ph2, or Ph3. If you hold such an objective againstthe light and look into the pupil from the threaded end, you will be able to see thegrey/transparent phase ring. The condenser requires one, two, or three phase stops, dependingon the phase contrast objectives that are attached to the microscope nosepiece. The requiredring diameter increases with the numerical aperture (high apertures require the maximumdiameter (Ph3), for example 0.9 in air or 1.3 with oil immersion). Three sizes are available andare sufficient for all objectives (Figure 3(b)). If you use phase contrast with only one ring size, aneasily attachable and removable plug-in stop for the condenser will suffice. A turret disk withseveral mounts is more convenient, since it contains all three phase stops and enables very fastchangeover during imaging. Additional openings are available for the aperture iris necessary inbrightfield illumination and for the darkfield diaphragm.



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The phase stops must be centered after they have been installed in the condenser so that theimage of the phase stop in the objective pupil corresponds exactly with the position of the phasering in the beam path (see Figure 4(c)). Centering is performed using two small wrenches (orscrewdrivers, depending upon the microscope model) on the turret disk of the condenser. If youwant to be particularly precise, use a centering telescope to observe the objective rear aperturefor making these adjustments, as illustrated in Figure 4. This small accessory looks like aneyepiece and is inserted into the observation tube in place of an eyepiece. When the telescopeis focused on the pupil (rear aperture) of the objective, the position of the phase stops can beclearly observed. Again, look into the objective pupil and bring the bright image of the condenserphase stop into coincidence with the phase ring of the objective. This is clearly shown in Figure4. In Figures 4(a) and 4(b), the phase stop is not aligned with the objective phase plate, while thestop and phase plate are in perfect congruence in Figure 4(c).

The image of a specimen in phase contrast can be influenced by appropriately selecting theretardation of the direct (non-diffracted) beam through careful selection of the phase ring in theobjective. Depending on the retardation value selected, objects with a higher refractive indexthan their surroundings appear either brighter or darker than their surroundings. This is alsocalled either positive or negative phase contrast. In modern microscopes, positive phase contrastis standard, where the darkness of object features increases with their refractive index. The effectsimulates absorption to the observer's eye in areas where a higher refractive index produceshigh contrast features. This impression is considered the optimum, particularly with cells andtissue in aqueous media because cell nuclei and organelles, for example, appear darker thanthe cytoplasm. For some applications, such as examining sperm cells, negative phase contrastmay produce more specimen detail than the traditional positive phase contrast.

Differential Interference Contrast

Differential interference contrast microscopy (DIC;; Figure 1(c)) requires plane-polarized light andadditional light-shearing (Nomarski) prisms to exaggerate minute differences in specimenthickness gradients and refractive index. Lipid bilayers, for example, produce excellent contrastin DIC because of the difference in refractive index between aqueous and lipid phases of thecell. In addition, cell boundaries in relatively flat adherent mammalian and plant cells, includingthe plasma membrane, nucleus, vacuoles, mitochondria, and stress fibers, which usuallygenerate significant gradients, are readily imaged with DIC. In plant tissues, the birefringent cellwall reduces contrast in DIC to a limited degree, but a properly aligned system should permitvisualization of nuclear and vacuolar membranes, some mitochondria, chloroplasts, andcondensed chromosomes in epidermal cells. Differential interference contrast is an importanttechnique for imaging thick plant and animal tissues because, in addition to the increasedcontrast, DIC exhibits decreased depth of field at wide apertures, creating a thin optical section ofthe thick specimen. This effect is also advantageous for imaging adherent cells to minimize blurarising from floating debris in the culture medium.

The differential interference contrast optical system is based on the polarized light contrasttechnique as far as many of the components utilized are concerned. DIC for a moderntransmitted light microscope is different than that in reflected light because two birefringent



prisms are used (see Figure 6), and the specimen's optical path difference is determined by the

product of the refractive index difference (between the specimen and its surrounding medium)

and the thickness (geometrical distance) traversed by a light beam between two points on the

optical path (Figure 5). Images produced in differential interference contrast microscopy are

characterized by a distinctive shadow-cast appearance, as if they were illuminated from a highly

oblique light source originating from a single azimuth. Unfortunately, this effect, which often will

render a specimen in a pseudo three-dimensional relief (Figure 6(d)), is frequently assumed by

uninformed microscopists to be an indicator of actual topographical structure.

