Optical Microscopy

Optical Microscopy 

Microscopy studies the enlargement of the image of the objects which are too small to be properly seen by the unaided eye. Microscopy fulfills its task by making use of the radiations (Fig 1) emitted, absorbed, transmitted, or reflected by the sample which is to be observed. The nature of the radiation specifies the type of microscopy such as optical microscopy, electron microscopy, x-ray microscopy, or acoustic microscopy etc. The visible part of electromagnetic spectrum is the type of radiation used by optical microscopy. Optical microscopy is the microscopic examination of materials through the optical microscope.

Fig 1 Electromagnetic waves

Rough magnifying glasses were used in ancient times, but the evolution of modern microscopes started in the 17th century. Although the first compound microscope was built by Hans and Zacharias Janssen in 1595, Antoni van Leeuwenhoek (1632–1723) managed to make lenses so good to achieve the amazing magnification of around 300x in their very simple microscopes. Because of the suggestions of the scientist Robert Hook around 1670, the instrument maker Christopher Cock in London built a very successful compound microscope. With this instrument, Hook was able to observe the cells. The Hook’s microscope can be regarded as the father of modern instruments.

The optical microscope, frequently referred to as the ‘light microscope’, is a type of microscope which uses visible light (Fig 1) and a system of lenses to magnify images of small samples. Optical microscopes are the oldest and simplest of the microscopes. It is a very important instrument for the study of microstructure, despite the evolution of sophisticated electron metallographic instruments. The sophisticated ‘scanning electron microscope’ (SEM) and ‘transmission electron microscope’ (TEM) are invaluable instruments as well. However they are to be used in conjunction with optical microscope, rather than as a substitute.

All examinations of microstructure begin with use of the optical microscope, starting at low magnification, such as 100×, followed by progressively higher magnifications to assess the basic characteristics of the microstructure efficiently. Majority of the microstructures can be observed with the optical microscope and identified based on their characteristics. Identification of questionable or unknown constituents can be aided by observation of their hardness relative to the matrix, by their natural colour, by their response to the polarized light, and by their response to the selective etchants. These observations are compared to the known details about the physical metallurgy of the material being examined. If doubt still remains or if the structure is too fine to observe, more sophisticated techniques are to be implemented.

The optical microscope can be used to examine as-polished or etched metallographic samples. Certain constituents are more readily observed as-polished, because they are not obscured by etching detail. Inclusions, nitrides, certain carbides, and inter-metallic phases can be readily observed without etching. Except for inclusions, the other phases can be more easily examined if some relief is introduced during final polishing. The samples are to be adequately prepared to ensure correct observation and interpretation of the microstructure without complications from artifacts. Samples which respond to the polarized light, such as materials with non-cubic crystal structures, are normally examined without etching. However, in majority of the cases, etching is to be performed to observe the microstructure. A general-purpose etchant is normally used first to reveal the grain structure and the phases present, followed by selective etchants which attack or colour specific phases of interest. Selective etchants are widely used for quantitative metallography, particularly if performed using an automated device. In either case, etching is to be carefully carried out to reveal the microstructure with clarity.

A microscope uses a very short focal length objective lens to form a greatly enlarged image. This image is then viewed with a short focal length eyepiece used as a simple magnifier. The basic imaging concept and structures of the optical microscopy is shown in Fig 2. The optical system of a microscope mainly includes an objective lens and an eyepiece. The purpose of an objective lens is to magnify an object so that it can be clearly observed by the user. During the observation, the sample is placed near the focal plane of the objective lens in the object space, and a magnified real image of the sample is first created on the intermediate plane. The intermediate plane is located on the focal plane of the eyepiece, thus the eyepiece is working as a magnifier to further magnify the image projected on the intermediate image plane. Finally, a magnified, virtual, inverted image is provided for the observer.

Fig 2 Optical principle of microscope imaging

The ability of an optical microscope to produce separable images of different points on an object is limited. The resolving power of a lens is a quantitative measure of this ability. Points closer than the limit of resolution cannot be distinguished as separate points. Ernst Abbe in 1873 first fixed the value of the minimal distance d between two adjacent points allowing them to be perceived as separated by the equation ‘d = l/2n sin A’, where ‘l’ is the wavelength of light, ‘A’ is one-half the angular aperture of the lens, and ‘n’  is the refractive index of the medium between the object and the lens.

At present, the smallest linear separation of two object points for which they can be resolved by an objective is fixed by the Rayleigh criterion given by equation ‘d = 1.22(l/2NA)’ where ‘l’ is the wavelength of light, and ‘NA’ is the numerical aperture of the objective. Both the Abbe criteria and the Rayleigh criteria are very similar, being the numerical aperture related to the imaging medium by NA = n sin A. The maximum value of sinA is 1 (A = 90 degrees), hence the theoretical maximal numerical aperture of an objective in air (n = 1) is NA = 1. Since a high NA is an essential requirement for high resolution, immersion optics has been developed. Samples can be imaged at a very short distance from the objective through immersion media having a different refractive index, like water (n = 1.33), glycerine (n = 1.47), or oil (n = 1.52).

