HOW TO ADJUST AN ANTIQUE MICROSCOPE FOR OPTIMAL TRANSMITTED BRIGHTFIELD ILLUMINATION

AUTHOR: Barry Sobel

EDITORS: James Solliday and Joseph Zeligs

INTRODUCTION:
Although many of us take illumination for granted, correctly adjusting illumination is very important for getting the best image when viewing a transmitted light subject through a microscope, old or new. If you have an antique microscope, you may want to use it like the original owner could. But simply putting a specimen on the stage and focusing the microscope while the mirror is pointed at some random source of light will not provide the best image your microscope is capable of. This page, and its links, provide a guide to allow you to get the best image feasible from your microscope.

Microscopes have always been designed to magnify objects. As improvements in the lenses took place, it became clear that improvements in the method of illumination was also important. Starting in the 19th century, not only did the types of illuminator improve, but the way it was used also did. Although there was a need to increase brightness of the illuminator(especially for photography), it became apparent that factors other than the brightness of illumination were important in achieving the best image. Although the optical design of objectives, especially correction of spherical and chromatic aberration is important in achieving the best image, those issues are covered elsewhere. Here we will concentrate on proper methods of illumination to achieve the best image with the objectives provided with the microscope.

The optimal method of illumination differs depending on the design of the microscope and the accessories available to the user. Microscopes with only a mirror, even with the best objectives, cannot deliver the quality image of a microscope with substage components. Furthermore, the use of a substage condenser will allow further refinement of the image. In this web page I will discuss how to achieve the best image depending on how the microscope is equipped. Before we discuss that though some discussion of the light source is in order.

A few terms need to understood before we begin. Glare or halation, refers to unwanted light reflections that reduce image quality and can be caused by reflections from the microscope's components or the sample itself. Resolution refers to the ability to see fine details. The latter is not the same as magnification, because although with proper adjustments a modern microscope will have more resolution with higher magnification, simply increasing magnification without proper adjustments of the illumination system will result in poorer resolution than if the setup was optimized. Resolution depends both on the numerical aperture of the objective and also on the illumination system. It turns out that the higher the magnification, the more important proper illumination is in achieving the best result. One also needs to understand that there may be up to three locations that need to be focused. These are the lamp filament, the condenser, and the focusing of the image of the object under study. Another thing to understand is that in microscopes so equipped there are two different diaphragms or apertures that need to be adjusted. These are the field diaphragm, closest to the light source, and the aperture diaphragm closer to the stage or condenser if so equipped. One needs to pay attention to what is being focused and which diaphragm is being adjusted in each step.

What do we mean by best image? This is the image which provides the most information for the situation at hand. This includes a bright enough image, best resolution, least interfering stray lighting (glare), and provision of even illumination across the field of view. When using a microscope, some characteristics are beyond our control and reside purely in the characteristics of the objective, the eyepiece, etc. On the other hand, proper adjustment of the substage, the mirror, and the illuminant(source of illumination) itself can also affect the quality of the image. Although chromatic aberration and spherical aberration are mostly determined by the objective, the other aberrations of glare, evenness of illumination, and brightness will be affected by how the illumination is adjusted. In some cases these adjustments are limited by the microscope itself; a microscope with no substage at all cannot be optimized the way one with variable substage apertures can. Nevertheless, one can take advantage of some variables even with no substage to give the best possible image for that microscope.

THE LIGHT SOURCE IS IMPORTANT
In the early days of microscopy lighting was simply daylight, or a candle. Later came oil lamps, and still later electric lamps. Although it may be of historical interest to use these illuminants, we will assume the user will use an electric lamp. There are two kinds of lamps that one could use. One is a simple lamp like a desk lamp or a simple illuminator that has no adjustments. The other is a lamp designed specifically for use with a microscope, either as a stand-alone unit or built into the microscope as in more modern equipment with a filament light bulb (not an LED). To obtain the best image with a microscope equipped with a mirror, the best result will be obtained by using a stand-alone illuminator designed for use with microscope. As of 2025, used stand-alone electric microscope illuminators are still readily available on sites like Ebay. When obtaining one, try to obtain one with adjustments, particularly a diaphragm. The ability to focus the light and center the light source is also important. The ability to regulate brightness is also a plus, although without this, neutral density filters can be used to reduce brightness when needed.

