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.

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.

The numbers written on an objective designate different optical characteristics and standards.

What do the numbers and abbreviations on an objective mean? Especially when buying used microscopes from research laboratories or hospitals a basic knowledge of the text written on the optics can become handy. You don’t want to buy things that you don’t need.

• A or ACHRO (depending on brand): This signifies that the objective is an achromat. This means that chromatic abberration was corrected for 2 colors (in contrast to the expensive APOchromatic lenses). Achromatic lenses are those most commonly found in education, they are the cheapest.
• PLAN: These objectives produce an image which is in focus from edge to edge. They are used for photographic work and are more expensive.
• PLANAPO: This refers to a planapochromatic objective. It produces a flat image (in focus from edge to edge) and it is has a chromatic abberration correction for 4 colors. Expensive and not needed for educational work.
• PLANFL: A Planfluorite objective. A bit less expensive than the planapochromats but also not as fully corrected.
• 160: This represents the standard tube length of 160mm. Objectives with this standard are interchangeable between manufacturers.
• 0.17: This represents the thickness of the cover slip to be used in mm. Coverslips with a deviating thickness will result is an image of lower resolution.
• 4, 10, 20, 40, 100: This represents the magnification of the objective. The total magnification is calculated by multiplying the magnification of the objective with the magnification of the ocular (eye piece), which is usually 10x. The magnification is also indicated by the ring colors:
  • red: 4x or 5x
  • yellow: 10x
  • green: 20x
  • blue: 40x, 50x or 60x
  • white: 100x
• OIL: This designates an oil immersion objectives. Do not immerse non-oil objectives into immersion oil!
• WI: Water Immersion. Here water is used instead of oil.
• 0.65 (etc): This is the numerical aperture. This value indicates the angle to which an objective is able to receive light. This value also determines the resolution of the system. For maximum resolution, the iris diaphragm should be set to a value equal or larger than the numerical aperture of the objective in use.
• NCG or NC: These abbreviations stand for “No cover glass”. These objectives are designed to be used without a cover glass. They are useful in the medical area where blood smears etc. are observed.
• LWD or ULWD: These abbreviations stand for “long working distance” or “ultra-long working distance”. These objectives are able to work with a large specimen-objective distance and are used for specific applications.
• P, POL or SF: These objectives are designed to be used for polarization microscopy. The objectives are strain-free (SF) and will therefore not modify the polarization of the light. They are not necessary for simple polarization microscopy conducted in classrooms.
• PL or NH: These are designation of objectives used for phase contrast microscopy. A PL (positive low) objective produces an image of a specimen which is darker than the background, a NH (negative high) objective produces an image which is brighter than the background.
• NIC or DIC: Nomarski Interference Contrast or Differential Interference Contrast objectives produce an image of a specimen which appears to be slightly 3 dimensional. If you use a filter to achieve oblique illumination, then the result will look similar.