For a well designed microscope, the spatial resolution is mainly determined by the objective lens. Although an eyepiece can also magnify the image, it cannot improve the resolving power of the microscopes. The spatial resolution of an optical microscope is given by the Rayleigh equation Ro = 0.62 l/n sin A, where ‘Ro’ is the minimum resolvable distance, ‘l’ is the wavelength of the light, ‘n’ is the refractive index of the medium between the lens and the object, and ‘A’ is one-half the angular aperture of the lens, and n sinA is the numerical aperture of the objective.

Based on the above equation, and considering the practical limitations namely (i) the use of visible light with the wavelength between 390 nm (nano meters) and 760 nm, (ii) the maximally reachable aperture with the half angle of 70 degrees to 75 degrees, and (iii) the requirement of using immersion methods with water or oil to increase of refractive index, the resolution of a conventional optical microscope cannot exceed 200 nm.

The optical microscope and the simplified optical wave path of the microscope are shown in Fig 3. The modern optical microscope is able to magnify an object by 1,500 times with the 200 nm limit in spatial resolution. The optical microscopes can be divided into many different types using a variety of criteria. For example, based on a lighting method, there are the transmission and reflection types of microscopes. In a transmission microscope, the light passes through transparent objects. In a reflection microscope, the light source installed on the top of the microscopic lens illuminates the non-transparent objects, and the reflected light is collected by the lens. The microscopes can also be differentiated based on the observation methods, including bright field microscopes, dark field microscopes, phase difference microscopes, polarized light microscopes, interference microscopes, and fluorescent microscopes.

Each microscope can use either the transmission or reflection approach. The bright field microscopes are the most popular and widely used of all microscopes. Using this type of microscope, the transmission (or absorption) ratio and reflection ratio of some observed objects vary according to the change of working environments. The amplitude of these objects varies with the change in lighting intensity. The colourless transparent objects are visible only when the phase of illuminated light changes. Because the bright field microscopes cannot change light phase, the colourless transparent samples are invisible when using this type of microscope.

Fig 3 Optical microscope and its principle

Microscope components

Optical microscopes vary considerably in cost and capability. Reflected light is used for the study of metals. Transmitted light microscopes are used to study minerals and polymers, which can also be examined using reflected light. Optical microscopes are also classified as ‘upright’ or ‘inverted’. These terms refer to the orientation of the plane of polish of the sample during observation. Since each configuration has certain advantages and disadvantages, selection is based on personal preference. The simplest optical microscope is the bench type (normally upright). Photographic capabilities can be added to some microscopes depending on the rigidity of the stand.

Different types of microscopes suitable for observation and photo microscopy are available. These can be rather simple units or full-scale research microscopes with assorted illumination modes, light sources, micro-hardness attachments, hot stages, and so on. Basic components of the optical microscope are given below and shown in Fig 3.

Illumination system – A variety of light sources for optical microscopy are available. The low-voltage tungsten filament lamp used primarily with bench microscopes has adequate intensity for observation, but not for photography. Altering of the current to the bulb controls the light intensity. Carbon-arc illumination systems, once common on microscopes have been replaced by arc or filament light sources. The xenon-arc light source is prevalent because of its high intensity and daylight colour characteristics. Light intensity, however, can be adjusted only by the use of neutral-density filters. Tungsten-halogen filament lamps are also widely used for their high intensity and high colour temperature. Light intensity can be controlled by varying the current or by use of neutral-density filters. Other light sources, such as the zirconium-arc, sodium-arc, quartz-iodine, or mercury-vapour lamps, are less common.

Condenser – An adjustable lens free of spherical aberration and coma is placed in front of the light source to focus the light at the desired point in the optical path. A field diaphragm is placed in front of this lens to minimize internal glare and reflections within the microscope. The field diaphragm is stopped down to the edge of the field of view. A second adjustable-iris diaphragm, the aperture diaphragm, is placed in the light path before the vertical illuminator.

Opening or closing this diaphragm alters the amount of light and the angle of the cone of light entering the objective lens. The optimum setting for this aperture varies with each objective lens and is a compromise among image contrast, sharpness, and depth of field. As magnification increases, the aperture diaphragm is stopped down. Opening this aperture increases image sharpness, but reduces contrast. Closing the aperture increases contrast, but impairs image sharpness. The aperture diaphragm is not to be used for reducing light intensity. It is to be adjusted only for contrast and sharpness.

Light filters – These are used to modify the light for ease of observation, for improved photo microscopy, or to alter contrast. Neutral-density filters are used to reduce the light intensity uniformly across the visible spectrum. Different neutral-density filters from around 85 % to 0.01 % transmittance are available. Majority of the optical microscopes have selection of at least two such filters.

Selective filters are used to balance the colour temperature of the light source to that of the film. This is frequently necessary for faithful reproduction of colour images, depending on the light source used and the film type. A green or yellow-green filter is widely used in black-and-white photography to reduce the effect of lens defects on image quality. Majority of the objectives, particularly the lower cost achromats, need such filtering for good results.

Polarizing filters are used to produce plane-polarized light (one filter) or crossed-polarized light (two filters rotated to produce extinction) for examination of non-cubic (crystallographic) materials. Materials which are optically anisotropic, such as beryllium, zirconium, alpha-titanium, and uranium, can be examined in the crossed-polarized condition without etching. A sensitive-tint plate can also be used with crossed-polarized light to enhance colouration.