SETTING UP THE ILLUMINATOR
Before using the illuminator it is important to be sure the light beam is properly centered. To adjust this, project the light on a wall from a distance without any filters or ground glass in the optical path if possible. The filament of the light bulb should be visible, though focusing may be needed to see it clearly. Check centration by rotating the part containing the bulb. The filament should rotate on its own axis and not move in an arc; if it moves in an arc the filament needs to be centered using the appropriate controls on the device. Once this is accomplished, if the device provides a lock for this adjustment that can be used to keep the filament centered until the bulb needs to be replaced. Recentering will be needed if the bulb is replaced. Once the filament is centered, proceed to the next step.

AIMING THE LIGHT AT THE MIRROR:
The light source should be at the height of the mirror or slightly above that. Start with the front of the light source about 6 inches from the mirror. Aim the light at the mirrror and direct the light up into the microscope so that the light is passing through the microscope to the eyepiece. Place a piece of paper on the mirror and center the light source on the center of the mirror.

USE OF A MICROSCOPE WITH NO SUBSTAGE:
In this case, after placing the slide with an object you want to observe, focus the microscope on the object. These microscopes usually have a concave mirror which will provide better lighting than a flat one in the absence of a condenser. Now with the object in view, if using a plane light source with no focus or diaphragm, the only other adjustment is changing the distance of the light from the microscope to achieve the best illumination of the field of view. The optimal distance can be determined by visual inspection but placing a pointed object immediately in front of the light source should be in focus in the field of view if illumination is properly adjusted. If the image is too bright the best method to reduce brightness is to reduce voltage to the illuminator if possible. Alternatives include reducing the diameter of the beam with the diaphragm on the illuminator(if provided) Placing neutral density filters at the light source can also reduce the brightness if needed.

IF YOU HAVE A SUBSTAGE WITH VARIABLE APERTURES BUT NO CONDENSER:
Next, we address the adjustment of a microscope that has no lensed condenser, but does have some way of adjusting the apertures. This can be a wheel of apertures, individual apertures fitting onto a substage holder, or little cylinders that fit into the center of a holder under the stage, each one with a hole designed to optimize illumination for a specific objective. Occasionally these apertures or stops are even labeled to indicate which objective they are to be used with, but more often they are not. In these systems, the best image is achieved by both using the correct size aperture, and optimizing the lighting with the mirror and source of illumination(the illuminant). In most cases, the aperture should be as close to the slide as possible; some microscopes have the wheel of apertures actually incorporated into the stage itself, with the apertures close to the surface of the stage. Some have a wheel attached directly to the underside of the stage, while others attach the wheel in such a way that it can be removed so other apparatus such as a polarizer or oblique illuminator can be substituted. The adjustment of the substage aperture is simple; with the eyepiece removed, use the aperture that fills the field of the back element of the objective, seen without the eyepiece to slightly less than full. The use of an optimized substage aperture and properly focused light source, will reduce glare and slightly improve contrast and resolution, especially as higher powers are used. Focusing the light here is the same as if there were no aperture adjustment.

MICROSCOPES WITH LENSED CONDENSERS:
When illuminating a specimen with just a mirror, it may be difficult to fill the entire field of view with concentrated light; even when optimized the field may not be completely filled or the illumination across the field uneven. What's more the illumination may not result in an optically sharp image regardless of how well focused the objective is. A condenser can provide, when used properly, both a brighter light source and a much better field of illumination with a sharper image. There are two ways to properly illuminate the specimen with an antique microscope equipped with a condenser. The first is called Nelsonian or Critical Illumination and the other is Kohler illumination. Modern microscopes sometimes use a proprietary method of illumination and in such cases the owner's manual would need to be consulted for how to provide optimal illumination. The Leitz/Leica Orthoplan is an example of such a proprietary design.

