Oblique Illumination

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- Oblique Illumination - Introduction

Anyone who has ever examined a coin in direct, bright light will have observed that the relief on the surface of the coin is very difficult to see under these illumination conditions, due to specular reflections from the surface of the coin. On the other hand, when incident light is arranged to "strike" the coin at a low glancing angle, the resulting shadow effect on one side and brightness on the other side (nearer the light) cause the relief detail of the coin to stand out in three-dimensional clarity.

A somewhat similar appearance is produced when examining microscopic specimens with a classical technique known as oblique illumination (sometimes referred to as anaxial illumination). Direct light restricted to a single azimuth of the condenser light cone is allowed to illuminate the specimen from one side only, as illustrated in Figure 1. The net effect is to reveal details in pseudo-relief from an otherwise almost invisible, colorless specimen. A typical configuration for oblique illumination in compound transmitted light microscopy is presented in Figure 1. Beneath the condenser lens system is positioned an opaque light stop having a small slit (or sector stop), which is removed from the central optical axis of the microscope. Illuminating light rays from the source pass through the slit and subsequently through the condenser lens element system from a single azimuth that is off-axis. The oblique light rays then illuminate the specimen from a highly specific angle, on one side, and enter the microscope objective through the front lens element near the edge of the aperture (very close to the lens mount). The focused light rays converge at the objective rear focal plane and are directed to the microscope intermediate image plane, where they are observed through the eyepieces.

Oblique lighting has caused the zeroth order to be moved to a position just within the periphery of the objective aperture (this effect can be observed with an eyepiece telescope or Bertrand lens focused at the rear focal plane of the objective). The consequence of oblique illumination is to shift the zeroth order of light passing through the specimen from the center to one peripheral side of the objective front lens element. This shift of the zeroth order to a single side enables one or more additional higher orders of the diffracted light to be included at the rear focal plane of the objective and contribute to formation of the image. Only diffracted orders on a single side of the zeroth order (often termed sidebands) are admitted to the objective and, because of the obliquity of illumination, diffracted orders on the other side miss the objective altogether. This concept is illustrated in Figure 2 utilizing ray traces and a simple lens to represent the microscope objective.

In general, when transparent specimens are illuminated in transmitted brightfield microscopy with a parallel bundle of light rays, the resulting images have poor visibility and lack any significant degree of contrast. This occurs because the sum of the light diffracted by each detail of the specimen is a quarter-wavelength out of phase with direct light passing through the specimen when both are recombined at the image plane. However, the diffracted light emanating from each specimen point contains sidebands (see Figure 2) on either side of the zeroth order undeviated light, which are offset from the quarter wave displacement by a defined margin. If one sideband of the diffracted light is prevented from reaching the image plane, constructive and destructive interference will occur between the remaining sideband and the direct light to produce a visible image at the intermediate image plane of the microscope. Oblique illumination is an excellent mechanism to achieve the sideband suppression necessary for the image formation process.

In Figure 2, the uppermost ray trace system identifies the pathway for axial light rays passing through the specimen and into the microscope optical system for a standard brightfield configuration. Symmetrical components of the diffracted light are admitted into the objective and produce complimentary patterns having no significant interference (either constructive or destructive) with the direct light components at the image plane, leading to images having poor contrast and visibility. With oblique illumination (the lower ray trace system in Figure 2), several of the diffracted component sidebands are not captured by the objective, but at least one higher order component is admitted along with the zeroth order (the second order sideband (D(-2)) is not included at the objective rear focal plane in the figure to increase the clarity of the illustration). Interference at the image plane produces an image of the specimen having significantly more contrast and visibility. The appearance of diffraction maxima at the objective rear focal plane for both axial and oblique illumination is also presented in the figure. Because only two maxima are necessary for image formation, oblique illumination will produce a visible image.

Oblique illumination is capable of resolving very fine specimen detail that is difficult to distinguish using conventional brightfield techniques. When oblique light rays strike the objective front lens at angle "ob" to the microscope optical axis, and the refractive index of the medium between the condenser front lens and the objective is n, the relationship between resolution, illuminating wavelength (λ), and refractive index can be described as:

Resolution (D) = λ/(n • sin (ob)) + Numerical Aperture (NA)

Some authors equate the term n • sin (ob) with the numerical aperture of illumination, but this is misleading because the equation is not intended to signify a full cone of light emanating from all azimuths, but is restricted to a beam of light produced by anaxial illumination from a single azimuth. In cases where specimen detail is so fine that the zeroth order undiffracted and first order diffracted sideband light are separated by a distance equal to the diameter of the objective aperture, the resolving power is twice as high as observed for axial transmitted illumination and is expressed by the equation:

Resolution (D) = λ/2NA

The optical conditions required for the microscope to conform with the equation above (when utilizing oblique illumination) are such that the fine details of specimen periodicity are limited by the resolving power of the objective. In many cases, specimen details that are resolved utilizing brightfield illumination are so severely lacking in contrast that they can scarcely be visualized or imaged. The net result of observing specimens with oblique illumination is often an increase in resolution (over that obtained in brightfield illumination with a closed condenser aperture diaphragm) and also the production of a shadowed, relief-like pseudo three-dimensional appearance in the image of the specimen.

Oblique Illumination Light Pathways

Examine how off-axis light rays can be collected by the microscope objective when illumination is shifted from axial (brightfield) to oblique (off-axis).

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Oblique illumination techniques are ideal for imaging a wide variety of unstained objects such as living cells, crystals, diatoms, and similar transparent or semi-transparent specimens. However, the resulting images must be viewed and interpreted with caution because the diffracted orders from one side have not contributed to the image formation. Potential false structures appearing in the image can seriously limit the usefulness of oblique light to examine and quantitatively describe previously unobserved specimen detail. This fact should always be taken into consideration when observing specimens with oblique illumination.

Achieving conditions necessary for oblique illumination, which has been employed to enhance specimen visibility since the dawn of microscopy, can be accomplished by a variety of techniques with a simple transmitted optical microscope. Perhaps the easiest methods are to offset a partially closed condenser iris diaphragm or the image of the light source. In former years, some microscopes were equipped with a condenser having a decenterable aperture iris diaphragm (illustrated in Figure 3; note that the lens system of the condenser should not be displaced from the center of the microscope optical axis). The device was engineered to allow the entire iris to move off-center in a horizontal plane so that closing the circular diaphragm opening would result in moving the zeroth order to the periphery of the objective rear focal plane. In advanced models, the entire diaphragm was rotatable around the axis of the microscope so that oblique light could be directed toward the specimen from any azimuth to achieve the best desired effect for a given specimen.