The objective lens – It forms the primary image of the microstructure and is the most important component of the optical microscope. The objective lens collects as much light as possible from the sample and combines this light to produce the image. The NA of the objective is a measure of the light-collecting ability of the lens. Light-collecting ability increases with the angle ‘A’. The setting of the aperture diaphragm alters the NA of the condenser and hence the NA of the system.

Objective lenses (Fig 4) are normally mounted on a nose piece turret which can accept four to six objectives. Some microscopes do not use nose piece turrets, and only one objective at a time can be placed on the vertical illuminator using a bayonet mount. The vertical illuminator contains a reflector or prism which deflects the light down the objective onto the sample surface. It normally holds the aperture and field diaphragms and filters as well. The vertical illuminator normally provides only one or two types of illumination, such as bright-field and dark-field illumination or bright-field and polarized light illumination. However, universal vertical illuminators are now available which provide all types of illumination with one vertical illuminator and one set of objectives.

The tube length is the length of the body tube from the eye line of the eyepiece to the objective thread. This length is not standardized and can vary. Majority of the objectives are designed for use with a certain tube length, normally 160 mm to 250 mm, and normally cannot be interchanged.

The most commonly used objective is the achromat, which is corrected spherically for one colour (normally yellow-green) and for longitudinal chromatic aberration for two colours (normally red and green). Hence, achromats are not suitable for colour photo microscopy. Use of a yellow-green filter and ortho-chromatic film yields optimum results. However, achromats do provide a relatively long working distance, that is, the distance from the front lens of the objective to the sample surface. Working distance decreases as magnification of the objective increases. Majority of the producers make long-working distance objectives for special applications, for example, in hot-stage microscopy. Achromats are strain free, which is important for polarized light examinations. Since they contain fewer lenses than other more highly corrected lenses, internal reflection losses are minimized.

Semi-apochromatic or fluorite objectives provide a higher degree of correction of spherical and chromatic aberration. Hence, they produce higher quality images than achromats. The apochromatic objectives have the highest degree of correction, produce the best results, and are more expensive. Plano objectives have extensive correction for flatness of field, which reduces eye strain, and are frequently found on modern microscopes.

With par-focal lens systems, each objective on the nosepiece turret is nearly in focus when the turret is rotated, preventing the objective front lens from striking the sample when lenses are switched. Many objectives also are spring loaded, which helps prevent damage to the lens. This is more of a problem with high-magnification objectives, because the working distance can be very small.

Certain objectives are designed for use with oil between the sample and the front lens of the objective. However, oil immersion lenses are rarely used, since the sample and lens are to be cleaned after use. However, they do provide higher resolutions than can be achieved when air is between the lens and sample. In the latter case, the maximum possible NA is 0.95, while the oil-immersion lenses produce a 1.3 NA to 1.45 NA, depending on the lens and the oil used. Magnifications from 25x to 160× are available. Use of oil also sharpens the image, which is valuable when examining low reflectivity specimens, such as coal or ceramics.

Fig 4 Types of objective lens and eyepiece

Eyepiece – It is also called ocular lens. Eyepiece (Fig 4) magnifies the primary image produced by the objective. The eye can then use the full resolution capability of the objective. The microscope produces a virtual image of the sample at the point of most distinct vision, normally 250 mm from the eye. The eyepiece magnifies this image, permitting achievement of useful magnifications. The standard eyepiece has a 24 mm diameter field of view while the wide-field eyepieces for plano-objectives have a 30 mm diameter field of view, which increases the usable area of the primary image.

The simplest eyepiece is the Huygenian eyepiece whose design was invented by C. Huygens. It consists of two plano-convex lenses with their convex surfaces facing the objective lens. It is satisfactory for use with low-power and medium-power achromat objectives. Compensating eyepieces are used with high NA achromat and the more highly corrected objectives. Because some lens corrections are performed using these eyepieces, the eyepiece is to be matched with the type of objective used.

Eye clearance is the distance between the eye lens of the ocular and the eye. For most eyepieces, the eye clearance is 10 mm or less which is inadequate if the person using the microscope wears glasses. Simple vision problems, such as near-sightedness, can be accommodated using the fine focus adjustment. Vision problems such as astigmatism cannot be corrected by the microscope, and glasses are to be worn. High-eye-point eyepieces are available to provide the eye clearance of around 20 mm necessary for glasses.

Eyepieces are normally equipped with various reticles or graticules for locating, measuring, counting, or comparing microstructures. The eyepiece enlarges the reticle or graticule image and the primary image. Both images are to be in focus simultaneously. Special eyepieces are also produced to permit more accurate measurements than can be made with a graticule scale. Examples are the filar-micrometer ocular or screw-micrometer ocular. Such devices can be automated to produce a direct digital readout of the measurement, which is accurate to around 1 micrometer.

A 10× magnification eyepiece is normally used, however to obtain standard magnifications, some systems needs other magnifications, such as 6.3×. Higher power eyepieces, such as 1×, 15×, 20×, or 25×, are also useful in certain situations. The overall magnification is found by multiplying the objective magnification, Mo, by the eyepiece magnification, Me (Fig 2). If a zoom system or bellows is also used, the magnification is to be altered accordingly.