Figure 6 illustrates the differential interference contrast beam path that is similar to that of

polarized transmitted light. In DIC (as compared to polarized light), the two birefringent prisms

(Figure 6(b)) are inserted into the optical train, one in the condenser and the second near the

objective pupil. The condenser prism performs a vectorial decomposition of the previously

linearly polarized light (Figure 6(c)) into two vibration directions that are perpendicularly

polarized to each other, and laterally shifts these partial beams in such a way that a small lateral

displacement of the wavefronts occurs where regions of thickness or refractive index vary. If the

two partial beams now pass through exactly the same structures, no further path difference will

occur in the specimen (Figure 5(a) and Figure 5(c)). However, if the two partial beams see

slightly different conditions, each of them will experience a slightly difference pathlength that

accompanies it on the remaining trip to the intermediate image plane (Figure 5(b)).

As discussed, the split DIC light beams enter and pass through the specimen where their wave

paths are altered in accordance with the specimen's varying thickness, slopes, and refractive

indices. These variations cause alterations in the wave path of both beams passing through

areas of any specimen details lying close together. When the parallel beams enter the objective,

they are focused above the rear focal plane where they enter a second DIC prism that combines

the two beams at a defined distance outside of the prism itself. This removes the shear (distance

between the waves) and the original path difference between the beam pairs. As a result of

having traversed the specimen, the paths of the parallel beams are not of the same length

(optical path difference) for differing areas of the specimen.

In order for the beams to interfere, the vibrations of the beams of different path length must be

brought into the same plane and axis. This is accomplished by placing a second polarizer

(analyzer) above the upper DIC beam-combining prism. The light then proceeds toward the

eyepiece where it can be observed as differences in intensity and color. The design results in

one side of a specimen detail appearing bright (or possibly in color) while the other side appears

darker (or another color). This shadow effect bestows the pseudo three-dimensional appearance

to the specimen that was described above. There are numerous advantages in DIC microscopy

as compared to phase contrast microscopy. With DIC, it is possible to make fuller use of the

numerical aperture of the system and to provide optical staining (color). DIC also allows the

microscope to achieve excellent resolution. Use of full objective aperture enables the

microscopist to focus on a thin plane section of a thick specimen without confusing images from



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above or below the plane. Annoying halos, often encountered in phase contrast, are absent inDIC images, and suitable achromat and fluorite objectives can be used for this work. It isimportant to keep in mind, however, since DIC is based on polarized light principles, highlybirefringent specimens or those embedded in birefringent materials should not be observedunder DIC.

Hoffman Modulation and VAREL Contrast

Often metaphorically referred to as "poor man's DIC", Hoffman modulation contrast (HMC) is anoblique illumination technique that enhances contrast in living cells and tissues by detection ofoptical phase gradients (see Figure 1(d)). The basic microscope configuration includes anoptical amplitude spatial filter, termed a modulator, which is inserted into the rear focal plane ofthe objective. Light intensity passing through the modulator varies above and below an averagevalue, which by definition, is then said to be modulated. Coupled to the objective modulator is anoff-axis slit aperture that is placed in the condenser front focal plane to direct oblique illuminationtowards the specimen. Unlike the phase plate in phase contrast microscopy, the Hoffmanmodulator is designed not to alter the phase of light passing through;; rather it influences theprincipal zeroth order maxima to produce contrast. Hoffman modulation contrast is not hamperedby the use of birefringent materials (such as plastic Petri dishes) in the optical pathway, so thetechnique is more useful for examining specimens in containers constructed with polymericmaterials. On the downside, HMC produces a number of optical artifacts that render thetechnique somewhat less useful than phase contrast or DIC for live-cell imaging on glasscoverslips.

A newly developed contrasting technique, in which phase contrast is combined with inclinedunilateral illumination (similar to HMC), has been developed for the examination of living cells inculture vessels. An additional stop shaped like a ring sector is used on the illumination side,permitting unilateral inclined illumination. Three illumination modes, unilateral darkfield, VAREL(variable relief) contrast, and inclined brightfield (similar to oblique illumination or HMC) are setby shifting the stop in the radial direction from the outside to the inside (see Figure 7) and asegment of a matching annulus is moved into position. This slit directs the source illumination topass through a second ring of high absorption on the objective aperture's periphery, whichsuppresses its intensity by an amount dependent upon the density of the outer ring. Thediffracted orders of light pass through the objective without being suppressed by the VAREL ring.You can also manipulate the position of the slit if you want to vary the extent the sourceillumination is suppressed.