In practice, utilizing a partially closed condenser iris diaphragm technique to achieve oblique lighting is plagued with problems similar to those hampering brightfield observation. The results are a general loss of resolution and superimposition of diffraction rings, which surround and confuse the interpretation of minute specimen detail. In addition, Becke lines and other undesirable optical effects originating from regions of the specimen that are not in exact focus complicate the image. To circumvent many of these problems, a combination of oblique illumination at large condenser numerical aperture, coupled to video contrast enhancement, has been utilized as an effective method to generate optical thin sections that strongly resemble those observed with differential interference contrast (DIC) microscopy.

Although oblique illumination may be configured by decentering the condenser aperture, a majority of the condensers currently sold by the microscope manufacturers do not have this capability. An alternative technique involves lateral displacement of the tungsten lamp filament from the microscope optical axis, as illustrated in Figure 4. The diagram in Figure 4(a) presents the image of a focused filament, which has been translated to the right by adjustment of the lamp position in the lamphouse at the rear of the microscope. When a diffusion filter is inserted into the light path (Figure 4(b)), the light is spread to fill the condenser aperture with a gradient of illumination having the proper orientation for viewing specimens with the oblique method. Optimum gray tones can be achieved, in most cases, by placing the filament image in a position to cover approximately half of the condenser aperture diaphragm (as illustrated in Figure 4). However, careful adjustment of the filament position and condenser aperture, coupled to the utilization of diffusion screens having differing opacities, should allow the microscopist a significant degree of latitude in order to fine-tune modulation of image contrast.

A more commonly employed alternative for establishing variable degrees of oblique illumination is the use of sector stops (Figure 5) placed just below the lower lens element and aperture diaphragm of the condenser. Many microscopes are equipped with a filter holder positioned near the bottom of the condenser, providing an ideal location for insertion of a sector stop. Illustrations of several sector stop designs are presented in Figure 5. In the past, sector stops were available from the microscope manufacturers, but they are increasingly difficult to find today. However, simple stops can be made from a section of opaque paper or cardboard and cut to fit the filter tray or taped to the underside of the condenser.

Each sector stop has a section that blocks light (labeled stop in Figure 5) and another area that allows only oblique light to pass through (labeled pass in Figure 5) and illuminate the specimen. The size of the stop and pass sections often vary with objective magnification and numerical aperture, and proper size determination is largely an experimental effort on the part of the microscopist to achieve the optimum balance. The sector stop can be rotated within the filter holder to illuminate the specimen from a variety of angles, or (more conveniently) a circular rotating stage can be employed to rotate the specimen through 360 degrees to achieve the desired oblique lighting effect. These suggestions can be employed as a guideline, but the individual microscopist should make final adjustments to both the condenser and sector stop configuration while observing the effects on the specimen image to optimize oblique illumination and enhance contrast and detail.

Oblique illumination can dramatically increase specimen contrast when compared to conventional brightfield techniques, as illustrated by the digital images presented in Figure 6. The specimen in Figure 6(a) is a bovine arterial cell that has been imaged using brightfield illumination with the condenser aperture adjusted for maximum contrast. It is obvious that the specimen is almost invisible and details are very difficult to distinguish in brightfield illumination. In contrast, when the specimen is illuminated obliquely using sector stops placed near the condenser aperture (Figure 6(b)), contrast is dramatically increased and many cellular details, including location of the nucleus and pseudopods, become visible. This particular specimen is difficult to image, regardless of the contrast enhancement technology employed by the microscopist (including phase contrast, differential interference contrast, darkfield, or Hoffman modulation contrast).

A somewhat easier specimen to image is the mouse kidney thick section presented in Figures 6(c) and 6(d). In brightfield illumination (Figure 6(c)), some specimen detail is visible, but specific features are difficult to distinguish and the overall image suffers from a serious lack of contrast. Utilizing sector stops to illuminate the specimen obliquely produces a significant improvement in contrast, as shown in Figure 6(d). In this digital image, minute specimen details become visible that were previously difficult to observe in brightfield illumination.

When compared to the darkfield technique, where the specimen is illuminated from all azimuths with highly oblique light, asymmetrical oblique illumination produces images whose character is highly dependent upon the incident angle of illumination. Images produced by oblique illumination are asymmetrical in the sense that edges lying perpendicular to the direction of incident illumination are made visible, while those that lie parallel (or close) to this direction are not. This concept is illustrated in Figure 7 for two specimens at different orientations with respect to the angle of incident oblique illumination. The specimen in Figures 7(a) and 7(b) consists of identical viewfields (but rotated by 90 degrees) obtained from a thin single-crystal wafer of lanthanum aluminate, a perovskite that is commonly employed as a substrate for epitaxial thin film deposition of high-temperature superconducting ceramics. Twinning in these crystals hampers confluent thin film formation and can have detrimental effects on the properties of resulting films. The digital image presented in Figure 7(a) depicts the pseudo-relief generated by twinning domains when the crystal is oriented with the longitudinal twin axis parallel to the oblique incident light rays. In contrast, when the crystal (and twin) axis is rotated by 90 degrees, so that it is perpendicular to the incident rays (Figure 7(b)), the twinning domains become readily apparent. This represents a spectacular display of specimen orientation restrictions on the textural effects observed under oblique illumination.

A similar, but less dramatic, result is obtained when observing semi-transparent goat hair specimens with oblique illumination at several orientation angles. When the long axis of the rod-like hair strands is oriented parallel to the incident oblique illumination (Figure 7(c)), structural details in the central portion and edges of the hair fibers is revealed. This detail is absent when the hair fibers are oriented perpendicular to the illumination axis (Figure 7(d)), and a significant difference is observed in the apparent thickness of the fibers between the two orientation angles. Fibers oriented parallel to the incident illumination appear to be much thicker than those oriented perpendicular to the light source. Thus, it is evident that the oblique illumination technique cannot be reliably employed to generate faithful measurement data from images gathered by this method.

Oblique Illumination

Specimens that are nearly transparent and colorless may be almost invisible when viewed in the stereomicroscope using traditional transmitted (diascopic) brightfield illumination techniques. This occurs because light diffracted by minute specimen detail is a quarter-wavelength out of phase with direct light passing through the specimen when both are recombined in the intermediate image plane, a classical phenomenon that seriously reduces contrast in brightfield images.

