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Glare and Eyestrain

Glare is a serious problem for the microscopist. Stray light bounces around between the objective and the cover slip and between the slide and the condenser lens, degrading the image. Keeping a dimmer for the room lights on the microscope table is helpful. If this cannot be worked out, keep a lamp on the worktable, and keep the room lights low.

Some microscopes have mirrors instead of lights. Never angle a mirror directly toward the sun; your eyes will be damaged when you look into the microscope. Angle the mirror toward an softer indirect light source. The sky is not the best, since it has a lot of uv light that damages your eye.

Most sources advise keeping both eyes open when looking into a monocular microscope -- advice more easily given than taken. Often, it is difficult to ignore the images that come from the free eye. If you are one of the lucky people who can simply close one eyelid without causing the other eyelid to squint, then do so. If you do tend to squint, there are some tricks that may help:
1) Remove bright, light colored objects from the free eye's field of view.
2) Dim the room lights or turn off the table lamp.
3) Hold the tube with your hand in a way that will block images that might come into the free eye.
4) Cut a light blocker from a rubber sheet to fit onto the microscope tube. The figure shows one of these, along with a rubber eyepiece protector that helps to keep the eyepiece from getting scratched.
5) Place the microscope on a dark table or place a sheet of black paper or cloth under the microscope.
6) Cover the free eye with your hand to get the microscope eye to take the lead. Once the microscope eye is in the lead, remove your hand.

Computer users are familiar with a kind of eyestrain that also affects microscope users. It is easy to stare hypnotically while trying to interpret a specimen. When this happens, natural eye blinking stops and the eyeball dries, causing discomfort. Be aware of the tendency and remember to blink regularly.

The younger microscopist may try to use her own eye's internal lens to accomodate the image (bring it into focus) instead of tweaking the focus adjustment knobs. This causes eye strain. Learn to let your eyes relax, look away from the microscope often, and use your hands to continuously adjust the focus.

People over forty often have eyes with internal lenses that are no longer flexible; continual adjustment by focus knob comes naturally because it is necessary. Indeed, you may find that the only time anything comes into perfect focus is while looking into microscopes or telescopes. Anyone with a cataract can adjust to a more normal color vision by adding a weak violet filter and cranking up the light intensity a bit. This compensates for the yellowness and light blocking characteristics of the aging internal lens of the eye.

Expensive eyepieces often provide more eye relief than cheap ones. Eye relief is the distance from the eyelens to the image of the aperture diaphragm. Eye relief determines the distance that the viewer's eye can be separated from the eyepiece when viewing a specimen. This distance can range from 6 to about 26 mm. An eyepiece with long eye relief allows the use of eyeglasses.

But we're talking about budget microscopes here. If the eyepiece does not provide exceptional eye relief, it is usually best to remove eyeglasses when looking into a microscope. Simply twist the focus adjustment knob to correct either myopia (nearsightedness) or hyperopia (farsightedness). The microscope will not correct for an astigmatic eye. However, those with only mild astigmatism will still find it best not to use eyeglasses with this kind of eyepiece. The eye will be much better positioned if it is near the eyepiece. If eyeglasses must be used, the eyepiece should have a rubber eyepiece guard to keep the eyeglass lens separated from the eyepiece lens. The two lenses easily scratch one another.

The Illumination System

We have seen how the microscope creates a large virtual image of a tiny object. To create the image, the light that travels through the specimen must be spread across a much wider area and the image becomes very dim. The more powerful the objective used, the more dim the image. This problem can only be overcome by directing more light to the specimen. Every microscope must have an illumination system that can provide this light. Many specimens also require special lighting setups. Arrangements of lamps, mirrors, light condensing lenses, filters, and diaphragm stops are used in building these setups.

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The Aperture Diaphragm

The aperture diaphragm is normally located just below the condenser. In some cases it is integral with the condenser housing. The diaphragm may be a wheel with different sized perforations or an iris of curved, interleaved plates that slide against each another. In the case of the wheel diaphragm, one turns the wheel to position different apertures in the optical path. The iris diaphragm is opened and shut by a lever. The infinite adjustability of the lever is preferable, but the wheel diaphragm works well enough.

Most manufacturers of economy microscopes encourage use of the aperture to control the amount of light reaching the specimen. This is understandable because neither a set of light filters nor a dimmer for the light is provided with these instruments. However, the microscope will produce much better images using a dimmer or filters. A design for a dimmer is presented in the chapter on light sources.

The aperture diaphragm should be used only for contrast adjustments. As the aperture becomes smaller, image contrast increases. If the aperture becomes too small, however, the image is ruined. We will postpone the discussion of just why this is so for later.

Different aperture settings will be required when switching between different illumination setups. Aperture adjustment is also needed with each change of objective. The aperture is the part of the optical system that the experienced microscopist most constantly changes. You, too, should acquire the habit of adjusting the aperture whenever a change is made in magnification or illumination.

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Filters

Various filters are required for different illumination setups. These filters are available from many sources.

