Different filters (patch stops) printed on overhead foil. The blue filter on the left is a commercial blue glass filter, on the bottom: the condenser with the 2 centering screws.

In a previous post, I already mentioned the making of patch stops from cardboard. For some background information (and more pictures) try these articles:

• Oblique Illumination
• Increasing Contrast using Optical Methods

Making Patch stops for dark-field illumination.

• Measure the diameter of the filter holder of your condenser.
• Using a program, such as PowerPoint or OpenOffice Impress to draw a circle, fill-color white, of the same diameter as the filter holder. You can adjust the size of the circle in the context menu.
• Draw a smaller black circle into the center. Copy-paste both circles and then change the size of the inner smaller circle. You want to make several filters to find the one that works best.
• Print the filters on overhead foil. Print with a laser printer. The overhead foils for laser printers are more heat resistant.
• Cut out the filters with a scissor
• Take a black marker and darken the black inner circle.
• For microscopy work, take two of these filters and place them on top of each other. This ensures that the central circle is completely black.
• Place the filter into the filter holder, completely open the condenser aperture diaphragm and the field diaphram (should you have one).
• Try out the different objectives and find the suitable filter/objective combination.

Making patch stops of oblique illumunation

The method is very similar to making patch stops for dark filed. In this case, light is only allowed to hit the specimen from one side only. This will produce a relief-like image.

• Draw a black and a white circle of the diameter of the condenser filter holder.
• Overlap the two circles, so that the white circle covers part of the black circle. The white circle should not reach the center of the black circle.
• Cut out and proceed as described for making a dark field patch stop.
• Again it is necessary to experiment to find the appropriate filter/objective combination.

Making Rheinberg filters

Maybe you want to show yellow specimens on a blue background. Take the dimensions of the dark-field patch stop and color the center yellow and the periphery blue (color printer!). You have to use intensive colors to achieve an effect. Try different color combinations.

A darkfield filter (patch stop) placed into the filter holder of the condenser. To the left and the right are the centering screws.

Potato starch grains. Left: darkfield image; Center: Brightfield, inverted colors; Right: Brightfield; The comparison shows that a darkfield image is not simply an inverted version of a brightfield image. Darkfield images have more sharply defined corners.

Maize. Left: darkfield image; Center: Brightfield, inverted colors; Right: Brightfield; The darkfield image possesses less contrast due to the opened aperture diaphragm and a different color representation.

To achieve a darkfield image, it is necessary to place a dark field filter (a “patch stop”) into the filter holder of the condenser. This filter prevents light of the lamp to directly enter the objective (therefore the background appears dark). The specimen will be illuminated from the side and will scatter some of the light to enter the objective. The specimen will appear bright on dark background.

It can be compared to dust floating in the air with sun shining in from the side through a window. The dust is illuminated by the sun and appears bright on dark background.

There are two possibilities to achieve a darkfield image:

• By using specialized darkfield condensers: This is the best but also the most expensive solution.
• By using a darkfield filter (a “patch stop”) which is placed into the filter holder of the condenser. It is possible to make the patch stop out of cardboard or a tin can using a cutting knife and scissors.

Advantages of darkfield microscopy:

• It is a simple procedure which can be used on live transparent specimens, specimens which normally need to be stained (and therefore killed).
• The images appear spectacular and are visually impressive.
• Darkfield microscopy even allows for the visualization of objects that are below (!) the resolution of the microscope. These objects will appear as bright spots on a dark background. It is not possible to see the shape of these objects, however.

Some possible disadvantages of darkfield microscopy:

• Darkfield microscopy is very sensitive to dirt and dust located in the light path.
• It is not suitable for all specimens. If the refractive index of a transparent specimen is similar to the surrounding medium, then the specimen light will pass right through the specimen and it will not be scattered into the objective.
• The intensity of the illumination system must be high so see the specimen properly.
• It is necessary to open the condenser aperture diaphragm, and this limits the effective use of the diaphragm.
• One patch stop is generally sufficient for low magnification work, but at a higher magnification the quality of the image drops. It may be necessary to experiment with different patch stop sizes for the different objectives.

The condenser aperture diaphragm can be controlled with a small horizontal lever (top). Left and right are the condenser centering screws. They are needed for adjusting Koehler illumination. Behind the left centering screw you can see the condenser focus knob.

Here the condenser aperture diaphragm is set to a value of 0.25, which is the recommended value for the objective in use. The depth of field is low, the resolution high, the contrast is low.

Here the condenser aperture diaphragm is set to a value of 0.1, which is the closed position. The depth of field and contrast are both high. The image appears crisp, but resolution is lower.

An improper setting of the condenser aperture diaphragm (especially at higher magnifications) can be the cause of much frustration both for teachers and students.