Figure 1 - Nikon Oblique Coherent Contrast Illumination Field

However, if the illumination is directed so that it originates from a single azimuth and strikes the specimen from an oblique angle, details in the specimen may be revealed with much greater contrast and visual clarity than when the light is allowed to pass directly through specimen features along the optical axis of the microscope. Phase and refractive index gradients in the specimen deflect the light rays by diffraction, reflection, and refraction, so that only the zeroth order (undiffracted) and one or two sidebands of diffracted light can recombine at the image plane. This produces a relief-like specimen pattern having regions displaying shadows and highlights, much like that observed with the differential interference contrast (DIC) technique in compound microscopes.

Presented in Figure 1 is a modern stereomicroscope illumination stand (the Nikon Oblique Coherent Contrast (OCC) diascopic model), which is designed to illuminate specimens through a transitional mechanism ranging from axial brightfield to highly oblique off-axis light rays that render transparent specimens visible in a scheme closely resembling darkfield. The stand contains both a high and low numerical aperture condenser enabling utilization of the entire stereomicroscope objective magnification (generally 0.5x to 2x) and numerical aperture (0.07 to 0.21) range. Oblique illumination is achieved by means of a sliding diaphragm that shields the center of the light beam to produce a partially coherent light source, which is projected obliquely onto the specimen, producing a high contrast image. The diaphragm position is controlled by means of a rotating knob that can be employed to adjust the obliquity of illumination.

Specimens are placed on the glass stage of the Nikon OCC diascopic stand and can be illuminated either in brightfield, darkfield, or varying degrees of oblique illumination, by rotating the diaphragm control knob. A series of digital images captured at successive levels of oblique illumination are illustrated in Figure 2 for a semi-transparent Nemathelminth (hookworm; Ancylostoma caninum) specimen. As is evident in Figure 2(a), the unstained hookworm is semi-transparent, and very little detail is revealed under brightfield axial illumination. However, when the sliding diaphragm is rotated into the light path, increasing degrees of oblique illumination can be achieved (Figures 2(b), 2(c), and 2(d)), with the most extreme position corresponding closely to darkfield illumination from a single azimuth. The change in specimen contrast possible with this illumination stand is most dramatically portrayed by comparing Figures 2(a) and 2(c), which were produced in brightfield and highly oblique coherent contrast, respectively.

Figure 2 - Hookworm (<em>Ancylostoma caninum</em>) in OCC Illumination

One of the major design criteria of the Nikon oblique stereomicroscope illumination stand is to enable enhancement of image contrast, such as might be achieved with iris diaphragms, while maintaining a high condenser numerical aperture capable of matching that displayed by apochromatic objectives. Many stereomicroscopes having the parallel or common main objective (CMO) design are coupled to illumination stands that are equipped with iris diaphragms, either built-in or available as accessories. Current Nikon CMO design stereomicroscopes compatible with OCC diascopic stands include the SMZ25/18, SMZ1270/1270i, and SMZ800N series. In order to increase depth of field, the diaphragm aperture is often reduced in size, which also serves to increase image contrast in diascopic illumination. Unfortunately, reducing the iris diaphragm size also renders the image-forming light rays more coherent, and compromises the resolving power of the microscope by reducing the working numerical aperture of the objective.

The oblique coherent contrast illumination system combines the effect of oblique illumination with the coherence enhancement obtained by reducing condenser aperture diaphragm size, and creates images in the stereomicroscope that have an appearance similar to differential interference contrast images produced by a compound microscope. Instead of using an iris aperture diaphragm, the oblique coherent contrast system employs a baffle that acts as a sliding diaphragm. The linear diaphragm mechanism behaves as an iris would if placed at the same position, except that the objective retains most of its resolving power. This can be achieved because the numerical aperture of the illumination cone is reduced, but that of the objective lens is not.

Interactive Tutorial - Oblique Coherent Contrast Illumination

Enhancing contrast in stereomicroscopy with transmitted light.

The illumination pathways that characterize the Nikon oblique stereomicroscope system are illustrated in Figure 3, which also presents the light pathway for normal brightfield (diascopic) conditions. A collector lens system focuses an image of the lamp filament onto a mirror positioned at a 45-degree angle with respect to both the lamp and specimen optical axes. The sliding diaphragm is a baffle that can be translated across the surface of the mirror to block light that would ordinarily pass directly through the specimen, ensuring that only oblique light is utilized to illuminate specimen detail. A condenser lens positioned above the mirror maintains the coherence of illumination by bringing the sharp edge of the baffle into focus at the rear focal plane of the objective. The rear pupil of the objective will be only partially filled with light, as determined by the sliding baffle position (functioning as an iris diaphragm).

With this system, the full numerical aperture of the objective is maintained. One of the advantages of the Nikon oblique system (over other oblique illumination techniques) is the ease of translating between brightfield, oblique, and darkfield illumination by means of a single adjustment knob, which controls the position of the sliding baffle. The appearance of the specimen with the baffle moved away from the mirror is equivalent to that seen in normal brightfield illumination. With the baffle moved up to the top of the mirror, the image will be similar to darkfield, and in between these extremes, true oblique illumination conditions are obtained. The degree of obliquity is easily adjusted to suit the specimen and the type of detail that is desired.

Figure 3 - Oblique Coherent Contrast Lightpaths

Oblique illumination is similar in many aspects to the darkfield technique except that, instead of the specimen being lighted from all directions at oblique angles, light is projected from only a single azimuth. A variety of illumination scenarios have been employed to provide oblique directional lighting for observation of specimens with the stereomicroscope (an example is presented in Figure 4). Simple diascopic bases are often equipped with a tilting mirror that can be adjusted to provide a certain degree of oblique illumination, but the light is not easily controlled and does not provide a uniform field of view. More complex microscope stands (or bases) have additional control possibilities, including tilting mirrors that are not restricted to a single axis and sliding mirror assemblies that can be inserted and removed from the light path. Some models use one or more sliding baffles to restrict the illumination geometry and ensure that only oblique light strikes the specimen.

Any of these techniques can provide acceptable to excellent results on a wide variety of specimens, but most require a significant degree of manipulation involving the illumination pathway, which is often difficult to quantitatively analyze or reproduce. Involving more art than science, the results achieved with oblique illumination in stereomicroscopy are highly dependent on the microscopist's skill, experience, and patience. A large number of lighting schemes have been developed, which further complicate the issues surrounding this technique for enhancing specimen contrast.

