Although much neglected and undervalued as an investigative tool, polarized light microscopy (Figure 1) provides all the benefits of brightfield microscopy and yet offers a wealth of information, which is simply not available with any other optical microscopy technique.

As well as providing information on absorption color and boundaries between minerals of differing refractive indices obtainable in brightfield microscopy, polarized light microscopy can distinguish between isotropic and anisotropic materials. The technique exploits optical properties of anisotropy to reveal detailed information about the structure and composition of materials, which are invaluable for identification and diagnostic purposes.

Isotropic materials, which include gases, liquids, unstressed glasses and cubic crystals, demonstrate the same optical properties in all directions. They have only one refractive index and no restriction on the vibration direction of light passing through them. Anisotropic materials, in contrast, which include 90 percent of all solid substances, have optical properties that vary with the orientation of incident light with the crystallographic axes. They demonstrate a range of refractive indices depending both on the propagation direction of light through the substance and on the vibrational plane coordinates. More importantly, anisotropic materials act as beam splitters and divide light rays into two parts (as illustrated in Figure 1). The technique of polarizing microscopy exploits the interference of the split light rays, as they are re-united along the same optical path to extract information about these materials.

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Birefringent Crystals in Polarized Light Explore how birefringent anisotropic crystals interact with polarized light in an optical microscope as the circular stage is rotated through 360 degrees.

Polarized light microscopy is perhaps best known for its geological applications--primarily for the study of minerals in rock thin sections, but it can also be used to study many other materials. These include both natural and industrial minerals whether refined, extracted or manufactured, composites such as cements, ceramics, mineral fibers and polymers, and crystalline or highly ordered biological molecules such as DNA, starch, wood and urea. The technique can be used both qualitatively and quantitatively and is an outstanding tool for materials science, geology, chemistry, biology, metallurgy and even medicine.

While an understanding of the analytical techniques of polarized microscopy may be perhaps more demanding than other forms of microscopy, it is well worth pursuing, simply for the enhanced information that can be obtained over brightfield imaging. An awareness of the principles of polarizing microscopy is also essential for the effective interpretation of differential interference contrast (DIC) microscopy.

Polarized Light

The wave model of light describes light waves vibrating at right angles to the direction of travel of light with all vibration directions being equally probable. This is "common" light. In plane-polarized light there is only one vibration direction (Figure 1). The human eye-brain system has no sensitivity to the vibration directions of light, and plane-polarized light can only be detected by an intensity or color effect, for example, by reduced glare when wearing polarized sun glasses.

The most widely used material is Polaroid^TM film. Invented by Land in 1932, Polaroid film consists of long chain polymers, treated with light absorbing dyes, and stretched so that the chains are aligned. Light vibrating parallel with the chains is absorbed while light perpendicular to the chains is transmitted.

There are two polarizing filters in a polarizing microscope - the polarizer and analyzer (see Figure 1). The polarizer is situated below the specimen stage usually with its permitted vibration direction fixed in the left-to-right, East-West direction, although this is usually rotatable through 360 degrees. The analyzer, usually aligned North-South but again rotatable on some microscopes, is sited above the objectives and can be moved in and out of the light path as required. When both the analyzer and polarizer are in the optical path, their permitted vibration directions are positioned at right angles to each other. In this configuration, the polarizer and analyzer are said to be crossed, with no light passing through the system and a dark field of view present in the eyepieces.

The polarizer and analyzer are the essential components of the polarizing microscope - but other desirable features include:

• A rotating specimen stage to facilitate orientation studies with centration of the objectives and stage with the microscope optical axis to make the center of rotation coincide with the center of the field of view.

• Strain free objectives – stress in assembly can produce optical effects under polarized light, a factor that could complicate observations.

• An eyepiece fitted with a cross wire graticule to mark the center of the field of view. Often, the cross wire graticule is substituted for a photomicrography graticule that assists in focusing the specimen and composing images with a set of frames bounding the area of the viewfield to be captured either digitally or onto film.

• A Bertrand lens – to enable easy examination of the objective rear focal plane, to allow accurate adjustment of the illuminating aperture diaphragm and to view interference figures, as presented in Figure 2.