CONDENSERS:
Usually there is a provision to add a stop underneath the condenser; in some cases there is a wheel of apertures, in some individual discs with different size apertures fit into a holder at the bottom of the condenser. In better condensers, the stop is variable, via an iris diaphragm. Early on, the optical condensers were not achromatic, but eventually achromatic and even aplanatic condensers were also supplied. Since achromatic-aplanatic condensers are most useful at the highest powers, these were not standard equipment except on the most expensive microscopes even in modern times. The optimization of illumination when using a condenser requires the ability to focus the condenser. In some less complex microscopes this may simply be moving the condenser housing up or down by hand either inside a substage ring or on a tailpiece. Rack and pinion focusing was provided and many better stands and later some microscopes had a vertically oriented screw focusing. Today the best microscopes use rack and pinion substage condenser focusing. The best methods of illumination, both developed in the late 19th century are Nelsonian(Critical), illumination and Kohler Illumination. These methods can be used only with microscopes equipped with a focusable substage condenser.

NELSONIAN ILLUMINATION:
When Nelsonian illumination is used the substage condenser is focused (using the substage focusing mechanism) on the light source so that when the object is in focus, so is the light source. To do this one could use the "edge" of the flame from a candle or oil lamp. Later on, this could be accomplished more accurately with electric lamps using a ribbon filament. This is known as critical or Nelsonian illumination, named after its proponent, Edward Miles Nelson who developed this in the 1880s. Once the light source is focused on the same image plane as the specimen, the aperture of the condense is adjusted (or changed in the case of disk stops) to just barely fill the field, which reduces glare and reflections. With lower magnification this does not eliminate the fact that the field may be brighter in the center of the field of view. This kind of focusing was made even more exact by the addition of a substage fine focusing adjustment in addition to the usual substage coarse focusing. The provision for such substage fine focusing can be found on several of the microscopes on this site including the Nelson-Curties No 1a and No 2 on this site, as well as the Watson Royal and Watson Grand Van Heurck on this website. Once the edge of the flame or the ribbon is in focus, the center of the flame or ribbon can be used to illuminate the field without the ribbon or flame edge superimposed on the image. With a large field of view however, the edge of the light source may still be visible. In this case very slight defocusing from perfect focusing of the condenser will result in an adequate image. It should be noted that when using an oil lamp source, it is best to used a flat wick rather than a round one to produce the most uniform source of light. If the image of the filament in a modern electric illuminator interferes with viewing the specimen, a frosted glass in front of the light source may solve the problem. Note here that we are assuming the use of a light source without facility to change its focus or aperture.

STEPS FOR CRITICAL OR NELSONIAN ILLUMINATION:
This requires a substage condenser lens system and variable aperture. It requires an illuminator with centered filament. 1.If you are using a separate illuminator, not built in to the micro scope, follow the instructions above for setting this up.
2.With the slide in focus, use the condenser focusing controls to focus the filament of the illuminator on the subject. 3.The problem with the filament superimposed on the subject can be solved as follows:
&nbsb;&nbsb;&nbsb;&nbsb;a)Defocus the substage condenser very slightly-a fraction of a mm, and just enough to make the filament image invisible OR &nbsb;&nbsb;&nbsb;&nbsb;b)Place a frosted glass filter immediately in front of the light source.

KOHLER ILLUMINATION:
The best type of illumination for use with an antique microscope from the 19th or 20th century is Kohler illumination. This technique provides optimum contrast and resolution. Since ribbon filaments are no longer available, it eliminates the image of the spiral filament being in the field of view. It also eliminates the need for a diffusing screen for those types of filaments to deal with this; a diffusing screen limits the spectrum of light and its grains may still appear in the image. Kohler illumination aligns the light rays for homogenous and even illumination. This requires two areas of aperture control, the aperture diaphragm, and the field diaphragm. The aperture diaphragm, is usually located just below the condenser lenses, whereas the field diaphragm is placed below that, closer to the light source. When a stand-alone illuminator is used, this field diaphragm is part of that illuminator. This type of illumination, invented by August Kohler in 1893, allows perfect uniform illumination even at almost all powers whereas Nelsonian illumination may be uneven with coil filaments. If Nelsonian illumination is carried out to perfect focus, the image of the lamp filament will be in focus while the object being studied is in focus. Both Nelsonian and Kohler illumination require all optical elements to be in the optical axis and well centered to each other. Many less expensive microscopes did not have the capacity to center the condenser, and this includes both antique microscopes and modern ones as well. Steps for Kohler illumination are outlined below.