Figure 4 - Oblique/Brightfield Stereomicroscope Stand

The mechanism by which oblique illumination enhances detail in otherwise almost invisible, colorless specimens is best understood by consideration of the oblique illumination technique as it is commonly described for the classical compound microscope. Direct light from one azimuth of the substage condenser illuminates the specimen from a single side. Oblique illumination is generally achieved by placing a slit or sector stop beneath the lower lens and aperture diaphragm of the condenser, allowing only oblique light passing through the narrow opening of the stop to illuminate the specimen. The effect of the oblique lighting is to shift the zeroth order of light passing through the specimen to the periphery of the objective lens aperture. The shift of the zeroth order to one side enables one or more additional higher orders (sidebands) of the diffracted light to be included at the rear focal plane of the objective and to contribute to the image formation. In many instances, the result is in an increase in optical resolution because the zeroth and some higher orders both contribute to image formation. In addition, the technique also produces an image having shadowed, high relief features that render the specimen pseudo three-dimensional in appearance.

When compared to the darkfield technique, where the specimen is illuminated from all azimuths with highly oblique light, asymmetrical oblique illumination produces images whose character is highly dependent upon the incident angle of illumination. Images produced by oblique illumination are asymmetrical in the sense that edges lying perpendicular to the direction of incident illumination are made visible, while those that lie parallel (or close) to this direction are not. This concept is illustrated in Figure 5 for two specimens at different orientations with respect to the angle of incident oblique illumination. The specimen in Figures 5(a) and 5(b) consists of identical viewfields obtained from a thin single-crystal wafer of lanthanum aluminate, a perovskite that is commonly employed as a substrate for epitaxial thin film deposition of high-temperature superconducting ceramics. Twinning in these crystals hampers confluent thin film formation and can have detrimental effects on the properties of resulting films. The digital image presented in Figure 5(a) depicts the pseudo-relief generated by twinning domains when the crystal is oriented with the longitudinal twin axis parallel to the oblique incident light rays. In contrast, when the crystal (and twin) axis is rotated by 90 degrees, so that it is perpendicular to the incident rays (Figure 5(b)), the twinning domains become readily apparent. This represents a spectacular display of specimen orientation restrictions on the textural effects observed under oblique illumination.

Figure 5 - Orientation Angle Effects of Specimen Appearance

A similar, but less dramatic, result is obtained when observing semi-transparent goat hair fibers with oblique illumination at several orientation angles. When the long axis of the rod-like hair strands is oriented parallel to the incident oblique illumination (Figure 5(c)), structural details in the central portion and edges of the hair fibers is revealed. This detail is absent when the hair fibers are oriented perpendicular to the illumination axis (Figure 5(d)), and a significant difference is observed in the apparent thickness of the fibers between the two orientation angles. Fibers oriented parallel to the incident illumination appear to be much thicker than those oriented perpendicular to the light source. Thus, it is evident that the oblique illumination technique cannot be reliably employed to generate faithful measurement data from images gathered by this method.

The apparent three-dimensional effect afforded by oblique illumination techniques does not represent the actual specimen geometry or topography, and should not be employed to conduct measurements of specimen dimensions. The true value of the oblique illumination image is in revealing transitions in refractive index or other optical path differences within the specimen that enable the morphology and internal structural arrangement to be more clearly understood. The technique can be applied to a variety of materials that appear nearly invisible or transparent in brightfield illumination and cannot be stained or otherwise chemically or thermally treated to enhance contrast. Study of living organisms and processes such as in vitro fertilization, glass or acrylic fibers, chemical crystals, and other unstained materials can be facilitated by the utilization of an easily controlled oblique illumination system.

Oblique Illumination -

or why knocking the mirror or condenser out of alignment sometimes helps!

by Dave Walker

A brief survey of the benefits of using off-axis lighting to view some subjects, and how it can be easily accomplished without cost for most microscopes. Also a look at some rather bizarre 19th century microscopes which took the use of oblique illumination a bit too far!

If, like the author, you own a microscope with a mirror for external lighting, you may well have accidentally discovered the benefit of oblique illumination for studying some subjects. Microscope mirrors are occasionally knocked out of alignment at some stage during viewing, with the result that the subject is lit with an off-axis light source. This oblique illumination is in fact a useful way of improving the visibility of some low contrast subjects like protozoa and the details on diatoms.

Essentially oblique illumination works by accentuating any phase gradients within a transparent specimen. It's an easy form of lighting to achieve up to medium powers (e.g. 40x objectives) with most microscopes with or without a mirror, and is a 'cheap and cheerful' technique the amateur can use to improve visibility when studying low contrast subjects, especially if you're not fortunate enough to possess phase contrast or high power dark field illumination.

To illustrate its effect, the image right shows part of the classic diatom Pleurosigma angulatum using a 40x achromatic objective (Russian, NA 0.65 dry). This is one of the cheapest 40x objectives you can buy, but is perfectly capable of resolving the dots i.e. the fine detail on the silica shell, but in normal brightfield this detail is quite difficult to see. Only the central 'raphe' (on the right of the image) and an edge of the diatom frustule at the left can be made out.

It's not resolution that's the problem, it's contrast. If the diatom is studied with an oblique patch stop in the filter tray (see below) the image right is seen. The dots which are fine markings on the diatom frustule are now clearly seen. (Incidentally I have to push my ancient image capture card to the limit to capture these sort of images, so they are a lot less convincing than the visual image down the microscope).

It's also possible to resolve the dots under dark-field with this objective, but I have never found using home-made patch stops for a 40X dry objective very easy and have achieved less than successful dark-field, so oblique illumination is an easier alternative to improve contrast.

In a second example, the image left and right shows the classic epithelial cells taken by scraping the inside of the cheek lightly with the finger nail and mounting the saliva under a cover slip (again using the 40x dry objective). The image left is in brightfield with the iris stopped down more than normal to improve contrast - it's an acceptable image but rather flat. The same view with the oblique patch stop gives a very pleasing depth to the cell especially the nucleus, cell granules and folding of the cell (again, the visual image is a lot clearer than in the captured image).

Oblique illumination also works well on single cells such as protozoa. One book I referred to (ref. 1) illustrates that the internal vacuoles are particularly distinctive in this sort of illumination.

If you haven't tried the technique, how can it be achieved? A few methods are given below.

