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Types of Illumination

Now we turn to the specifics of setting up the microscope for the various types of illumination. More information about the specimen becomes available as it is viewed using different illumination setups, since each setup accentuates different aspects of the specimen.

Make a careful record of any setup that works well. Once you know how to set your microscope up for a specialized kind of illumination, returning to that setup will be easy. With each setup, try to determine what is happening to the light as it travels through the optical system.

Brightfield Illumination

This is the kind of illumination that most microscopes come ready to use -- basic transmitted light illumination. Resolution is high and colors are true. Brightfield is a good illumination for thin, stained tissue sections, and for most other stained specimens. Because economy microscopes excel at brightfield illumination, owners of these microscopes should become adept at staining, embedding, and sectioning of specimens. These techniques will be discussed later.

For many specimens, brightfield illumination is the wrong choice. Brightfield gives very few clues about the three-dimensional shape of the object under observation. Unstained transparent objects do not create enough contrast, and often can not be seen at all because they do not stand out from the liquid or resinous mounting medium.

For research microscopes, the basic brightfield setup for objectives powered higher than 4X is called Köhler illumination. True Köhler illumination cannot be achieved on just any microscope, but it can be approximated by the following method:
1) Put a slide with a familiar specimen on the stage, look into the microscope, and focus on the slide.
2) If the microscope has a focusing condenser, close the field diaphragm, look into the microscope, and change the condenser focus until the outline of the field diaphragm appears sharply on the specimen. If the microscope has no field diaphragm, improvise one as discussed earlier. If the microscope has a fixed condenser, you may be able to achieve this step by experimenting with magnifiers under the stage.
3) If the lamp has a focusing lens and no diffusing filter, use this lens to focus the image of the lamp filament on the plane of the closed aperture diaphragm. It is easier to use a small mirror than to lower your head to look at the bottom of the diaphragm. If the light can be moved, make sure the illumination is centered by removing the eyepiece and looking down the body tube at the back focal plane of the objective. Replace the eyepiece.
4) Open the aperture diaphragm completely. Center the field diaphragm carefully, then open it just past the field of view.
5) Remove the eyepiece and look down into the tube at the objective. Close the aperture diaphragm until about one third of the diameter of the bright circular field is blocked out. Replace the eyepiece.

Setting the field diaphragm is important because the aperture diaphragm cannot do its job properly if light is allowed to reach it at too great an angle. Glare and low contrast result.

The explanation for the setting of the aperture diaphragm is more complex. Diffraction is the phenomenon of light waves bending as they pass near the edge of an obstruction.

In addition to reflecting and refracting light, structures in the specimen diffract light. The best resolution results when the objective can capture all of the rays diffracted from a structure and reunite them with the undiffracted light in the image. This allows for maximum interference between diffracted and undiffracted wavefronts. Interference between wavefronts causes light and dark area contrast. Assuming that the field diaphragm is set correctly, diffracted light leaves the specimen at greater angles than the transmitted light, and the ratio of diffracted light to transmitted light entering the objective increases as the aperture diaphragm becomes smaller. An equal ratio between the amount of diffracted and transmitted light is needed. If the aperture is too small resolution suffers; if it is too large, the specimen shows little contrast. The correct setting gives the most contrast possible without halos and other unwanted image artifacts.

Anytime you experiment with lenses beneath the stage, both the field and aperture diaphragms may need to be reset. With most microscopes, illumination must be reset with each change of objective. For objectives 4X and below, insert a diffuser, remove the top condenser lens, or insert a negative lens just below the condenser. Judge the result visually.

Polarized Illumination

Special polarization microscopes are used by scientists studying inorganic materials of all kinds including metal alloys, ceramics, metals, minerals, and crystals. These microscopes have rotating stages and slots or internal sliders for the placement of the polarizing components. They also have specialized strain-free optics that do not interfere with polarizing contrast or add to the polarization.

Surprisingly good polarization effects can be created using a standard microscope. All that is needed is a couple of pieces of polarizing material. Polarizer experimentation kits can be ordered from suppliers. Camera shops and optics suppliers sell higher quality polarizing filters that are designed for use with cameras but work well with microscopes.Such a simple microscope conversion is not sufficient for advanced tasks in chemical and petrological microscopy, but will provide many clues to the nature of your specimen that are invisible under any other kind of illumination.