Microscopes with only a mirror and aperture diaphragm with the condenser can still be set up for Kohler illumination. The easiest way to do this is to use an electric light source with a built-in iris diaphragm. This is the field diaphragm. Variable voltage is also helpful for adjusting the brightness of the field. This means you can still set up an antique microscope with a mirror for Kohler illumination. Many of the major manufacturers supplied such illuminants starting in the early 20th century. Examples by Bausch & Lomb and Nikon are quite common. The chief disadvantage of these is the bulbs became very hot. This was true even with modern microscopes with built-in provision for Kohler illumination until the advent of LED lighting. Many microscopists have experienced a burn by accidentally touching these older light sources or even their housings, so care is advised when using them!

STEPS FOR SETTING UP KOHLER ILLUMINATION:
The adjustments for Kohler illumination are more complicated than the other methods, but are easy to do and easy to learn.
1. If using an illuminator separate from the microscope, adjust the filament centration and center the light beam on the mirror as noted above.
2. With a built-in light source, first center the light at the field diaphragm-the diaphragm closest to the light source-usually at the bottom of the microscope. Placing a piece paper over it, center the light source so that it is in the center of the opening when it is fully open. This adjustment is usually at the light source but in some cases, as the Wild M20 microscope, there is a sliding lens in the base that is also adjustable by hand, and also assists in centering the light source. This centering will be refined later.
3.With the microscope equipped with an ordinary condenser such as an Abbe condenser, the low power (usually 10X) objective is turned into position.
4.Next, a slide with a specimen is placed on the stage and brought into focus.
5.Now, with the condenser raised to its highest point, open both the field diaphragm and the aperture diaphragm so that they are open just enough to fill the field of view with light.
6.The next step is to center the condenser. First, if there is a ground glass diffuser on the light source and it can be removed or otherwise taken out of the optical path, do so. If it cannot be removed, treat the ground glass as the filament would be.
7.After the illumination in the field is centered and even, and continuing with the slide in focus, move the slide so that a clear area (without the specimen) of the slide under the coverslip is in view. Now reduce the size of the field diaphragm until its edges are in view. Now focus the condenser on edges of the field diaphragm.
Now use the adjustments provided-usually of the condenser, to center it in the field of view. Once the condenser is centered, the field diaphragm should be opened to just beyond the field of view.
8.The next step is to focus the lamp filament either at the plane of the aperture diaphragm or at the back element of the objective. In many cases the former is not feasible as the aperture diaphragm will not be easily accessible. This step is skipped if the microscope has no filament, such as a fiberoptic light source. In these cases an educated guess can be made though using the same technique. To focus the filament, one can place a piece of paper on the aperture diaphragm if accessible and center it there, or remove the eyepiece and look at the back focal plane of the objective, either with a phase telescope or Bertrand lens. If these are not available, a pinhole eye-cap can be used or is easily fabricated and the image magnified with a hand lens held above the pinhole if needed. Now adjust the light housing until the filament is both centered, using its provided adjustments.
Note that if the illuminator has a built-in condenser lens that can be taken out of axis, the filament may not be seen except with the condenser lens IN the axis of illumination.
After the filament is focused and properly centered, adjust the aperture diaphragm at the bottom of the condenser. Generally this should be closed to about 75% of the field of view with the eyepiece removed. With histology slides, the aperture diaphragm should be matched to just slightly less than the numerical aperture of the objective if the diaphragm is calibrated. But for urinalysis, if phase contrast is not available, closing the aperture diaphragm a bit further may increase contrast (at the expense of resolution). Generally closing it to less than 2/3 of the field will degrade the imaged. For a description of the optical explanation for using Kohler illumination please see the Zeiss-FSU website.