Knock the mirror!

As commented above, if you have a microscope with a mirror, moving the mirror (plane side) so that light is shone off-axis can achieve the effect. This one of the few techniques for improving contrast that the owner of a simple microscope without a condenser can achieve i.e. unlike dark-field or phase contrast. I first spotted the effect myself as a ten year old with a no-frills toy microscope, and I got into the habit of rocking the mirror on its axis when studying transparent live pond-life to see if more details were revealed.

Simple stop for the condenser filter tray

If you have a condenser and filter tray, a simple stop can be made as shown left. Just cut out a piece of opaque card to fit the filter tray and experiment with various sizes of sector cut out of the disc for a given objective to allow light from an angle to reach the subject. Normal axial illumination should be setup first. As for dark field, the condenser iris should be fully open and the condenser focus adjusted to get the best effect.

You may need to experiment with the size of the notch for a given objective. The orientation of the notch with respect to the subject should also be changed to see what achieves the best effect. If you have a rotating stage this is easier than rotating the patch stop in the filter tray. If the filter tray is a swing out type, also experiment with the extent the filter is swung in, as well as varying the orientation of the notch in the filter tray.

Condenser with oblique illumination

Buying a condenser specifically for oblique illumination is probably not justified, but this is a feature found in some condensers. My Russian Biolam microscope is supplied with a modest Abbe condenser, but for critical work I use the better quality Russian aplanatic condenser (N.A. 1.4). As a bonus this also has an oblique illumination feature, see image right. (Supplementary low power lens N.A. 0.3 also shown).

The iris can be moved up to 10mm off-axis in opposite directions, which also rotates in the mount to change the orientation of the light to the subject. The latter feature, as mentioned above for the oblique patch stop, is important as the off-axis lighting often works best in a specific orientation to a subject e.g. for non-symmetric diatoms like Pleurosigma.

Historical footnote

In the late19th century but before the advent of high numerical aperture objectives, oblique illumination was quite popular and some makers developed microscopes especially to exploit the benefits of the technique. One of the first models was shown by Zentmayer of Philadelphia in 1876 at an Exposition in this city. It had a swinging substage, i.e. the whole substage assembly swung about an axis in the same plane as the stage. An image of this microscope is in the Moody Medical Library On-line (item 1.028 in catalogue). (This site illustrates a wonderful selection of historical microscopes from the famous Moody collection, most makers are represented).

Some makers designed microscopes that took oblique illumination to rather extreme limits. The classic example is the Ross Radial microscope built by the famous English maker Ross to a design by Wenham. This is a superb example of quality engineering and optics by a respected maker, but a rather bizarre microscope nevertheless. Almost every conceivable plane and axis of the microscope limb, body and substage has the facility to be tilted or rotated (see ref. 2 or similar history of microscopes where this model is often illustrated). An image of this microscope is also in the Moody Medical Library On-line (item 1.031 in catalogue).

The technique fell out of favour towards the end of the 19th century as high numerical aperture objectives became available with the ability to resolve the fine detail of diatoms etc. with good axial illumination. A firmer understanding of the requirements for a critical image with axial illumination were also realised.

I wasn't sure how well the technique is used nowadays given the many other ways of improving contrast. However, a web search for 'oblique illumination' shows that some of the mainstream microscope makers mention this form of lighting as a feature in some of their models (particularly metallurgical), so presumably it does still have a worthwhile role. The oblique patch stop is certainly a cheap and cheerful way for the amateur to improve contrast for some subjects, especially at medium powers where patch stops for dark-field get trickier to make.

I was also intrigued to see that at least one manufacturer, 'Edge', market a microscope with 'patented Multiple Obliquetm Illumination' which apparently gives 3D images. Visit the Edge web site for details and read the theory behind the technique for 3D imaging.

Comments on the article to the author Dave Walker welcomed.

References

1) 'Under the Microscope' by A. Curry, R. F. Grayson and G. R. Hosey, (Blandford Press, UK, 1982), Chapter 8. Now out of print, but worth looking out for in second-hand bookshops as it gives clear explanations of many techniques and principles, especially the use of polarised light.

2) 'The Microscope Past and Present' by S. Bradbury. Pergamon Press, UK, 1968, pp. 180-183. A compact but thorough overview of the history of the microscope and it's development. Illustrates and discusses the Ross Radial and Ross / Zentmayer microscope and the fad for oblique illumination.

Introduction to Oblique Illumination

Anyone who has ever examined a coin in direct, bright light will have observed that the relief on the surface of the coin is very difficult to see under these illumination conditions, due to specular reflections from the surface of the coin. On the other hand, when incident light is arranged to "strike" the coin at a low glancing angle, the resulting shadow effect on one side and brightness on the other side (nearer the light) cause the relief detail of the coin to stand out in three-dimensional clarity.

A somewhat similar appearance is produced when examining microscopic specimens with a classical technique known as oblique illumination (sometimes referred to as anaxial illumination). Direct light restricted to a single azimuth of the condenser light cone is allowed to illuminate the specimen from one side only, as illustrated in Figure 1. The net effect is to reveal details in pseudo-relief from an otherwise almost invisible, colorless specimen. A typical configuration for oblique illumination in compound transmitted light microscopy is presented in Figure 1. Beneath the condenser lens system is positioned an opaque light stop having a small slit (or sector stop), which is removed from the central optical axis of the microscope. Illuminating light rays from the source pass through the slit and subsequently through the condenser lens element system from a single azimuth that is off-axis. The oblique light rays then illuminate the specimen from a highly specific angle, on one side, and enter the microscope objective through the front lens element near the edge of the aperture (very close to the lens mount). The focused light rays converge at the objective rear focal plane and are directed to the microscope intermediate image plane, where they are observed through the eyepieces.

Oblique lighting has caused the zeroth order to be moved to a position just within the periphery of the objective aperture (this effect can be observed with an eyepiece telescope or Bertrand lens focused at the rear focal plane of the objective). The consequence of oblique illumination is to shift the zeroth order of light passing through the specimen from the center to one peripheral side of the objective front lens element. This shift of the zeroth order to a single side enables one or more additional higher orders of the diffracted light to be included at the rear focal plane of the objective and contribute to formation of the image. Only diffracted orders on a single side of the zeroth order (often termed sidebands) are admitted to the objective and, because of the obliquity of illumination, diffracted orders on the other side miss the objective altogether. This concept is illustrated in Figure 2 utilizing ray traces and a simple lens to represent the microscope objective.