Stage – A mechanical stage is provided for focusing and moving the sample, which is placed on the stage and secured using clips. The stage of an inverted microscope has replaceable centre-stage plates with different size holes. The polished surface is placed against the hole for viewing. However, the entire surface cannot be viewed, and at high magnifications it is not be possible to focus the objective near the edge of the hole due to the restricted working distance. In case of upright microscope, the sample is placed on a slide on the stage. Since the polished surface is to be perpendicular to the light beam, clay is placed between the sample bottom and the slide. A piece of lens tissue is placed over the polished surface, and the sample is pressed into the clay using a leveling press. However, pieces of tissue can adhere to the sample surface. An alternative, particularly useful with mounted samples, is to use a ring instead of tissue to flatten the sample. Aluminum or stainless steel ring forms of the same size as the mounts (flattened slightly in a vise) seat on the mount rather than the sample.

The upright microscope allows viewing of the entire surface with any objective, and the operator can see which section of the sample is being viewed. It is a useful feature when examining specific areas on coated samples, welds, and other samples where specific areas are to be examined. For mounted samples, an auto-leveling stage holder for mounts can eliminate leveling of samples on clay.

The stage is to be rigid to eliminate vibrations. Stage movement, controlled by x- and y- micrometers, is to be smooth and precise and hence rack and pinion gearing is normally used. Many stages have scales for measuring the distances in the x- and y- directions. The focusing controls frequently contain rulings for estimating vertical movement. Some units have motorized stages and focus controls.

A circular rotatable stage plate can facilitate polarized light examination. Such stages, common for mineralogical or petrographic studies, are graduated to permit measuring the angle of rotation. A rectilinear stage is normally placed on top of the circular stage.

Stand – Bench microscopes need a rigid stand, particularly if photo-microscopy is performed on the unit. The various pieces of the microscope are attached to the stand when assembled. In some cases, the bench microscope is placed on a separate stand that also holds the photographic system.

Lens defects

Many lens defects result from the laws of reflection and refraction. The refractive index of a lens varies with the wavelength of light, and the focal length of the lens varies with the refractive index. Hence, focal length changes for different colours of light. A separate image for each wavelength present is focused at different distances from the lens. This is longitudinal chromatic aberration (Fig 5). Moreover, magnification varies with focal length, altering the size of the image. This is lateral chromatic aberration (Fig 5). These differences are to be eliminated to produce colour photographs. Since achromats have limited corrections for these problems, they are to be used with yellow-green light filtering to obtain sharp images. Spherical aberration (Fig 5) occurs when light from a point object on the optical axis is more strongly refracted at the centre or at the periphery of the lens, producing a series of focal positions in which the point image appears as a circle of finite area. This can be minimized by using an aperture which restricts use of the objective to the central portion. Lens design can also correct part of this problem.

Since the image surface of optimum focus is curved, compensating eyepieces with equal but opposite curvature are used to produce a flat image. Other problems, such as coma and astigmatism, can impair image quality unless corrected.

Fig 5 Lens defects


To see micro-structural detail, the optical system is needed to produce adequate resolution, or resolving power, and adequate image contrast. If resolution is acceptable but contrast is lacking, detail cannot be observed. In general, the ability to resolve two points or lines separated by a distance ‘d’ is a function of the wavelength, ‘l’, of the incident light and the numerical aperture, NA, of the objective. This follows the equation ‘d = k.l / NA’, where k is 0.5 or 0.61. Fig 6 shows this relationship for k = 0.61 and four light wavelengths. Other formulas have also been reported. The equation does not include other factors which influence resolution, such as the degree of correction of the objectives and the visual acuity of the person looking through the microscope. It is based on the work of Abbe under conditions not present in metallography, such as self-luminous points, perfect black-white contrast, transmitted-light examination, an ideal point-light source, and absence of lens defects.

Using the equation in previous paragraph, the limit of resolution for an objective with an NA of 1.4 is around 0.2 micrometers. To see lines or points spaced 0.2 micrometers apart, the required magnification is to be determined by dividing the resolving power of the objective by the resolving power of the human eye, which is difficult to determine under observation conditions. Abbe used a value of 0.3 mm at a distance of 250 mm which is the distance from the eye for optimum vision. For light with a mean wavelength of 0.55 micrometers, the needed magnification is 1,100 times the NA of the objective. This is the origin of the 1,000 NA rule for the maximum useful magnification. Any magnification above 1,000 NA is termed ‘empty’, or useless.

Strict adherence to the 1,000 NA rule is to be questioned, considering the conditions under which it has been developed, which are certainly far different from those encountered in metallography. According to the Abbe analysis, for a person using optical microscope with optimum 20/20 vision and for optimum contrast conditions and a mean light wavelength of 550 nm, the lowest magnification which takes full advantage of the NA of the objective is 550 times the NA. This establishes a useful minimum magnification to use with a given objective. It has been suggested that the upper limit of useful magnification for the average person using optical microscope is 2,200 NA, not 1,000 NA.

Fig 6 Relationship between the resolution and depth of field with numerical aperture

Depth of field

Depth of field is the distance along the optical axis over which image details are observed with acceptable clarity. Those factors which influence resolution also affect depth of field, but in the opposite direction. Hence, a compromise is to be reached between these two parameters, which become more difficult as magnification increases. This is one reason light etching is preferred for high-magnification examination.