One way of explaining polarized light uses the wave theory of light. This theory assumes that light is a wave traveling away from the light source. Normally, light moving through the microscope will be in waves that are oriented in random directions. Think of a sheet of polarizing material as a sheet of tiny slits that will only allow waves oriented in a single direction to pass through. Let's say that we have a polarizer oriented so that only east-west oriented waves pass upward. Then place another polarizer above the first. This second polarizer is called the analyzer. If the analyzer is oriented so that only north-south waves can pass, then none of the light that strikes it will pass through. This is so because all of the light striking the analyzer will be in the form of east-west waves. The only way that we can get any light to pass through the analyzer is to place some material between it and the first polarizer that will alter some of the east-west light so that it is north-south oriented. Figure 2.19 shows such a set-up. (Polarization can also be explained with the particle theory of light. The various parts of the system under this theory affect the direction of proton particles' spin instead of the direction of waves.)

Molecules in many substances, both mineral and biological, have the ability to alter polarized light. These are called birefringent molecules. The name comes from their ability to create double refraction. Two refracted beams are created from one beam by a material that has two different refractive indices. Only the birefringent parts of the specimen are visible through crossed polarizers. These parts will be displayed in interference colors -- colors created by the interaction of the polarized light with the birefringent molecules and the analyzer.

The figure demonstrates the setup for polarization with a standard microscope. On the left we see a user looking at a specimen that contains birefringint molecules between two polarizers. To the right, the rest of the microscope is added to the picture. The user simply rotates the analyzer until the background of the specimen appears darkest, at which point the polarizer and analyzer will be crossed. Be careful not to let the analyzer scratch against the eyepiece; use a rubber eyepiece guard.

If the polarizers are not completely crossed, a hybrid of brightfield and polarization will occur. One will see the usual objects that are visible under brightfield illumination with birefringent parts highlighted.

Some polarization kits contain retarders that can be placed anywhere between the polarizer and analyzer to alter the character of the polarization. Retarders are made of birefringent material. Cellophane can be substituted if no other retarder is available. Try retarders both below the specimen and between the analyzer and eyepiece. A 1/4 cycle retarder will change the linearly polarized light to circularly polarized light. The retarder splits the linear beam into two linear beams that have the same wave length and intensity, but are polarized at a 90 degree angle to one another and travel in the same direction. (Normally with birefringent materials, the two waves will leave at different angles.) The phases of these two beams are retarded to different extents. This means that the wavefronts of the two beams no longer coincide. Leaving the retarder, the beams, which are traveling in the same direction, recombine to form a single beam. The net result is that the direction of polarization of the emerging beam rotates. A 1/2 cycle retarder simply shifts the direction of polarization by 90 degrees. Other retarders produce elliptical polarization. Devices that allow continuously adjustable retardation are called compensators. These are only available on specialized microscopes. However, many variations of polarization can be reached by rotating two retarders in relation to one another.

Circularly and eliptically polarized light will interact with the analyzer in different ways than linearly polarized light. It is possible, for instance, to show polarization effects in objects on a light background. Retarders and compensators can also be used to locate weakly birefringent parts of the specimen.

Beneath the stage, always place polarizing filters on top of all other filters and other components. Acrylic sheeting, plastic filters, acetate, and stressed glass will change the polarization of light as it passes through. For the same reason, polarization will not work well if your specimen is in a plastic petri dish, on a plastic slide, or under a plastic cover slip.

Polarized illumination is worth trying when looking at any kind of fiber, hair, bone, chitin, muscle, crystal, protozoan, fish scale, botanical section, or mineral. Plastic and cellophane films are also interesting when stretched or crushed and used as specimens. In the section on crystals, we will discuss another way to use polarization.

Centrally Stopped Setups: Darkfield, Annular Brightfield, and Rheinberg Illumination

Darkfield illumination presents a bright specimen on a dark background. Research microscopes are equipped with special darkfield condensers. Happily, it is possible to improvise darkfield illumination on almost any compound microscope.