In general, when transparent specimens are illuminated in transmitted brightfield microscopy with a parallel bundle of light rays, the resulting images have poor visibility and lack any significant degree of contrast. This occurs because the sum of the light diffracted by each detail of the specimen is a quarter-wavelength out of phase with direct light passing through the specimen when both are recombined at the image plane. However, the diffracted light emanating from each specimen point contains sidebands (see Figure 2) on either side of the zeroth order undeviated light, which are offset from the quarter wave displacement by a defined margin. If one sideband of the diffracted light is prevented from reaching the image plane, constructive and destructive interference will occur between the remaining sideband and the direct light to produce a visible image at the intermediate image plane of the microscope. Oblique illumination is an excellent mechanism to achieve the sideband suppression necessary for the image formation process.

In Figure 2, the uppermost ray trace system identifies the pathway for axial light rays passing through the specimen and into the microscope optical system for a standard brightfield configuration. Symmetrical components of the diffracted light are admitted into the objective and produce complementary patterns having no significant interference (either constructive or destructive) with the direct light components at the image plane, leading to images having poor contrast and visibility. With oblique illumination (the lower ray trace system in Figure 2), several of the diffracted component sidebands are not captured by the objective, but at least one higher order component is admitted along with the zeroth order (the second order sideband (D(-2)) is not included at the objective rear focal plane in the figure to increase the clarity of the illustration). Interference at the image plane produces an image of the specimen having significantly more contrast and visibility. The appearance of diffraction maxima at the objective rear focal plane for both axial and oblique illumination is also presented in the figure. Because only two maxima are necessary for image formation, oblique illumination will produce a visible image.

Oblique illumination is capable of resolving very fine specimen detail that is difficult to distinguish using conventional brightfield techniques. When oblique light rays strike the objective front lens at angle "ob" to the microscope optical axis, and the refractive index of the medium between the condenser front lens and the objective is n, the relationship between resolution, illuminating wavelength (l), and refractive index can be described as:

Resolution (D) = l/(n • sin (ob)) + Numerical Aperture (NA)

Some authors equate the term n • sin (ob) with the numerical aperture of illumination, but this is misleading because the equation is not intended to signify a full cone of light emanating from all azimuths, but is restricted to a beam of light produced by anaxial illumination from a single azimuth. In cases where specimen detail is so fine that the zeroth order undiffracted and first order diffracted sideband light are separated by a distance equal to the diameter of the objective aperture, the resolving power is twice as high as observed for axial transmitted illumination and is expressed by the equation:

Resolution (D) = l/2NA

The optical conditions required for the microscope to conform with the equation above (when utilizing oblique illumination) are such that the fine details of specimen periodicity are limited by the resolving power of the objective. In many cases, specimen details that are resolved utilizing brightfield illumination are so severely lacking in contrast that they can scarcely be visualized or imaged. The net result of observing specimens with oblique illumination is often an increase in resolution (over that obtained in brightfield illumination with a closed condenser aperture diaphragm) and also the production of a shadowed, relief-like pseudo three-dimensional appearance in the image of the specimen.

Interactive Tutorial

Oblique Illumination Light Pathways

Examine how off-axis light rays can be collected by the microscope objective when illumination is shifted from axial (brightfield) to oblique (off-axis).

Oblique illumination techniques are ideal for imaging a wide variety of unstained objects such as living cells, crystals, diatoms, and similar transparent or semi-transparent specimens. However, the resulting images must be viewed and interpreted with caution because the diffracted orders from one side have not contributed to the image formation. Potential false structures appearing in the image can seriously limit the usefulness of oblique light to examine and quantitatively describe previously unobserved specimen detail. This fact should always be taken into consideration when observing specimens with oblique illumination.

Achieving conditions necessary for oblique illumination, which has been employed to enhance specimen visibility since the dawn of microscopy, can be accomplished by a variety of techniques with a simple transmitted optical microscope. Perhaps the easiest methods are to offset a partially closed condenser iris diaphragm or the image of the light source. In former years, some microscopes were equipped with a condenser having a decenterable aperture iris diaphragm (illustrated in Figure 3; note that the lens system of the condenser should not be displaced from the center of the microscope optical axis). The device was engineered to allow the entire iris to move off-center in a horizontal plane so that closing the circular diaphragm opening would result in moving the zeroth order to the periphery of the objective rear focal plane. In advanced models, the entire diaphragm was rotatable around the axis of the microscope so that oblique light could be directed toward the specimen from any azimuth to achieve the best desired effect for a given specimen.

In practice, utilizing a partially closed condenser iris diaphragm technique to achieve oblique lighting is plagued with problems similar to those hampering brightfield observation. The results are a general loss of resolution and superimposition of diffraction rings, which surround and confuse the interpretation of minute specimen detail. In addition, Becke lines and other undesirable optical effects originating from regions of the specimen that are not in exact focus complicate the image. To circumvent many of these problems, a combination of oblique illumination at large condenser numerical aperture, coupled to video contrast enhancement, has been utilized as an effective method to generate optical thin sections that strongly resemble those observed with differential interference contrast (DIC) microscopy.

Although oblique illumination may be configured by decentering the condenser aperture, a majority of the condensers currently sold by the microscope manufacturers do not have this capability. An alternative technique involves lateral displacement of the tungsten lamp filament from the microscope optical axis, as illustrated in Figure 4. The diagram in Figure 4(a) presents the image of a focused filament, which has been translated to the right by adjustment of the lamp position in the lamphouse at the rear of the microscope. When a diffusion filter is inserted into the light path (Figure 4(b)), the light is spread to fill the condenser aperture with a gradient of illumination having the proper orientation for viewing specimens with the oblique method. Optimum gray tones can be achieved, in most cases, by placing the filament image in a position to cover approximately half of the condenser aperture diaphragm (as illustrated in Figure 4). However, careful adjustment of the filament position and condenser aperture, coupled to the utilization of diffusion screens having differing opacities, should allow the microscopist a significant degree of latitude in order to fine-tune modulation of image contrast.

A more commonly employed alternative for establishing variable degrees of oblique illumination is the use of sector stops (Figure 5) placed just below the lower lens element and aperture diaphragm of the condenser. Many microscopes are equipped with a filter holder positioned near the bottom of the condenser, providing an ideal location for insertion of a sector stop. Illustrations of several sector stop designs are presented in Figure 5. In the past, sector stops were available from the microscope manufacturers, but they are increasingly difficult to find today. However, simple stops can be made from a section of opaque paper or cardboard and cut to fit the filter tray or taped to the underside of the condenser.