Examination modes

For achieving the resolution capability of the selected objective, image contrast is to be adequate. Image contrast depends on sample preparation and optics. Differences in light reflectivity from the sample surface produce amplitude features visible to the eye after magnification. Phase differences created by light reflection are to be rendered visible by the use of phase-contrast or interference-contrast attachments to the microscope.

Bright-field illumination – Bright-field vertical illumination, the most widely used method of observation, accounts for the vast majority of micrographs taken. In operation, light passes through the objective and strikes the sample surface perpendicularly. Surface features normal to the incident light reflect light back through the objective to the eyepieces, where the surface features appear bright. Surfaces oblique to the light beam reflect less light to the objective and appear darker, depending on their angle.

Oblique illumination – With some microscopes, it is possible to decentre the condenser assembly or the mirror so that the light passing through the objective strikes the sample surface at a non-perpendicular angle. Roughness on the sample surface casts shadows, producing a three-dimensional appearance. This allows determination of features which are in relief or are recessed. However, very little obliqueness can be introduced, since this technique causes lighting to become non-uniform and reduces resolution.

In dark-field illumination – In dark field illumination, the light reflected from obliquely oriented features is collected, and the rays reflected from features normal to the incident beam are blocked. Hence, the contrast is essentially reversed from that of bright field illumination; that is, features which are bright in bright-field illumination appear dark, and features normally dark appear bright. This produces very strong image contrast, with the oblique features appearing luminous. Under such conditions, it is frequently possible to see features not visible using bright-field illumination. This method is particularly useful for studying grain structures. However, the low light intensity makes photo-microscopy more difficult, a problem lessened by the use of automatic exposure-control devices.

Polarized light – Polarized light, as used in metallography, has normally been limited to observation of certain optically anisotropic metals, such as beryllium, alpha-titanium, zirconium, and uranium, which are difficult to etch but respond well to the polarized light when properly polished. Before development of the electron micro-probe analyzer (EMPA) and energy dispersive spectroscopy (EDS), polarized light examination was an integral part of the method for identifying inclusions. Since the development of these instruments, polarized light has been used less frequently for this purpose, since identification with the EMPA or EDS techniques is more definitive. Most metallurgical microscopes now use synthetic Polaroid filters. The ‘polarizer’ is placed in the light path before the objective, and the ‘analyzer’ is placed in the light path after the objective, normally just below the eyepiece.

Light consists of transverse waves vibrating in all directions at right angles to the direction of propagation. These vibrations occur symmetrically around the direction of propagation and are unpolarized. When light passes through a polarizing filter, the vibrations occur in only one plane in the direction of propagation, and the light is termed plane polarized. This plane changes as the filter is rotated. When the analyzer filter is placed in the light path, plane polarized light passes through it if the plane of vibration of the light is parallel to the plane of vibration of the analyzer. If the plane of vibration of the analyzer is perpendicular to that of the light the light does not pass through, and extinction results. When plane-polarized light is reflected from the surface of an isotropic metal (any metal with a cubic crystallographic structure, such as iron), then passes through the analyzer in the crossed position (plane of vibration perpendicular to that of the plane-polarized light), the image is extinguished, or dark. However, in practice, since the metallurgical microscope does not produce perfectly plane-polarized light, complete extinction does not occur. This is not a serious problem, since polarized light is used only in a qualitative manner in metallography. Strain-free objectives, normally achromats, are to be used. Fluorite or apochromatic objectives are unsuitable. A strong white-light source is needed to produce accurate colour effects.

If an optically anisotropic, polished metal is placed under the light beam with the polarizer and analyzer crossed, the microstructure is revealed. The quality of sample preparation is very important, and the surface is to be perpendicular to the light path. Rotation of the sample under the beam changes light intensity and colour. Since it is difficult to set the polarizer and analyzer in the crossed position accurately when an anisotropic sample is in place unless the crossed positions are marked on the polarizer and the analyzer, it is best to find this position first using an isotropic sample.

When plane-polarized light strikes an anisotropic metal surface, reflection occurs as two plane-polarized components at right angles to each other. The directions vary with crystal structure. The strength of these two perpendicular reflections can change, and a phase difference exists between them. These differences vary with each metal and depend on the crystal orientation. No reflection is obtained when the basal plane of hexagonal or tetragonal crystals is perpendicular to the light beam. Maximum reflectance occurs when the principal symmetry axis of the crystal is perpendicular to the light beam. The resultant image is predominantly influenced by these orientation effects with phase differences are of little significance.

When the analyzer is crossed with respect to the polarizer, rotation of plane-polarized light from the anisotropic surface allows the light to pass through the analyzer, producing an image in which each grain has a different light intensity and colour, depending on its crystal orientation relative to the light beam. As the stage is rotated, each grain changes four times in intensity from light to dark during a 360 degree rotation. If the phase difference is appreciable, the light is elliptically polarized, the difference in intensity in each grain with rotation is less, and extinction is not observed. Colour images are obtained when the reflected plane-polarized light varies with wavelength. When little colour is present, a sensitive tint plate inserted between the polarizer and the objective enhance colouration.