• A slot to allow the insertion of compensators/retardation plates between the polarizers, which are used to enhance optical path differences in the specimen. In most modern microscope designs, this slot is placed either in the microscope nosepiece or an intermediate tube positioned between the body and eyepiece tubes. Compensation plates inserted into the slot are then situated between the specimen and the analyzer.

Polarizing microscopy can be used both with reflected and transmitted light. Reflected light is useful for the study of opaque materials such as mineral oxides and sulphides, metals and silicon wafers (Figure 3). Reflected light techniques require a dedicated set of objectives that have not been corrected for viewing through the coverslip, and those for polarizing work should, again, be stress free.

Illustrated in Figure 3 is a series of reflected polarized light photomicrographs of typical specimens imaged utilizing this technique. On the left (Figure 3(a)) is a digital image revealing surface features of a microprocessor integrated circuit. Birefringent elements employed in the fabrication of the circuit are clearly visible in the image, which displays a portion of the chip's arithmetic logic unit. The polished surface of a ceramic superconducting tape (Yttrium-1,2,3) is presented in Figure 3(b), which shows birefringent crystalline areas with interference colors interspersed in a matrix of isotropic binder. Metallic thin films are also visible with reflected polarized light. Figure 3(c) illustrates blisters that form imperfections in an otherwise confluent thin film of copper (about 0.1 micron thick) sandwiched over a nickel/sodium chloride substrate to form a metallic superlattice assembly.

Careful specimen preparation is essential for good results. The method chosen will depend on the type of material studied. In geological applications the standard thickness for rock thin sections is 25-30 micrometers. Specimens can be ground down with diamond impregnated wheels and then hand finished to the correct thickness using abrasive powders of successively decreasing grit size. Softer materials can be prepared in a manner similar to biological samples using a microtome. Slices between one and 40 micrometers thick are used for transmitted light observations. These should be strain-free and free from any knife marks. Specimens are mounted between the slide and the coverslip using a mounting medium whose composition will depend on the chemical and physical nature of the specimen. This is particularly significant in the study of synthetic polymers where some media can chemically react with and cause structural changes to the material being studied.

Making Use of Anisotropy

Different levels of information can be obtained in plane-polarized light (analyzer out of the optical path) or with crossed polarizers (analyzer inserted into the optical path). Observations in plane-polarized light reveal details of the optical relief of the specimen, which is manifested in the "visibility" of boundaries, and increases with the increase of refractive index across them. Differences in the refractive indices of the mounting adhesive and the specimen determine the extent to which light is scattered as it emerges from the uneven specimen surface. Materials with high relief, which appear to stand out from the image, have refractive indices, which are appreciably different from that of the mountant. Immersion refractometry is used to measure substances having unknown refractive indices by comparison with oils of known refractive index.

Examinations of transparent or translucent materials in plane-polarized light will be similar to those seen in natural light until the specimen is rotated about the optical axis of the microscope. Then observers may see changes in the brightness and/or the color of the material being examined. This pleochroism, that is, variation of absorption color with vibration direction of the light, depends on the orientation of the material in the light path and is a characteristic of anisotropic materials only. An example of a material showing pleochroism is crocidolite, more commonly known as blue asbestos. This effect helps in its identification.

Polarization colors result from the interference of the two components of light split by the anisotropic specimen and may be regarded as white light minus those colors that are interfering destructively. Figure 2 illustrates conoscopic images of uniaxial and biaxial crystals observed at the objective rear focal plane. Interference patterns are formed by light rays traveling along different axes of the crystal being observed. Uniaxial crystals (Figures 2(a) and 2(b)) display an interference pattern consisting of two intersecting black bars (termed isogyres) that form a Maltese cross-like pattern. When illuminated with white (polarized) light, birefringent specimens produce circular distributions of interference colors (Figure 2), with the inner circles, called isochromes, consisting of increasingly lower order colors (see the Michel-Levy interference color chart, Figure 4). A common center for both the black cross and the isochromes is termed the melatope, which denotes the origin of the light rays traveling along the optical axis of the crystal. Biaxial crystals display two melatopes (Figure 2(c)) and a far more complex pattern of interference rings.

The two components of light travel at different speeds through the specimen and have different refractive indices, or refringences. Birefringence is the numerical difference between these refringences. The faster beam emerges first from the specimen with an optical path difference (OPD), which may be regarded as a "winning margin" over the slower one. The analyzer recombines only components of the two beams traveling in the same direction and vibrating in the same plane. The polarizer ensures that the two beams have the same amplitude at the time of recombination for maximum contrast.