For low power objectives -- up to about 20X or so -- just place a round, opaque object, centered above the light, somewhere below the condenser. Such an opaque object is called a stop. (The term "stop" does not refer only to light blocks in the center; the various diaphragms on the microscope are also stops.) A nickle or dime often works (protect the light's lens from being scratched by placing a sheet of transparent material on the lamp housing), or a disk cut from any opaque sheet. The figure shows a side view of such a setup. With this kind of jury-rigged darkfield illumination, all light striking the condenser will be at the periphery of the light path. Without the stop a solid cone of light exits the condenser; with a darkfield stop the condenser will produce a hollow cone of light. The result is that all light striking the objective lens will be deflected from the specimen; no light striking the objective lens will be coming directly upward from the light source.

When trying to set up darkfield illumination, be sure that both the field diaphragm and the aperture diaphragm are sufficiently open so that light can reach the periphery of the condenser. The image of the lamp filament should be on the plane of the specimen (critical illumination) instead of on the aperture diaphragm as in Köhler illumination.

If the microscope has an Abbe condenser, a stop in the filter holder may work for objectives as powerful as 40X. For microscopes fitted with a fixed condenser, it is also possible to improvise darkfield illumination for higher power objectives. Place a stand magnifier with a lens of wide diameter, or any high power magnifying lens, near the condenser. (This will make more horizontal rays available to the fixed condenser.) A filter caddy can be used, or the magnifier can be set on the lamp housing. Experiment with different stops centered on top of the magnifier's lens.

Place a familiar slide on the stage. Look into the microscope and slide the magnifier around on the caddy or light until the darkest spot is found. If the stop is too big, everything will be black. If the stop is too small, the background will never become completely dark. If the stop is exactly the right size, objects will appear bright against a dark background. To make the job easier, have a selection of stops pre-cut, and have several magnifiers of different powers available. Each magnifier must be tried with each stop, and must be tried at different distances from the condenser. Once the right combination is found, record it for future use. Finding the right combination is a pain, but having darkfield illumination with a 40X objective is well worth the trouble.

The above procedure should work, but if you still cannot get dark field illumination from your 40X objective, try stopping down the objective's NA. Specialized darkfield microscopes have objectives with smaller NA than would normally be desirable. This allows the angle of rays coming from the condenser to be a bit less steep. Objectives are also available with a collar that adjusts an internal iris diaphragm. These objectives are quite expensive.

A homemade light stop can be fashioned to fit onto the back of any objective. Use any material that is black, opaque and will not release fibers or particles into the objective. Cut a disk the size of the back of the objective. In the middle of the disk, cut a hole that is smaller than the back opening of the objective. (Several hole sizes may have to be tried.) Very carefully, to avoid damaging the inside of the objective, glue the stop to the back of the objective with two or three tiny drops of rubber cement, or attach it with double sided tape. When finished using the stop, peel it from the back of the objective and carefully remove any remaining rubber cement with a ball of dried cement. Yes, switching between brightfield and darkfield illumination using this kind of setup is tedious. Hopefully, you can get by without it.

Some manufacturers used to supply funnel stops, which could be inserted into the back of an objective, reducing its NA and reducing any stray light inside the objective. If a small lathe is available a funnel stop can be turned from black plastic. Make a tube with an outer diameter that flares at one end so that it takes the shape of a T when seen from the side. The top of the tube is slightly wider than the objective's opening to prevent the tube from falling into the objective. The outer diameter of the bottom part of the T should be the diameter of the objective's back opening. Experiment as in the previous paragraph to determine the tube's hole size. Do not allow the tube to be so long that it touches a lens when inserted into the objective.

Darkfield illumination with a 100X objective cannot be improvised by any simple method. If you must have darkfield at this magnification, you will have to buy a darkfield condenser and possibly a 100X objective with a built in diaphragm. Before investing in such a condenser, be sure that your microscope's substage hardware can accept it.

Darkfield illumination is more than a simple value reversal of brightfield illumination. Only diffracted, reflected or refracted light from objects in the specimen can reach the eye. Since most of the light that reaches the eye is light scattered from the specimen, the light source should be very bright. Removal of the light's frosted filter as discussed earlier may be helpful. Darkfield illumination, along with polarization and several other types of illumination, is an optical contrasting technique. Borders between objects with different refractive indices are strongly represented by reflected and diffracted light in the absence of axially transmitted light. The darkfield effect is sometimes more three-dimensional, and many structures that are invisible under brightfield illumination are distinctly visible. Some objects that are too small for the microscope to resolve as an image will show up as bright spots of diffracted light.

