Field diaphragm is wide open. Reflections from the side of the tube are very strong.

Field diaphragm is half open. The reflections are less.

Field diaphragm is closed. Only the direct light is able to reach the camera.

Taking a picture of the tube with a webcam. Any camera with a small lens would also have done the job.

Advantages of Köhler illumination for photography

Köhler illumination increases the contrast of a photomicrograph because it reduces stray light and glare caused by reflections inside the microscope. On the right side you can see images taken through a trinocular head with a web cam. The more that the field diaphragm is closed, the less the reflections coming from the side of the tube. The bright spot in the center is the light which comes directly (unreflected) from the light source. In order to see a picture, it would be necessary to remove the lens from the webcam and project the image directly on the sensor of the webcam. In this case, the lens was left on to be able to see the side of the tube.

For more background info on Köhler illumination, you may be interested in the following two posts:

• Advantages of Koehler Illumination
• Adjusting Koehler Illumination

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.

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.