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Polarizer Rotation and Birefringence Discover how specimen birefringence is affected by the angle of polarizer when observed in a polarized light microscope.

There is constructive and destructive interference of light in the analyzer, depending on the OPD on the specimen and the wavelength of the light, which can be determined from the order of polarization color(s). This relies on the properties of the specimen, including the thickness difference between the refractive index and the birefringence of the two beams, which has a maximum value dependent on the specimen and on the direction of travel of light through the specimen. Optical path differences can be used to extract valuable "tilt" information from the specimen.

Superimposed on the polarization color information is an intensity component. As the specimen is rotated relative to the polarizers, the intensity of the polarization colors varies cyclically, from zero (extinction) up to a maximum after 45 degrees and back down to zero after a 90-degree rotation. That is why a rotating stage and centration are provided, which are critical on a polarizing microscope. Centration of the objective and stage ensures that the center of the stage rotation coincides with the center of the field of view, a great convenience, as anyone who has tried to manage without it will know.

Whenever the specimen is in extinction, the permitted vibration directions of light passing through are parallel with those of either the polarizer or analyzer. This can be related to geometrical features of the specimen, such as fiber length, film extrusion direction, and crystal faces. In crossed polarizers, isotropic materials can be easily distinguished from anisotropic materials as they remain permanently in extinction (remain dark) when the stage is rotated through 360 degrees.

To help in the identification of fast and slow beams, or to improve contrast when polarization colors are of low order, such as dark grey, accessory plates can be inserted in the optical path. These will cause color changes in the specimen, which can be interpreted with the help of a polarization color chart (Michel-Levy chart; see Figure 4). These charts show the polarization colors provided by optical path differences from 0 to 1800-3100 nanometers together with birefringence and thickness values. The wave plate produces its own optical path difference. When the light passes first through the specimen and then the accessory plate, the OPDs of the wave plate and the specimen are either added together or subtracted from one another in the way that "winning margins" of two races run in succession are calculated. They are added when the slow vibration directions of the specimen and accessory plate are parallel, and subtracted when the fast vibration direction of the specimen coincides with the slow vibration direction of the accessory plate. If the slow and fast directions are known for the accessory plate (they are usually marked on the mount of commercially available plates), then those of the specimen can be deduced. Since these directions are characteristic for different media, they are well worth finding out and are essential for orientation and stress studies.

The strengths of polarizing microscopy can best be illustrated by examining particular case studies and their associated images. All images illustrated in this section were recorded with a Nikon Eclipse E600 microscope equipped with polarizing accessories, a research grade microscope designed for analytical investigations.

Identification of Asbestos Fibers

Asbestos is a generic name for a group of naturally occurring mineral fibers, which have been widely used, for example, in insulating materials, brake pads and to reinforce concrete. They can be harmful to health when inhaled and it is important that their presence in the environment be easily identified. Samples are commonly screened using scanning electron microscopy and x-ray microanalysis, but polarizing microscopy provides a quicker and easier alternative that can be utilized to distinguish between asbestos and other fibers and between the major types asbestos – chrysotile, crocidolite and amosite. From a health care point of view, it is believed that the amphibole asbestos varieties (crocidolite and amosite) are more harmful than the serpentine, chrysotile.

Plane-polarized light provides information about gross fiber morphology, color, pleochroism and refractive index. Glass fibers will be unaffected by rotation under plane-polarized light while asbestos fibers will display some pleochroism. Chrysotile asbestos fibrils may appear crinkled, like permed or damaged hair, under plane-polarized light, whereas crocidolite and amosite asbestos are straight or slightly curved. Chrysotile has a refractive index of about 1.550, amosite 1.692 and crocidolite, 1.695.

With the use of crossed polars it is possible to deduce the permitted vibration direction of the light as it passes through the specimen, and with the whole wave plate, a determination of the slow and fast vibration directions (Figure 5). Under crossed polars, chrysotile shows pale interference colors - low order whites (Figure 5(a)). When a full wave plate is added (530-560 nanometers), the colors are transformed. Aligned Northeast-Southwest, the wave plate is additive and gives blue and yellow in the fiber (Figure 5(b)). When aligned Northwest-Southeast (Figure 5(c)) the plate is subtracting to give a paler yellow fiber with no blue. From this it is possible to deduce that the slow vibration direction is parallel with the long axis of the fiber. Amosite is similar in this respect.