Leeuwenhoek was secretive about his methods of observation, but a passage in one of his early letters suggests that he had hit on a method of darkfield illumination for his simple lenses. He likens a view of blood corpuscles to sand grains scattered on black silk -- a perfect description of a low power darkfield view of a blood smear. The use of darkfield illumination would explain how he got a clear view of certain objects before other microscopists were able to see them at all.

There is one serious disadvantage to darkfield illumination; there is little undiffracted light to interact with the diffracted light. This results in decreased resolution. Annular brightfield illumination can be set up in much the same way as darkfield illumination, except that the size of the stop is reduced so that a few of the directly transmitted rays can reach the objective. The startling color and value contrasts of darkfield illumination are traded for increased resolution. Because the light does not need to be as steeply angled as with darkfield illumination, you may even be able to improvise annular brightfield illumination with the 100X objective.

Rheinberg illumination is also closely related to darkfield illumination. The light stop is replaced by colored filter material. Surrounding this filter are filters of one or more colors that contrast with the color of the central filter. The central filter will be the same size as a darkfield stop would be for the objective in use. Work out your darkfield setup for each objective before trying to work out the corresponding Rheinberg setup.

Rheinberg illumination combines normal brightfield illumination in one color with darkfield illumination in another color. When light from the center filter strikes the condenser, it will throw a small, solid cone of light upward after focusing in the specimen's plane. Surrounding this cone will be a hollow cone of light from the surrounding filters. At the focus in the plane of the specimen, the light from the center filter will be moving roughly straight upward, while light from the surrounding filters will arrive at an angle. The background will be lit by the center filter while three-dimensional objects will pick up light from the peripheral filters.

The central filter must be quite dark or the background will not contrast with the objects in the specimen. In fact, a very useful hybrid of Rheinberg and darkfield uses an opaque central light stop surrounded by two or more colors.

The colored filter material can be assembled into a Rheinberg filter by using tiny drops of cyanoacrylate glue. Or, the components can be sandwiched between sheets of plastic or glass. Each filter can be built to work with a particular objective and specimen color. Because of this, your collection of Rheinberg filters may grow quite large.

Rheinberg illumination seldom provides any more real information than is supplied by darkfield illumination. Rheinberg does, however, make beautiful color effects and spectacular photographs possible.

The central disk of a Rheinberg filter can be made of polarizing material instead of a color filter. The specimen is viewed through an analyzer. The only light that will reach the eye by transmitting straight up through the specimen will be light that passes through birefringent particles in the specimen. Any other light reaching the eye will be from the colored outer filters.

Centrally stopped illumination setups are often improved if the slide is oiled to the condenser. Use type B slide oil, which is thicker than type A. The oil makes the background as dark as possible; otherwise some fogging and loss of light will occur as light bounces around between the condenser and the slide. There are, however, disadvantages to oiling the slide to the condenser. Because the light will be traveling at different angles, you may have to reconfigure the illumination setup. Also, it is inconvenient to clean oil from the stage when changing illumination setups, and it is inconvenient to clean the oil from slides when changing specimens.

A final point needs to be made about both darkfield and Rheinberg illumination: they are aesthetically pleasing to work with. For instance, the tiny protists that inhabit pond water look slimy under brightfield illumination. Under darkfield illumination they are transformed into animate jewels, swimming about in a glittery field of plankton and bacteria. When using one of these lighting techniques, it is easy to lose yourself for hours watching the antics of these tiny creatures. No novice microscopist should miss the experience.

Oblique Setups

Oblique brightfield illumination is like annular brightfield illumination, but most of the light will be coming from one side. Oblique darkfield illumination is likewise similar to darkfield illumination. Oblique brightfield illumination gives a strong three dimensional effect. The effect is not the same as would be seen with the specimen lighted from above, and to this extent the effect is not real. Oblique brightfield illumination is easier to set up with high power objectives than homemade darkfield illumination, and it provides very high resolution in one direction. Assuming that a phase microscope or interference contrast microscope is not available, you will want to become well acquainted with oblique illumination for your microscope. This will require some experimentation.