Each sector stop has a section that blocks light (labeled stop in Figure 5) and another area that allows only oblique light to pass through (labeled pass in Figure 5) and illuminate the specimen. The size of the stop and pass sections often vary with objective magnification and numerical aperture, and proper size determination is largely an experimental effort on the part of the microscopist to achieve the optimum balance. The sector stop can be rotated within the filter holder to illuminate the specimen from a variety of angles, or (more conveniently) a circular rotating stage can be employed to rotate the specimen through 360 degrees to achieve the desired oblique lighting effect. These suggestions can be employed as a guideline, but the individual microscopist should make final adjustments to both the condenser and sector stop configuration while observing the effects on the specimen image to optimize oblique illumination and enhance contrast and detail.

Oblique illumination can dramatically increase specimen contrast when compared to conventional brightfield techniques, as illustrated by the digital images presented in Figure 6. The specimen in Figure 6(a) is a bovine arterial cell that has been imaged using brightfield illumination with the condenser aperture adjusted for maximum contrast. It is obvious that the specimen is almost invisible and details are very difficult to distinguish in brightfield illumination. In contrast, when the specimen is illuminated obliquely using sector stops placed near the condenser aperture (Figure 6(b)), contrast is dramatically increased and many cellular details, including location of the nucleus and pseudopods, become visible. This particular specimen is difficult to image, regardless of the contrast enhancement technology employed by the microscopist (including phase contrast, differential interference contrast, darkfield, or Hoffman modulation contrast).

A somewhat easier specimen to image is the mouse kidney thick section presented in Figures 6(c) and 6(d). In brightfield illumination (Figure 6(c)), some specimen detail is visible, but specific features are difficult to distinguish and the overall image suffers from a serious lack of contrast. Utilizing sector stops to illuminate the specimen obliquely produces a significant improvement in contrast, as shown in Figure 6(d). In this digital image, minute specimen details become visible that were previously difficult to observe in brightfield illumination.

When compared to the darkfield technique, where the specimen is illuminated from all azimuths with highly oblique light, asymmetrical oblique illumination produces images whose character is highly dependent upon the incident angle of illumination. Images produced by oblique illumination are asymmetrical in the sense that edges lying perpendicular to the direction of incident illumination are made visible, while those that lie parallel (or close) to this direction are not. This concept is illustrated in Figure 7 for two specimens at different orientations with respect to the angle of incident oblique illumination. The specimen in Figures 7(a) and 7(b) consists of identical viewfields (but rotated by 90 degrees) obtained from a thin single-crystal wafer of lanthanum aluminate, a perovskite that is commonly employed as a substrate for epitaxial thin film deposition of high-temperature superconducting ceramics. Twinning in these crystals hampers confluent thin film formation and can have detrimental effects on the properties of resulting films. The digital image presented in Figure 7(a) depicts the pseudo-relief generated by twinning domains when the crystal is oriented with the longitudinal twin axis parallel to the oblique incident light rays. In contrast, when the crystal (and twin) axis is rotated by 90 degrees, so that it is perpendicular to the incident rays (Figure 7(b)), the twinning domains become readily apparent. This represents a spectacular display of specimen orientation restrictions on the textural effects observed under oblique illumination.

A similar, but less dramatic, result is obtained when observing semi-transparent goat hair specimens with oblique illumination at several orientation angles. When the long axis of the rod-like hair strands is oriented parallel to the incident oblique illumination (Figure 7(c)), structural details in the central portion and edges of the hair fibers is revealed. This detail is absent when the hair fibers are oriented perpendicular to the illumination axis (Figure 7(d)), and a significant difference is observed in the apparent thickness of the fibers between the two orientation angles. Fibers oriented parallel to the incident illumination appear to be much thicker than those oriented perpendicular to the light source. Thus, it is evident that the oblique illumination technique cannot be reliably employed to generate faithful measurement data from images gathered by this method.

The apparent three-dimensional effect afforded by oblique illumination techniques does not represent the actual specimen geometry or topography, and should not be employed to conduct measurements of specimen dimensions. The true value of the oblique illumination image is in revealing transitions in refractive index or other optical path differences within the specimen that enable the morphology and internal structural arrangement to be more clearly understood. The technique can be applied to a variety of materials that appear nearly invisible or transparent in brightfield illumination and cannot be stained or otherwise chemically or thermally treated to enhance contrast. Study of living organisms and processes such as in vitro fertilization, glass or acrylic fibers, chemical crystals, and other unstained materials can be facilitated by the utilization of an easily controlled oblique illumination system.

Refractive Index Determination by Oblique Illumination

Oblique illumination is sometimes utilized as an alternative to the Becke line test to determine whether the refractive index of a specimen is higher or lower than that of the surrounding medium. If the specimen is mounted in a medium of lower refractive index, shading that results from the anaxial illumination will appear on the side opposite to that from which the light enters the specimen, and vice versa, as illustrated in Figure 8. For both diagrams presented in Figure 8, two equal sized oblique light rays are depicted entering the specimen through the surrounding medium at the same angle of incidence. At point A on the left-hand diagram, the light is spread over a larger area of the specimen than at point B, so that the area near point A on the specimen appears darker than the area near point B. Under these conditions, one side of the specimen will appear shaded or somewhat darker than the other side when viewed through the microscope eyepieces (A' and B' in the upper left portion of Figure 8). This is the case when the specimen refractive index is higher than that of the surrounding medium.

The opposite effect occurs when the specimen has a lower refractive index than that of the surrounding medium (right-hand side of Figure 8). In this case, the shaded or darker side of the specimen will be on the side that is nearest to the oblique light sector stop. When the specimen and the surrounding medium have identical refractive indices, then the specimen will be transparent (or invisible) and will have no refractive effects on the oblique illumination. The sensitivity of this refractive index determination technique is highly dependent on the condenser focal length, the iris diaphragm position, and the geometry of sector stops (if employed). In general, the best results are obtained when the condenser is carefully focused and an even field of illumination is achieved.