Crocidolite displays blue colors, pleochroism and murky brown polarization colors and has its fast vibration direction parallel with its length. In summary, identification of the three asbestos fiber types depends on shape, refractive indices, pleochroism, birefringence, and fast and slow vibration directions.

Uncovering the History of Rock Formation

Phyllite - An examination of geological thin sections using polarizing microscopy, as well as providing information on component minerals, can reveal a great deal about how the rock was formed. Phyllite, a metamorphic rock, clearly shows the alignment of crystals under the effects of heat and stress. Small-scale folds are visible in the plane-polarized image (Figure 6(a)) and more clearly defined under crossed polars (Figure 6(b)) with and without the wave plate. The crossed polars image reveals that there are several minerals present--quartz in grey and whites and micas in higher order colors. The alignment of the micas is clearly apparent. Addition of the wave plate (Figure 6(c)) improves contrast for clear definition in the image.

Oolite - Oolite, a light gray rock composed of siliceous oolites cemented in compact silica, is formed in the sea. The mineral's name is derived from its structural similarity to fish roe - caviar! It forms in the sea when sand grains are rolled by gentle currents over beds of calcium carbonate or other minerals. These minerals build up around the sand grains and subsequent cementation transforms the grains into coherent rock. The thin sections show the original quartz nuclei (Figure 7(a-c)) on which the build up of carbonate mineral occurred.

In plane-polarized light (Figure 7(a)), the quartz is virtually invisible having the same refractive index as the cement, while the carbonate mineral with a different refractive index shows high contrast. The crossed polarizer image (Figure 7(b)) shows quartz grains in grays and whites and the calcium carbonate in the characteristic biscuit colored, high order whites. The groups of quartz grains in some of the cores reveal that these are polycrystalline and are metamorphic quartzite particles. When a full-wave retardation plate is inserted into the optical path (Figure 7(c)), optical path differences become apparent in the specimen, and contrast is enhanced.

Natural and Synthetic Polymers

During the solidification of polymer melts there may be some organization of the polymer chains, a process that is often dependent upon the annealing conditions. When nucleation occurs, the synthetic polymer chains often arrange themselves tangentially and the solidified regions grow radially. These can be seen in crossed polarized illumination as white regions with the black extinction crosses. When these spherulites impinge, their boundaries become polygonal. This can be clearly seen in crossed polars but not under plane-polarized light.

The addition of the whole wave plate (Figure 8(a)) confirms the tangential arrangement of the polymer chains. The banding occurring in these spherulites indicates slow cooling of the melt allowing the polymer chains to grow out in spirals. This information on thermal history is almost impossible to collect by any other technique. Nucleation in polymer melts can take place as the result of accidental contamination or contact with a nucleating surface and can lead to substantial weakening of the product. Identification of nucleation can be a valuable aid for quality control.

Other polymers may not be birefringent (evidenced by the polycarbonate specimen illustrated in Figure 8(b)), and do not display substantial secondary or tertiary structure. In other cases, both biological and synthetic polymers can undergo a series of lyotropic or thermotropic liquid crystalline phase transitions, which can often be observed and recorded in a polarized light microscope. Figure 8(c) illustrates a birefringent columnar-hexatic liquid crystalline phase exhibited by DNA at very high concentrations (exceeding 300 milligrams/milliliter).

Nylon Fibers - Observations under plane-polarized light (Figure 9(a)) reveal refractive index differences between the fiber and the mountant and the presence of opacifying titanium dioxide particles. The image under crossed polars (Figure 9(b)) shows third order polarization colors and their distribution across the fibers indicates that this is a cylindrical and not a lobate fiber useful in predicting mechanical strength. The use of the quartz wedge (Figure 9(c)) enables the determination of optical path differences for birefringence measurements.

In summary, polarizing microscopy provides a vast amount of information about the composition and three-dimensional structure of a variety of samples. Virtually unlimited in its scope, the technique can reveal information about thermal history and the stresses and strains to which a specimen was subjected during formation. Useful in manufacturing and research, polarizing microscopy is a relatively inexpensive and accessible investigative and quality control tool, which can provide information unavailable with any other technique.