The stop used for darkfield illumination can be moved slightly off center to achieve oblique illumination. A surprisingly large number of other stop shapes work as well. The figure shows several possible stops. Filters can be stacked in any configuration below or on top of these stops. The stop at the bottom right of the illustration is made by cutting colored filter material so that it overlaps, eventually ending up with so many layers that it is opaque at the right side of the filter.

The oblique lighting effect is strongest when the stop is near the condenser and when the light strikes the specimen most horizontally. However, angling the microscope mirror so that its light is no longer centered also produces oblique illumination. This can be handy for a quick switch of illumination between brightfield and oblique.

For microscopes with no condenser or a fixed condenser, oblique illumination can be achieved by use of a substage magnifier moved off center. Some microscopes have centerable condensers. In this case, it helps to shift the condenser off center. For microscopes that have a focusing condenser that is not centerable, the lower lens of the condenser can sometimes be removed and repositioned off center on a caddy. If the microscope has a rotating diaphragm instead of an iris, one of the holes can be moved a bit off center below the fixed condenser.

To use the stops, put them on the filter caddy or lamp housing, in the holder, or on a magnifier and slowly move them about until the best effect is achieved. An evenly lit background is sometimes desirable, particularly for photography, but the best way to see the most detail is to leave part of the field of view in shadow. Move the shadow's edge around, bringing strong contrast to different objects in the specimen.

Experimentation is the only way to discover which setups work best with your microscope. Do not stop experimenting until you have found both kinds of oblique illumination: darkfield oblique and brightfield oblique. The darkfield version should look the same as normal darkfield illumination except with the light coming from one side. As we would expect, darkfield oblique illumination is more difficult to obtain when using powerful objectives than brightfield oblique illumination.

Reflected Illumination

Reflected (or incident) illumination is achieved by shining a light down onto the specimen from above. Some research microscopes can send light down the body tube toward the specimen. The objective is used as a condenser on the downward trip, and as an magnifier after the light reflects from the specimen. The highest power oil immersion condensers can be used with this method.

The next best method uses a special microscope illuminator. The illuminator stands beside the microscope, shining the light down onto the specimen. Such an illuminator can get enough light onto the specimen without causing too much glare from light bouncing around on the stage. Some illuminators use a condensing lens in front of a lamp, others use a fiber optic tube with a lens on the end. Ring illuminators, circular fluorescent tubes that encircle the objective, are also available.

Using these illuminators, reflected light can be used with objectives of up to about 20X. With objectives of shorter focal length, glare becomes too much of a problem, and not enough light reaches the specimen. All of these illuminators are expensive, but not as expensive as microscopes that are set up to use the objective as a condenser.

A single lamp will give better clues to the three dimensional shape of an object; all of the shadows will be on one side. If the shadows become too deep, it may be necessary to use two lamps shining down from opposite directions or a ring illuminator. Multiple illuminators also work better at higher magnifications; they get more light to the specimen.

It is easy to mix reflected light with transmitted light viewing. Very interesting effects result when the transmitted light is filtered to be one color and the reflected light is filtered another (often complementary) color. This effect is reminiscent of Rheinberg illumination, but different structures are accentuated by the multicolored light.

A dimmer is less useful for the reflected light illuminator than for the microscope's transmitted light. Usually, it is hard to get enough reflected light to the specimen, so the transmitted light is the one that must be dimmed to get the proper lighting mix.

An unmodified table lamp can be used for reflected light illumination if a mask with a notch is leaned against the objective, and the lamp's light is directed through the notch. This will eliminate some glare. The light will be weak, and this method is only useful to about 6X objectives. Experiment with different notch sizes and shapes.

A better solution is to buy a commercial illuminator, or make the following project to create an inexpensive microscope illuminator by modifying a common table lamp.

WARNING: Most table lamps specify a 60 watt bulb. However, the following modification requires that the lamp housing be enclosed, causing heat to be trapped. This could cause a fire hazard, and it could cause the shape of a plastic magnifier to distort. A glass lens or an all glass magnifier should be used. Drill some holes in the lamp housing in a position that will not cause glare on the stage or in your eyes. This will help to release heat. A small spotlight bulb is preferable to a normal round light bulb. Use a low power bulb -- 20 or 30 watts, and turn the lamp off when you are not at the microscope table. Also keep flammable materials away from the lamp.