Oblique Illumination in Reflected Light

Reflected or incident light oblique illumination, although largely displaced by differential interference contrast and darkfield techniques, is often useful for examination of metals and other specimens in the field of metallurgy. A comparison between the basic configurations for transmitted and reflected light oblique illumination is presented in Figure 9. The diagram in Figure 9(a) illustrates the principal optical axes for axial (brightfield), oblique, and darkfield transmitted illumination. A simple lens simulates the microscope objective and yellow arrows represent hypothetical light rays oriented at the respective angles necessary to achieve the desired illumination mode. The angle q refers to one-half the objective angular aperture. Light rays that are directed along the microscope optical axis are representative of axial illumination, while those lying between the axis and the limiting angle q are captured by the objective front lens and form oblique illumination. Light rays having an incident oblique angle larger than q do not enter the objective and represent darkfield illumination.

Principal light paths for reflected light or epi-illumination are presented in Figure 9(b). Axial (brightfield) epi-illumination proceeds in a direction parallel to the microscope optical axis from the vertical illuminator to the half mirror, where it is reflected through the objective lens system and onto the specimen. In contrast, oblique epi-illumination is reflected onto the specimen by a 45-degree prism or mirror mounted at the edge of the objective. These light rays pass through the periphery of the objective front lens element and strike the specimen obliquely, but are reflected, refracted, and diffracted back through the objective. As is the case in Figure 9(a), the value q represents the one-half objective angular aperture through which all rays of axial and oblique illumination are directed. Light rays having a higher angle represent darkfield epi-illumination, which can originate from an external light source or an annular mirror or lens system that surrounds the objective. In Figure 9, only one imaging ray is illustrated for each illumination mode, but in reality, these light rays will propagate in all directions.

Oblique reflected or epi-illumination is suitable for the examination of periodic or linear structures on the surface of metals, ceramics, glasses, polymers, and other opaque specimens. For optimum results, the azimuth of illumination should be positioned at a 90-degree angle to the longitudinal direction of the structures under observation. The technique is very useful for examination of relief in surface structures (especially at lower magnifications), and can be utilized to demonstrate whether a structure on the surface of the specimen is a cavity or protrusion by determining the position of shadows relative to the illumination azimuth. In reflected oblique illumination, specimens should be examined through the complete range of inclination angles and azimuth directions available with the instrumentation to obtain the best possible analysis. If possible, a rotating circular stage should be employed, and the specimen rotated through a 360-degree angle to capture all essential surface detail. It should be noted that because oblique illumination can produce artifacts such as color fringes and directional contrast, the microscopist should compare oblique images to those obtained in brightfield illumination before attempting to interpret the results.

Advanced Oblique Illumination Techniques

Several microscopy techniques related to oblique illumination, including phase contrast and Hoffman modulation contrast, take advantage of fundamental optical parameters of the microscope. Specifically, these methods are based on the fact that the condenser and objective lens apertures are in principal conjugate planes and that the illuminating beam arising from each point of the condenser aperture is diffracted at a different angle (according to its spatial frequency) by the specimen. Another basic concept, utilized by these techniques, is the fact that the objective rear aperture is the Fourier optical plane of the microscope.

In Hoffman modulation contrast microscopy, a sophisticated derivative of the oblique illumination technique, the condenser aperture is fitted with a slit mask having the slit positioned near the edge of the aperture. The objective rear focal plane contains a second, complementary mask, termed a modulator, which consists of three zones having differing degrees of transmittance. Oblique illumination passing through the condenser slit is projected onto the central zone of the modulator and forms an image of the slit in this zone. The central zone has an optical density allowing only 15 percent of the light entering the zone to be transmitted through the modulator, while a darker (but smaller) zone transmits only 1 percent of the light. The largest area of the modulator plate comprises the third zone, which is transparent, and passes essentially all of the incident light (minus that lost due to reflection and absorption). The objective modulator plate (mask) thus attenuates the zeroth-order light undiffracted by the specimen (that passing through the zone of 15 percent transmission) while the light diffracted toward the dark sector (1 percent transmission) is blocked. The light diffracted by the specimen away from the dark sector of the mask passes unchanged, resulting in a shadow-cast image similar in appearance to that produced by differential interference contrast, which enhances gradients of refractive indices or of optical path differences in the specimen.

A related technique, known as single-sideband edge enhancement or SSEE utilizes an adjustable half-stop in the condenser front focal plane to occlude a portion of the condenser aperture. A complementary spatial filter located in the objective conjugate rear focal plane attenuates and controls the phase shift of the undiffracted light beam transmitted through the microscope. The degree of attenuation and the phase shift of the background (direct) wave, relative to light scattered and diffracted by the specimen into the aperture region obscured by the half-stop, are adjustable. Images generated by this technique are again similar in appearance to those obtained by differential interference methods, but the single-sideband approach, unlike DIC, does not require polarization optical elements to sandwich the specimen. The system can be adjusted to detect extremely minute phase differences in specimens, as well as anisotropies of refractive indices at high resolution. The technique takes advantage of the fact that only one of the two diffraction sidebands must be captured in order to gain the information regarding the spatial frequency in the specimen that gives rise to that particular diffraction angle. As presented in Figure 10, single-sideband edge enhancement contrast provides a high-spatial-frequency modulation transfer function superior to that provided by other popular modes of contrast generation.

Conclusions

The optical effects that occur when a microscope objective is illuminated at its margin vary considerably according to lens quality, correction for aberration, and the specimen characteristics. When the illumination beam falls partially outside the objective aperture, as it does in oblique illumination, the result is a reduction of zeroth order light intensity relative to the sideband intensity, and a diffraction pattern is generated at the edge of the objective aperture. For this reason, the illuminating cone should be restricted to less than the full objective aperture to avoid diffraction artifacts (such as interference fringes). Careful experimentation with condenser illumination methodology should ensure an even illumination field at restricted apertures.

There are several advantages to oblique illumination when compared to differential interference contrast and phase contrast. Birefringence inherent in a specimen does not confuse oblique illumination images, as is the case with DIC, but phase details are clearly observed in directionally shadowed images from the oblique illumination. A good example is vertebrate axons, which are more clearly imaged with oblique illumination due to their highly birefringent myelin sheaths that create excessive contrast in DIC. In addition, equipment costs are significantly less for oblique illumination than with either DIC, phase contrast, or Hoffman modulated contrast, because the only requirement is a properly configured brightfield microscope. In some cases, oblique illumination techniques enable the microscopist to image deeper into tissue layers than is possible with DIC. Finally, resolution is not compromised over that attainable with brightfield, and contrast can be dramatically enhanced.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.