Isotropic metals can be examined using crossed-polarized light if the surface can be rendered optically active by etching, staining, or anodizing. Procedures have been developed for several metals, however, all etched surfaces do not respond to polarized light. Normally, the etch s to produce etch pits or facets in each grain to cause double reflection at these features. Grains with different crystal orientations produce differently oriented pits or facets, yielding different degrees of elliptical polarization and hence varying light intensity. Anodizing produces a thick oxide film on the sample surface and irregularities in the film lead to double reflection.

Although the polarization response of anodized samples has been attributed to optical anisotropy of the film, experimentation has shown that the effect is due to film surface irregularities. Tint etchants produce surface films which result in interference colours which can be enhanced using polarized light. In general, best results are achieved when the analyzer is shifted slightly from the crossed position. In addition to its use in examining inclusions, anisotropic metals (antimony, beryllium, bismuth, cadmium, cobalt, magnesium, scandium, tellurium, tin, titanium, uranium, zinc, and zirconium, for example), and etched / anodized/ tint-etched cubic metals, polarized light is useful for examination of coated or deformed metals. Phase identification can also be aided in some cases. The internal structure of graphite nodules in cast iron is vividly revealed using polarized light. Martensitic structures are frequently better revealed using polarized light, which illustrate lath martensite in a high-strength iron-base alloy.

Phase contrast illumination – It permits examination of subtle phase variations in microstructures with little or no amplitude contrast from differences in the optical path at the surface (reflected light) or from differences in the optical path through the sample (transmitted light). Differences of height as small as 0.005 micrometers can be detected. Application of phase-contrast illumination in metallography has been limited. The technique needs a separate set of objectives and a special vertical illuminator.

Interference-contrast illumination – Differential interference-contrast illumination produces images with emphasized topographic detail similar to those observed using oblique illumination. Detail which is invisible or faintly visible using bright-field illumination can be revealed vividly with interference-contrast illumination. Examples of the topographic detail which can be revealed using differential interference-contrast illumination are the relative hardness of the constituents or the nature of the etching process, that is, which areas or constituents are attacked by the etchant. In some cases, other aspects of the structure can be revealed which are invisible or faintly visible in bright-field illumination.

Interference techniques – Several interference techniques are used to measure height differences on samples. Interference fringes on a perfectly flat surface appear as straight, parallel lines of equal width and spacing. Height variations cause these fringes to appear curved or jagged, depending on the unit used. The interference microscope divides the light from a single point source into two or more waves which are superimposed after traveling different paths. This produces interference. Two-beam and multiple-beam instruments are the two basic types of interferometers used. The measurements are based on the wavelength of the light used. Two-beam interferometers can measure height differences as small as ‘l’/20; multiple-beam interferometers, as small as ‘l’/200.

The Linnik-type interferometer is a two-beam reflecting microscope which uses non-polarized light. A beam-splitting prism produces two light beams from a monochromatic light source. One beam travels through the test piece objective to the test piece surface and is reflected back through the objective to the eyepiece. The other beam travels through the reference objective, strikes an optically flat reference mirror, and returns to the beam splitter, then to the eyepiece. If the path difference between the two beams is not equal or not a multiple of ‘l’/2, interference occurs and contour lines are formed which indicate locations of equal elevation. The height difference between adjacent fringes is ‘l’/2.

The Tolansky multiple-beam interferometer produces interference between many light beams by placing a reference mirror which is partially transmitting and partially reflecting very near the sample surface but slightly out of parallel. The reference mirror has a known reflectivity selected to approximate that of the surface. Light passes through the reference mirror and strikes the sample surface, is reflected by the sample surface, and interferes with the rays reflected between the reference mirror and the sample. The fringes produced by the multiple-beam interferometer are sharper than those from the two-beam interferometer, which accounts for the greater accuracy. The distance between the fringes is also ’l’/2. Elevations produce displacements of the fringes from parallel alignment. The displacement is compared to the distance between the fringes to obtain height measurements.

Light-section microscopy – The light-section microscope, also used to measure surface topography, complements interference techniques. Roughness differences from 1 micrometer to 400 micrometers can be measured, which is useful in examining machined surfaces and for measurement of surface layers or films. In operation, a slit is placed near the field iris in the illumination system and is imaged by an objective as a light line on the surface to be measured. Oblique illumination is used with a dark background. The light band is observed using a second objective which is identical to the first. The objectives are 45 degrees to the sample surface and 90 degrees to each other. A reticle in the eyepiece is used for measurements, or they are made on photographs. Vertical resolution is not as good as with interferometers, but lateral resolution is better.

Auxiliary techniques

Several special devices can be used with the optical microscope to get additional information. These techniques are described below.

Micro-hardness testing – Micro-indentation hardness data can be obtained by adding indenter attachments to the microscope. Single-purpose units also are made by many manufacturers of hardness test equipment. Loads are normally made from 1 g (gram) to 1,000 g, although some manufacturers have units for low loads (0.05 g to 200 g). Knoop or Vickers indenters can be used.

Hot-stage microscopy – Hot-stage microscope cells are available from several manufacturers. Single-purpose units can also be used. Cold-cell attachments have also been produced, but have rather limited use in metallography. The hot-stage microscope has been used to study phase transformations on heating or cooling or at constant temperature. Examination of reactions in the hot-stage microscope cell needs use of long-working-distance objectives, since the sample is held within the cell. Moreover, since the cell window is quartz, the objectives is to be quartz-corrected, especially those with magnifications of 20× or more.