Use a lamp that is tall enough to shine down on the microscope stage at a an angle of 45°. Also be sure that the lamp housing is deep enough to completely enclose the bulb.

Tools and materials:

Table lamp (clip-on or adjustable);
Needle nosed pliers;
Wire cutting pliers;
Coping saw with metal cutting or plastic
cutting blade;
Lens or magnifier with short focal length;
Metal funnel;
Wire (sheathed solid core electrical wire is easy to
work with, but coat hanger wire is more springy).

Wedge the magnifier or lens into the small end of a funnel. The funnel should have a flared-end diameter that is slightly larger than the lamp housing diameter. If the lens' or magnifier's shape is not right, use epoxy or screws to secure it. Drill two small holes near the flared edge of the funnel and fashion wire as a spring to hold the funnel to the lamp housing as shown in the illustration. Cut the funnel tip off just past the place where the magnifier has wedged. Drill a few small holes toward the back of the lamp housing to help release heat. Most time will be spent fiddling with the wire. The trick is to shape the wire in such a way that it needs to be sprung forcefully outward to attach the funnel or base plate. Halogen lamps can be modified in the same way as a normal table lamp, but they cost more. If colored light is desired, a filter holder can be fabricated between the magnifier and the bulb, or a filter can be leaned against the objective. You will want the focus point of the light to be on or near the specimen.

Other Specialized Illuminations

Several other illuminations are available on research microscopes and specialized microscopes. Phase contrast and interference contrast each exploit different methods to make phase shifts in light visible. Objects in the specimen that would normally be transparent can cause such phase shifts, which get translated into value or color contrasts.

Both of these methods are valuable in observing transparent specimens that cannot be easily stained. Phase contrast creates a halo around objects that lowers resolution, but some workers still prefer it to interference contrast because of the stronger contrasting effects. A few reasonably priced microscopes are now equipped with phase contrast, but interference contrast is still limited to research microscopes.

Modulation contrast uses spatial filtering instead of phase shifts to produce contrast in transparent specimens. It is a closer cousin to oblique illumination, but the effect is stronger and the background is more evenly lit. A rudimentary modulation contrast can be improvised, but this involves the use of photographic reduction and, for some microscopes, a machine shop. Interested parties can use Deborah Scarff's article (listed in references to this chapter) as a guide to the modification. Condensers and objectives for modulation contrast are also available from Modulation Optics Inc..

Fluorescence microscopy has also become important in recent years. It takes advantage of the fact that certain materials, when bombarded with one color of light (usually ultraviolet) glow in another color (often in the visible spectrum). Some natural objects and special stains have this characteristic. Fluorescence microscopy is carried out using either transmitted or reflected light. Fluorescence microscopes are expensive. However, one company now sells a special incident light fluorescence objective that connects to its own light source by a fiber optic cable. This objective simply screws into any DIN nosepiece. The cost is around two thousand dollars. Be sure that you can get access to fluorescent stains before moving into fluorescence microscopy.

Illumination summary

Following is a table of commonly used illumination methods and notes whether they can be used with an inexpensive microscope:

	4X	10X	40X	100X
polarized	yes	yes	yes	yes
darkfield	yes	yes	possibly, with experimentation	special condenser only
Rheinberg	yes	yes	possibly, with experimentation	no, special condenser not available
incident from side at 45°	yes	yes	special lamp only	no
incident through objective	special scope only	special scope only	special scope only	special scope only
brightfield	yes	yes	yes	yes
annular brightfield	yes	yes	yes	possibly, with experimentation
darkfield oblique	yes	yes	possibly, with experimentation	possibly, with special condenser used off-center
brightfield oblique	yes	yes	yes	yes
fluorescence	special scope or objective	special scope or objective	special scope or objective	special scope or objective
phase contrast	special scope	special scope	special scope	special scope
modulation contrast	yes, with modifications	yes, with modifications	yes, with modifications	yes, with modifications
interference contrast	special scope	special scope	special scope	special scope

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