• Students may attempt to find the focus with the condenser aperture diaphragm all the way open. This is difficult if the sample is very thin or weakly stained or the microscope is not equipped with parfocal objectives. Remember, an open condenser aperture diaphragm results in a low depth of field.
• Students may not see anything at all when working with high magnifications because the image is too dark. In this case the diaphragm is closed too much. The diaphragm should not be used to control the amount of light, but for some specimens or magnifications there may simply be no way around this especially if the lamp is not very powerful.

Many beginners are place an overly strong emphasis on magnification. Many think that they are able to see more at a higher magnification. But especially at higher magnifications the role of the condenser diaphragm becomes more important.

I recommend the following steps:

• Instruct the students to completely close the condenser aperture diaphragm when starting to use the microscope.
• They should then rotate the low power objective (4x) into position and find the focus with the coarse focus knob. The larger depth of field and higher contrast makes it easier for the students to focus the specimen.
• When switching to a higher magnification, the students should start to gradually open the condenser aperture diaphragm, to observe the differences in image quality. At the same time they have to adjust the light intensity with the dimmer to prevent glare.
• Students should be made aware that the condenser aperture diaphragm should be adjusted to the numerical aperture value which is printed on the objective. Opening the diaphragm further will not increase image quality, but may result in glare.
• If the sample is thick, strongly stained or pigmented then the diaphragm has to be opened to allow more light to pass through the specimen. As a consequence, the depth of field becomes smaller. It is then necessary to use the fine focus adjustment knob to focus through the different layers of the specimen.

The Koehler diaphragm is centered and in focus. The adjustment is correct.

The Koehler diaphragm is centered but out of focus. Raise or lower the condenser to focus the diaphragm.

The Koehler diaphragm is off-center. Turn the centering screws on the condenser to move the aperture into the center.

Koehler illumination was developed by August Köhler (1866-1948). This illumination principle greatly enhances the quality of the microscopic images (especially photographs). The illumination principle offers the following advantages:

• It illuminates the specimen uniformly without the need of a diffuser.
• It only illuminates the part of the specimen which is actually observed (at a higher magnifications a smaller section of the specimen). This reduces the heating of the specimen.
• It reduces internal reflections. This improves the contrast in photomicrographs.

The Koehler illumination must be adjusted before observation:

1. Rotate a low power objective (eg. 4x or 10x) into position. This will increase the field of view.
2. Insert a slide with a specimen and focus it.
3. Adjust the field iris diaphragm (the diaphragm of the light source) in such a way that its edges become visible. The field of view is reduced this way, only a small round part of the specimen is visible.
4. Raise or lower the condenser (not the stage!) and bring the edges of the field iris diaphragm (not the condenser aperture diaphragm) into focus. The focus of the specimen is not changed. Now both the edge of the iris diaphragm and and the specimen should be in focus. If the height of the condenser is not properly adjusted, then dust of the lamp will come into focus and disturb the image.
5. There are two condenser centering screws/knobs at the side of the condenser. Turn these knobs to bring the field into the center of view.
6. Now you can open the field diaphragm and start regular microscopic observation.
7. When doing photographic work, open the field diaphragm only as far as necessary. Opening it further will increase internal light reflections and result in a lower contrast. You need to observe the edges of the field diaphragm through the camera viewfinder. It may also be necessary to refocus the specimen when looking through the camera.

Left: a closed condenser diaphragm (set to a low value); Right: an open condenser diaphragm (set to a high value). Both condensers are shown from the bottom side.

An opened condenser diaphragm increases the angle of the light beam.

A closed condenser diaphragm decreases the angle of the light beam. Notice that opening and closing does not change the width of the beam where it exits the condenser.

Many modern course microscopes are equipped with a condenser and an associated condenser diaphragm. The purpose of the condenser is to concentrate the light onto the specimen, its diaphragm regulates resolution, contrast and depth of field. There is a trade-off to consider:

• When the condenser diaphragm is closed, then the depth of field and contrast increase and
• the image will lose resolution and becomes darker.

It is up to the microscopist to find the optimum setting of the aperture diaphragm, but for optimum resolution the setting of the diaphragm should be more or equal to the numerical aperture of the objective (this value is printed on the objective).

Many beginning microscope users prefer to generally close the aperture diaphragm all the way. The image possesses more contrast and subjectively appears more crisp. The image looks less “washed-out” The increased depth of field also makes it easier to find the plane of focus.

There is, however, the danger of introducing optical artifacts:

• Dust grains on the cover slip or on the optical surfaces start to become more pronounced and may give the impression that they are part of the specimen.
• Structures become more pronounced than they actually are.
• The larger depth of field may result in some structures covering up other structures that are in front of, or behind them.
• The larger depth of field causes structures overlap more and it becomes more difficult in determining the layer in which they are located.
• Last but not least, the maximum possible resolution of the objective is not used.