SUMMARY OF SETTING UP A MICROSCOPE FOR KOHLER ILLUMINATION WITH AN INCANDESCENT LIGHT SOURCE:
1. GROSSLY CENTER THE LIGHT SOURCE WHERE IT PROJECTS OUT OF THE BOTTOM OF THE MICROSCOPE OR THE STAND-ALONE ILLUMINATOR:
      a. REMOVE GROUND GLASS OR DIFFUSER SCREEN IF PRESENT
      b. IF USING A STAND-ALONE ILLUMINATOR AND MIRROR, PROJECT THE IMAGE ON A WALL AND ADJUST THE LAMP CENTERING CONTROLS TO BE SURE THE FILAMENT IS CENTERED.
     c. IF USING A BUILT-IN ILLUMINATOR, PLACE WHITE PAPER OVER THE FIELD DIAPHRAGM AND CENTER THE LIGHT SOURCE USING THE LAMP CENTERING CONTROLS.
      d. IF USING A STAND-ALONE ILLUMINATOR, PLACE IT ABOUT 6.5 INCHES AWAY FROM THE MIRROR AT THE MIRROR HEIGHT OR SLIGHTLY HIGHER AND CENTER THE IMAGE ON THE MIRROR WITH A PIECE OF PAPER ON THE MIRROR.
2. IF USING A MIRROR, ADJUST IT TO BE SURE THE LIGHT IS CENTERED IN THE FIELD WHILE LOOKING THROUGH THE MICROSCOPE EYEPIECE(S).
3. BE SURE THE SUBJECT ON THE SLIDE IS IN FOCUS-DO NOT CHANGE THIS FOR THE REST OF THE SETUP.
4. CENTER THE CONDENSER(APERTURE) DIAPHRAGM BY USING THE SUBSTAGE CENTERING CONTROLS (IF PROVIDED) OR IF NEEDED THE SLIDING CENTERING DEVICE ON THE BASE (WILD MICROSCOPES)
5. CLOSE THE FIELD DIAPHRAM UNTIL YOU CAN SEE ITS EDGES AND USE THE SUBSTAGE FOCUSING CONTROL TO FOCUS ON ITS EDGES BY RAISING OR LOWERING THE CONDENSER. AFTER THIS IS DONE, OPEN IT TO JUST BEYOND THE FIELD OF VIEW.
6. CENTER THE FILAMENT AT THE APERTURE DIAPHRAGM OR WITHOUT THE EYEPIECE, FOCUS THE FILAMENT IN THE BACK FOCAL PLANE OF THE OBJECTIVE BY USING THE LAMP FOCUSING CONTROLS USING A PHASE TELESCOPE OR PINHOLE EYE CAP VISUALIZE THE BACK FOCAL PLANE OF THE OBJECTIVE. NOTE: IF THERE IS A CONDENSING LENS THAT CAN BE BROUGHT IN OR OUT OF THE LIGHT PATH (e.g. VANOX), IT SHOULD BE IN.
7. IF PHASE CONTRAST IS NOT BEING USED, AND EXAMINING URINE, ADJUST THE APERTURE DIAPHRAGM FOR MAXIMUM CONTRAST WHILE MAINTAINING ADEQUATE ILLUMINATION AND RESOLUTION, OTHERWISE, THE APERTURE DIAPHRAGM IS PARTLY CLOSED TO BETWEEN MAINTAIN THE APERTURE AT 66 TO 80% OF THE FIELD.
8. IF POSSIBLE, ADJUST ILLUMINATION BRIGHTNESS BY VARYING THE BULB BRIGHTNESS, NOT CHANGING THE DIAPHRAGMS; IF THIS IS NOT ADJUSTABLE, USE NEUTRAL DENSITY FILTERS OVER THE LIGHT SOURCE TO REDUCE BRIGHTNESS.

FILTERS:
Blue and or Green filters were often used in microscopy, especially in photography. A blue filter, the most common used, will correct the color of an artificial light source, and even natural light whereas a green filter will increase contrast. These issues are more important in determining the true color and for photography, especially with higher power objectives. For different reasons, blue light will slightly improve resolution, while green light will increase contrast. Green filters improve the image in phase contrast work in particular.