Camera shops carry a number of useful filters. Neutral density filters can be used to control light intensity without changing the light's color. If a dimmer for the microscope light is not available, these filters are a necessity. If you take color photographs through the microscope, they will be needed even if you have a dimmer. Most camera shops also carry colored gel filters and quality polarizing filters that can be used to create special effects.

Some suppliers offer books of colored plastic films that can be used as filters. Almost any colored film can be used. Be alert to this possibility when visiting drugstores, office supply stores, and grocery stores.

You can buy filters ready-made for several specialized illuminations. These may not be right for your microscope or your specimens. Lighting is something that requires experimentation. No set of filters provided by the manufacturer is going to be just right for every specimen.

Supports for Filters

Every microscope needs some kind of filter holder. These can be improvised easily for microscopes that do not come with one. If a microscope has a built-in light, filters can simply be placed on top of it. Filters, polarizers, opaque light stops, and magnifiers can be set on the supporting sheet. You can buy a microscope lamp with a filter holder for a microscope that has a mirror but no filter holder. You can also construct a filter caddy as shown in the figures. The first can be made with sheathed, solid core electrical wire using a pair of pliers. The second can be constructed by making cut-outs from a cardboard box or plastic carton. The inside of this type caddy should be painted flat black. This will keep it from scattering light, creating glare. Lay a sheet of glass or acrylic across the top of the completed support. Clear acrylic sheeting can be found in hobby stores and some hardware stores. Buy the thinnest sheet that is stiff enough to span the support. A coping saw can be used to cut the acrylic. Glass and glass cutters are tricky to work with, but glass is better if you will be working with polarized illumination (which will be discussed in detail later).

WARNING: Always dress cut-glass edges with emory paper so that they will not be sharp, and wear gloves and eye protection while cutting glass and dressing edges. Young persons should seek help from an adult.

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Condensers: General Information

The resolution of a microscopic image depends not only on the NA (Numerical Aperture) of the objectives, but also on the NA of the condenser. Both must be high. A fixed condenser (a condenser without a changeable focus) with NA of about .65 is usually adequate for objectives of powers as high as 40X (which will also have NA of about .65). More powerful objectives produce dim images of low resolution with this kind of condenser. Focusing condensers (Abbe, achromatic, aplanatic, or achromatic-aplanatic) have higher numerical apertures and produce better resolution with powerful objectives. The Abbe condenser is standard. Achromatic condensers improve on the Abbe condenser by correcting some chromatic aberration. Aplanatic condensers focus light in a single plane. Achromatic-aplanatic condensers add both improvements. The resolution formula can be used to show why the higher numerical aperture is better in the following examples.

Assume we have a 1.25 NA oil immersion objective and a .65 NA condenser and illuminate the specimen with .00042mm violet light. The resolution equation gives:
(1.2 * .00042mm) / (1.25 + .65) = .000265mm.
Assume we exchange the .65 fixed condenser for a 1.25 NA Abbe condenser. The resolution equation now gives:
(1.2 * .00042mm) / (1.25 + 1.25) = .000201mm.
This is a .000064mm improvement in resolution, about 1/4 better. For best resolution we want a condenser with an NA that matches the NA of the objective. If condenser NA is lower than objective NA, resolution suffers; if condenser NA is higher than objective NA, the result is scattered light and glare, which also harms resolution.

Owners of fixed condenser microscopes can make good use of the fact that any positive lens with a wide diameter placed beneath the fixed condenser will create a composite condenser. For instance, a large diameter stand magnifier is handy to use for condensing the light column before it reaches the fixed condenser, intensifying the light available to the specimen. If a single lens is used, a plano-convex lens (flat on one side and convex on the other) with the flat side down will introduce the least aberration. A bit of care must be used in centering the new lens or magnifier.

The focal length of the new composite condenser will not be the same as that of the fixed condenser alone. If the lens or magnifier is powerful enough, the following fix will work. Close the field diaphragm to a very small opening and focus on a familiar slide with the 10X objective. Adjust the distance between the magnifier or lens and the fixed condenser until a sharp edged image of the field diaphragm appears. Several magnifiers may have to be tried. (The section on brightfield illumination will more fully explain basic microscope setup.)

Lenses, Stand Magnifiers, and Loupes as Condensers

Economy microscopes usually come with inadequate substage lighting facilities. Photo shops and microscope suppliers sell lenses, stand magnifiers and loupe magnifiers that can be useful in improving the illumination systems of these microscopes. Stand and loupe magnifiers consist of two lenses mounted in a tube. Loupes, designed to be held in an eye socket, have a more narrow base than stand magnifiers; otherwise they are the same.

It is convenient to keep a collection of lenses and magnifiers on hand. Magnifiers are useful for inspecting small objects, and for inspecting objective lenses. Lenses and magnifiers can be placed on filter caddies or lights and used either as condensers or as additions to simple substage condensers on microscopes without focusing condensers. They can also be used as condensers for the table lamp modifications to be discussed in the section on reflected illumination.

The focus of a stand magnifier usually falls at about the plane of the bottom of its base. When using one of these magnifiers as a condenser, this characteristic may need to be modified. You can shorten the magnifier with a coping saw. Before beginning, mark around the base so that the cut will be straight.