Techniques other than chemical etching are to be used to view phase changes. Grain boundaries are to be thermally etched if the sample is held at a constant temperature in the vacuum. Grain-boundary grooving is easily observed using bright field illumination. Phase transformations are visible by the relief produced at the surface. Hence, shear reactions, such as those produced by martensite or bainite formation, are most easily observed. Other phase transformations are more difficult or impossible to observe. Transformations can be photographed in situ, for which motion picture cameras are normally used.

Special stages – These are available in a variety of configurations. Auto leveling stages for mounted samples are a typical example. Universal tilting stages have also been made for rapid manipulation of rough, irregular samples. Special stages have also been designed for handling small objects. A number of stages have been made for performing in situ experiments. Basic studies of solidification have been performed by in situ observation of the freezing of low-melting-point organic materials, such as camphene, which solidify like metals. Observation of the recrystallization of low-melting-point metals and alloys has been similarly observed. Special stages have been used to observe the progress of electrolytic polishing and etching. Cells have also been used for in situ examination of corrosion processes. Stages have been designed to observe a variety of processes involving static or dynamic stress, and devices have also been designed to permit physical extraction of inclusions.

Hot-cell microscopy – Metallographic preparation of radioactive materials needs remote-control preparation using specially designed hot cells. Special microscopes have been designed for use with the hot cell.

Field microscopy – When the microstructure of a component or large object which cannot be cut and moved to the laboratory is to be examined, portable laboratory equipment, made by several manufacturers, can be used to polish a section in situ. A portable microscope can be sometimes used to examine and photograph the microstructure. If this cannot be done, replicas can be made and examined using an optical microscope or an electron microscope.

Comparison microscopes – The need occasionally arises to compare two microstructures. Normally, this is carried out by placing micrographs from each sample side-by-side, but it can also be performed using special microscopes. A bridge comparator is used to combine images from two bench microscopes for simultaneous viewing.

Television monitors – Projection microscopes can be used for group viewing, but it is more common to display the microstructure on a black-and-white or colour monitor. A number of high-resolution closed-circuit systems are available.

Clean-room microscopy – The study of small particles is influenced by dust contamination during viewing. Hence, such work is to be performed in a clean box, clean bench, or clean room which is specially made to provide a dust free environment.

Image analyzers – The increased use of quantitative metallography, particularly for characterization of inclusions, has promoted development of automated image analysis systems based on television principles. Phases or constituents of interest are detected primarily by differences in light reflectivity which produce gray-level differences on the monitor. Majority of the stereological measurements can be made using these systems. Considerable automation has been achieved using automated stages and powerful minicomputers. Although these devices can be quite expensive, they have stimulated interest in stereology and its application to structure-property correlations.

Features are detected on as-polished or etched samples, depending on the nature of the feature of interest. If etching is needed, selective techniques are normally used. Field and feature-specific measurements are utilized. Field measurements measure all the detected features simultaneously, as in volume fraction measurements. In feature-specific measurements, each separate particle is measured sequentially. This procedure is normally used for shape and size measurements.

Some structures do not lend themselves to accurate measurements using such systems. For example, quantification of fracture surface detail cannot be performed using an automatic image analyzer, since the device cannot separate fracture features by gray level. Many transmission electron micrograph structures also cannot be analyzed using these devices. For such structures, semi-automatic tracing devices can be used with the operator performing detection with a light pen or stylus. These lower-cost systems can be used for nearly any stereological measurement. Because of the greater time needed for detection, they are less suitable for measurement problems which need sampling of many fields.


Prior to the development of photographic attachments, microstructures were to be sketched. Although the need for such documentation is no more there, sketching remains useful as a teaching method. Photo-microscopy is important in metallography, since the photo-micrograph can faithfully reproduce the detail observed for others to view. With the equipment presently available, high-quality micrographs are easily produced. However, this needs careful attention to sample preparation, etching, and use of the microscope. Reproduction of false microstructures is all too common and has caused inaccurate interpretations, rejection of good materials, and faulty conclusions in failure analyses.

Historically, darkroom photographic procedures have been most prevalent. Since the introduction of instant photographic processes such as Polaroid, however, many photo-micrographs have been made using these materials, taking advantage of their speed and efficiency. However, image reproduction is sacrificed, and the process is to be repeated for each extra copy. Use of an automatic exposure device is necessary with instant process film to minimize waste. Traditional darkroom photographic methods need more effort, but yield better micrographs. Considerable automation in wet darkroom processes is possible, but frequent use of photo-microscopy is needed to justify the cost of such equipment.

Obtaining good micrographs needs adequate image contrast and resolution, uniform focus over the entire field, uniform lighting, and adequate depth of field. The light source is to be properly aligned, and the system is to be free of vibration. The yellow-green filter is to be employed to correct lens defects. The optics is to be clean, and the field and aperture diaphragms are to be adjusted correctly. The microscope is focused in a variety of ways, depending on the model. Several film formats can be used, such as plates, sheet film of different size, or 35-mm roll film. The magnification at the film plane is to be known. This is a simple procedure if the only variables are the objective and eyepiece magnification, but is more difficult when using a zoom system or bellows. A stage micrometer can be utilized to determine the true magnification.