Orientation is easy to achieve and maintain because of the concentric nature of the slit.Furthermore, because of the VAREL ring's location in the objective, VAREL and phase contrastrings can be combined in the same objective. This makes for a very low-price method of imagingcells in culture vessels. The problems of imaging in both traditional and routine tissue cultureapplications can be resolved with this method, it is a successful option for the examination ofliving objects (micromanipulation), and a range of structures with varying optical thickness are



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easily imaged with VAREL. Even culture vessels with a curved bottom allow a useful image to

be produced. Phase contrast alone sometimes fails in such cases because a curved chamber

base acts like a lens and impairs the superimposition of phase rings. A slider contains two of the

mentioned sectors to allow illumination to be performed from the left or right, as required. This

makes it possible to contrast cells even near the edges of the holes in microtiter plates.

Darkfield Illumination

The methodology surrounding darkfield microscopy, although widely used for imaging

transparent specimens throughout the 19th and 20th Centuries, is limited in use to physically

isolated cells and organisms (as presented in Figure 1(e)). In this technique, the condenser

directs a cone of light onto the specimen at high azimuths so that first-order wavefronts do not

directly enter the objective front lens element. Light passing through the specimen is diffracted,

reflected, and/or refracted by optical discontinuities (such as the cell membrane, nucleus, and

internal organelles) enabling these faint rays to enter the objective. The specimen can then be

visualized as a bright object on an otherwise black background. Unfortunately, light scattered by

objects removed from the focal plane also contribute to the image, thus reducing contrast and

obscuring specimen detail. This artifact is compounded by the fact that dust and debris in the

imaging chamber also contribute significantly to the resulting image. Furthermore, thin adherent

cells often suffer from very faint signal, whereas thick plant and animal tissues redirect too much

light into the objective path, reducing the effectiveness of the technique.

Recently, a renewed interest in transmitted darkfield microscopy has arisen due to its

advantages when used in combination with fluorescence microscopy. Darkfield microscopy is a

specialized illumination technique that capitalizes on oblique illumination, as described above,

to enhance contrast in specimens that are not imaged well under normal brightfield illumination

conditions. An artificial dark background is created in the microscope using an annular stop in

the condenser (see Figure 8). Although the condenser optics then illuminate the sample, they do

so with a hollow inverted cone of light that passes through the specimen at extremely oblique

angles at all azimuths so that only the light interacting with specimen features is able to enter the

microscope objective to form a bright image of the specimen superimposed onto a dark

background. To create the high angles, it is necessary for the objective aperture to be smaller

than the inner aperture of the illuminating light cone (see Figure 8(a)). However, objectives with

an integrated variable iris diaphragm are also available to shutter out the indirect light even if it

falls into the aperture cone of the objective (as illustrated in Figure 8(b)). This permits the use of

very high apertures for darkfield illumination. Darkfield microscopy is an excellent tool for

biological and medical investigations. It can be effectively used at high magnifications to image

living bacteria, or at low magnifications to view and image cells, tissues, and whole mounts.

Polarized Light

Polarized light microscopy is a contrast-enhancing technique that dramatically improves the

quality of an image acquired with birefringent materials when compared to other techniques such

as brightfield and darkfield illumination, phase contrast, differential interference contrast,

fluorescence, and Hoffman modulation contrast. Polarized light microscopes possess a high



degree of sensitivity and can be used for both qualitative and quantitative studies targeted at awide range of anisotropic specimens. Qualitative polarizing microscopy is quite popular inpractice, however, the quantitative aspects of polarized light microscopy, which are principallyapplied in crystallography, represent a much more difficult subject that is primarily used bymineralogists, geologists, and chemists. The steady advances achieved over the past few yearsin highly sensitive polarized light microscopy have allowed biologists to examine the birefringentcharacter of several anisotropic sub-cellular assemblies.