The magnifier can be raised by shimming with pieces of cardboard tubes from aluminum foil or wrapping paper rolls. You can also modify the height of a filter caddy to raise or lower a magnifier.

If their lenses are plastic, stand magnifiers and loupes will deform if left in a strong light path long enough to heat. Magnifiers with glass lenses do not present this problem. Glass lenses can also be mounted in all sorts of configurations inside a cardboard tube. The tube should be painted flat black on the inside. Small strips of cardboard can be bent into a curve and glued into the tube to hold the lenses in place.

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Light Condensers: Curved Mirrors

Many economy microscopes that have mirrors do not come with a condenser. There is simply a hole in the stage over which the glass slide that holds the specimen is placed. The mirror has a flat side and a concave side. The concave surface of the mirror condenses any parallel light rays that strike it by reflecting them onto a small area in the plane of the specimen. The curved surface is normally used with powerful objectives. The flat surface, which does not concentrate light, is used for low power objectives. Some microscopes fitted with mirrors do have a condenser. In this case, the plano mirror is used, even for the high power objectives, unless the condenser is removed. Owners of old fashioned instruments with mirrors will be pleased to learn that, while a mirror may at times be aggravating to adjust, it is more versatile than a light fixed into the microscope's base. The user has a wide choice of illuminators (which are required at high magnification), and the mirror tilts in all directions, allowing either even or oblique lighting of the specimen.

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Light Sources: Light Dimmers

Whether the microscope uses a built-in light or a mirror with illuminator, the lamp needs a dimmer. Lamp dimmer switches are available. A lamp dimmer switch can easily be wired into a three-line extension cord and set on the table near the microscope. The microscope is plugged into the extension cord, and the extension cord is plugged into the wall.

Lights

The figure shows the components of a microscope light. Above the bulb there is a filter, then a lens, and finally a diaphragm (a field diaphragm, not to be confused with the aperture diaphragm, which is located just beneath the stage or condenser). Imagine this setup resting on its side. This would be an illuminator for a microscope fitted with a mirror. Only expensive microscope lights have an adjustable field diaphragm and focusable lenses. There are ways, however, to make the economy microscope's lighting system almost as flexible.

Adding a homemade field diaphragm is simple. Just cut some disks from some opaque sheet material (exposed film, black paper, or aluminum foil). Cut various sized holes in the centers of these disks. One of these disks can then be centered above the mirror on the caddy or set on the light. Optics suppliers also sell iris diaphragms. An iris can also be salvaged from an old camera or microscope.

When choosing a size for the opening of the field diaphragm, the aperture diaphragm should be wide open. Different illumination setups require different opening diameters for the field diaphragm, but as a rule the aperture diaphragm must not be set until after the field diaphragm is set.

We have discussed using magnifiers below the stage to get more light to the specimen, but have not yet exhausted the ways of doing this. Because the microscope light is not the same color as natural light, a filter to compensate for the color change is present in the lamp housing. This filter is sometimes frosted so that the specimen will be lighted evenly; otherwise an image of the bulb's filament would be visible. At other times a separate frosted filter is present. When using objectives of 40X or higher power, the frosted filter is no longer necessary. The diameter of the field of view of the high power objective is smaller than the width of the image of the lamp filament.

More light is needed by a high power objective, and more light can reach it without the frosted filter. In cheaper lamp housings, the lens and diffuser are clamped together in such a way that the filters cannot be separated from the lens without a special wrench and considerable trouble -- more trouble than you will want to spend between changes of objective. However, the two can be easily removed together. This is another situation that calls for experimentation with lenses beneath the stage, in this case to replace the lamp housing lens. Because the light will be the wrong color without the diffuser and filter combination, a non-diffusing filter of the same color should be used. An infrared and ultraviolet filter protects the eyes.

Microscopes with mirrors require an artificial light source when an oil immersion objective is being used. High quality illuminators are available, equipped with focusing lenses and apertures. A table lamp can be used if it is equipped with a spotlight bulb, but it is up to the microscopist to modify the light with lenses and apertures as required. For one type of illuminator, the mirror is removed altogether, and the illuminator is pushed into the horseshoe shaped base beneath the stage. This kind of illuminator is rudimentary but usable and inexpensive.

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Stages

Many microscopes have a mechanical stage as an option. These are only necessary for specialized work that requires the recording of locations on a slide, microbial colony counts, or some other kinds of microscopic measurement (micrometry). If you do most of your work with objectives powered below 40X, a mechanical stage will sometimes get in the way more than it will help. In fact, even stage clips are unnecessary unless an objective of higher power is being used. It is often convenient to use only one of the stage clips, swinging the other off the stage. This makes it easy to move the slide around with the thumb of one hand while using the other hand to adjust focus. Use the clip on the side opposite the hand that will be moving the slide. Be sure, however, to use both clips with the oil immersion objective. At such high NA, clips or a mechanical stage stabilize the slide, preventing it from damaging the objective.

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