A range of black-and-white and colour films is available for darkroom or instant techniques. The manufacturers of these films document film characteristics. Black-and-white films are normally used due to their lower cost. They show better contrast control, are easier to process, and are normally quicker to use than colour films. Colour film has some important uses for which its cost is justified. In traditional black-and-white photography, a negative image is produced first and is used to produce a positive image of the microstructure on suitable paper. The micrograph lasts for many years without any apparent change. Selection of the negative film is based on the format available, colour sensitivity, contrast, resolving power, speed, graininess, and exposure and development latitudes.

Some black-and-white films are not sensitive to the entire visible spectrum. Orthochromatic films are sensitive to all colours except orange and red. Panchromatic films are sensitive to all colours, although they emphasize blue and de-emphasize yellow. A yellow filter can be used to reduce this colour bias.

Orthochromatic films can be developed under dark red light, but panchromatic films need total darkness. Orthochromatic films are very good for photo-microscopy, particularly when a yellow-green filter is inserted to correct lens defects.

Film speed is a critical variable only when illumination is low, as in polarized light, interference-contrast, or dark-field illumination. Orthochromatic film has a medium contrast which is adequate for most structures. Contrast can be enhanced with a high-contrast film. The resolving power of a film defines its ability to record fine details in the image. Hence, a high-resolving-power film is desirable. Graininess depends on the size of the silver grains in the emulsion, the developer used, and the development time and temperature. High-speed films are grainier than low-speed films, making them less suitable for enlarging. Contact printing is preferred. It needs a large film size, but saves enlargement time. It produces better images and eliminates re-determining the magnification of the print. A fine-grain film provides the best resolution.

When a negative is exposed, there is an allowable range of exposures which produces a useful, printable negative. Wide exposure latitude is quite valuable. Each film includes information on its characteristic relationship between exposure time and density. The exposure selected is to be on the linear portion of the density-time curve. A good, dense negative allows suppression of some of the fine image defects during printing. An underexposed negative greatly restricts printing and normally results in a poor print. Development of negatives is rather simple and involves use of a developing solution, a stop bath, a fixing solution, as well as washing and drying.

The correct exposure is most easily determined using a built-in exposure meter. If this is not available, a test exposure series can be made. This is accomplished by pulling out the film slide completely and exposing the entire film for a time judged to be considerably shorter than that needed. The slide is then inserted so that it covers around 10 mm to 20 mm of the film, and the exposure is repeated. This is repeated incrementally until the slide is fully inserted, covering the film. After development, the correct time can be assessed based on the density of the negative in each band.

Alternatively, the step exposure can be performed using an instant film of the same speed, saving the darkroom time. Majority of the black-and-white films are contact printed. The negative is placed emulsion side up on the contact printer, and a suitable paper is placed emulsion side down over the negative. The printer is closed, and light is passed through the film onto the paper. The print is developed, stopped, fixed, washed, and dried. Print contrast is controlled by the type of paper and development time. Print contrast types vary from extra-soft (flat) to extra-contrast (grades 1 to 5). Number 3 paper is used most frequently. Number 4 paper is used to increase contrast, and No. 2 paper to reduce contrast.

Instant process films eliminate the darkroom work, thus hastening the process. Polaroid prints use the diffusion-transfer reversal process. Development begins when the film is removed from the camera after the exposure. The action of pulling the film out of the camera crushes a pod containing the viscous, caustic developer and spreads it over the film. Black-and-white films develop rapidly while the colour prints need slightly more time. Some of the Polaroid films have very high speeds, an advantage in dim lighting. Some prints are to be coated with a neutralizing stabilizer / protective varnish to prevent staining and fading. Also available are instant films which produce a negative and a positive print. This negative is to be cleared, but a darkroom is not required. Polaroid films used in microscopy are all panchromatic. They are available as roll film, film packs, or sheets. Exposure times are to be more accurately controlled to get good prints than with traditional wet-process films.


Examination and photography are frequently needed for such objects as macro-etched disks and broken parts. Examination can be performed visually or with the aid of a simple hand lens or stereo-microscope. Macro-photography can be performed using majority of the cameras, perhaps aided by the use of close-up lens attachments, a bellows, or a macro-lens. Many stereomicroscopes can be equipped with cameras for photography while some takes stereo-pairs. A few manufacturers offer camera stands for macro-photography. Some metallographs also have low-magnification objectives which can perform certain types of macro-photography.

Macro-photography utilizes magnifications from less than 1× to 50×. Most laboratories, especially those engaged in failure analyses, have various cameras, light sources, and stereo-viewers to cover the wide range of objects photographed. Correct lighting is necessary to emphasize details and provide even illumination without glare or reflection. Adjustment of lighting needs some experimentation and experience. Available lighting includes flood lamps, rings, coaxial, or fiber optics. A light box is useful for eliminating shadows, but considerable creativity is needed to achieve good results.

Depth of field and resolution are important variables. Many of the objects to be photographed are three-dimensional, which needs a certain depth of field and proper lighting to reveal shape and texture. Depth of field varies with the aperture diaphragm lens setting, the magnification, and the focal length of the lens. Stopping down the aperture improves depth of field, but decreases image brightness and clarity. Depth of field also increases as magnification decreases and focal length increases. For magnifications below 5×, focal lengths of 100 mm or more are preferred. Shorter-focal-length lenses are used for higher magnifications.

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