Polarized light microscopy (Figure 1(f)) is conducted by viewing the specimen between crossedpolarizing elements inserted into the optical train before the condenser and after the objective.Assemblies within the cell having birefringent properties, such as the plant cell wall, starchgranules, and the mitotic spindle, as well as muscle tissue, rotate the plane of light polarization,appearing bright on a dark background. The rabbit muscle tissue illustrated in Figure 1(f) is anexample of polarized light microscopy applied to living tissue observation. Note that thistechnique is limited by the occurrence of birefringence in living cells and tissues, and has yet tobe fully explored. As mentioned above, differential interference contrast operates by placing amatched pair of opposing Nomarski prisms between crossed polarizers, so that any microscopeequipped for DIC observation can also be employed to examine specimens in plane-polarizedlight simply by removing the prisms from the optical pathway.

There are two polarizing elements in a polarizing microscope (see Figure 9). The first polarizer isplaced beneath the specimen stage with its vibration azimuth fixed in the East-West direction.Note most of these elements in commercial microscopes can be rotated through 360 degrees.Another polarized (the analyzer), which is usually aligned with a vibration direction orientedNorth-South (but again rotatable on some microscopes), is placed above the objective and canbe moved into and out of the light path as needed. When both the polarizer and analyzer areinserted into the optical path, their vibration azimuths are positioned at right angles to each other.In this configuration, the polarizer and analyzer are crossed, with no light passing through theoptical system and a dark background present in the eyepieces (Figure 9(a)). Linearly polarizedlight waves are generated by polarizers that filter out a privileged plane from the statisticalconfusion of vibrational directions prevailing in natural light. The two orthogonal components oflight (ordinary and extraordinary waves) travel at different speeds through the specimen and, asa result, observe different refractive indices, a phenomenon known as birefringence (derivedfrom the terms double or bi refraction). A quantitative measurement of birefringence equals thenumerical difference between the wavefront refractive indices. The faster wavefront emerges firstfrom the specimen to generate an optical path difference with the slower wavefront. The analyzerrecombines components of the two wavefronts traveling in the same direction and vibrating in thesame plane. This polarizing element ensures that the two wavefronts have the same amplitudeat the time of recombination for maximum contrast.

The appropriate arrangement for polarized light imaging is relatively easy to implement onvirtually any microscope. The polarizer beneath the condenser (near the aperture diaphragm)ensures that the specimen is illuminated with linearly polarized wavefronts that pass through the



condenser (refer to Figure 9(a)). The analyzer (a second polarizer), which is oriented with the

azimuth arranged at an angle of 90 degrees to that of the polarizer, is located behind the

objective. The tube lens forms the intermediate image, which is projected through the eyepieces

or onto a camera imaging plane. If no specimen is placed on the microscope stage, the scene

observed through the eyepieces will remain completely dark. When illuminated, many

specimens turn the vibration direction of the polarized light out of the plane produced by the

polarizer. Such specimens consist mainly of birefringent materials, in which the refractive index

depends on the vibration direction of the incident light. This is mainly the case with crystals, such

as starch or minerals (Figure 9(c)), but also occurs with fibers (Figure 9(d)) and polymers. If such

materials are viewed under the polarization microscope between the crossed polarizer and the

analyzer, bright areas can be seen in the image because light is partially transmitted by the

analyzer.

The polarized light microscope optical train can also include an auxiliary element known as a

lambda or retardation plate (Figure 9(a) and 9(b)) that is used in quantitative analysis. Inpolarized light, this lambda plate converts contrast into colors. As in phase contrast, optical path

differences give rise to colors, although this time with polarized light and birefringent material in

the specimen. The path differences generated leads to an extinction of certain wavelengths in

the light through interference (only certain colors remain from the white light and create beautiful,

colored pictures). Polarizing microscopy offers an immense amount of information about the

composition and three-dimensional structure of a range of samples. Practically unlimited in its

scope, the technique can reveal information about thermal history and the strains to which a

specimen was subjected during formation. Valuable in manufacturing and research, polarizing

microscopy is relatively inexpensive and an accessible research and quality control tool that can

provide information unavailable with any other technique.

Contributing Authors

Rudi Rottenfusser - Zeiss Microscopy Consultant, 46 Landfall, Falmouth, Massachusetts, 02540.

Erin E. Wilson and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr.,The Florida State University, Tallahassee, Florida, 32310.

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