Easy Kohler Illumination Method
ACADEMIC SKILLS CENTRE (ASC)
A. Silverberg
Completion of a Kohler illumination method is required before a microscope can be used
efficiently. The Kohler method is designed to achieve maximum resolution and light
focussing when observing an image using a microscope.
To understand how an image is generated from a microscope, it is essential to grasp how
a light path is generated. A path of light is created by a small halogen lamp located at the
base of a microscope. The light is directed upwards by mirrors to the condenser. The
condenser filters light to remove long wavelengths of light, thereby, permitting only the
shorter wavelengths to proceed throughout the system and hence, improve resolution.
When the condenser is positioned correctly, the light focuses onto the specimen held by
the specimen holder. The correct positioning of light is achieved using a Kohler
illumination method. Furthermore, the shorter wavelength of light is passed through the
objective lens, which produces a magnified image of the specimen. The eyepiece lenses
further magnify the image which is projected onto the eye’s retina.
Kohler Illumination Procedure: Detailed Steps
1) Place a specimen slide onto the microscope stage and secure between the
specimen holders. Refer to Appendix A for a breakdown of operational parts.
2) Ensure the microscope is turned on and the light source is adjusted to half of the
possible brightness, otherwise the user will experience visual fatigue and
discomfort.
3) Using the eyepiece portion, set the interpupillary distance to ensure comfort.
4) Place the 10X objective lens into position and focus the specimen using the coarse
adjustment knob.
5) Each eyepiece requires adjustment according to an individual’s sight
requirements. To achieve an eyepiece adjustment:
a. Close the right eye. Use the fine adjustment knob to sharpen the image.
b. Close the left eye. Focus is obtained using the Diopter ring (Figure 1).
The Diopter ring is normally located on the right eyepiece and moves both
in the counter clockwise and clockwise directions to achieve the necessary
focus requirements. Slowly, turn the Diopter ring to focus the right eye
with the left eye closed.
Figure 1: Location and Movement of the Diopter Ring
6) Close the field diaphragm ring and the condenser aperture diaphragm ring. A
small circle of light will be visible. The circle of light needs to be centred in the
field of view. If not, adjust the condenser screws as necessary to centre the circle
of light. Recommendation: adjust the condenser screws slowly!
7) If you do not see a circle of light, this indicates the microscope is extremely “out
of Kohler”. To correct the misalignment, open the field diaphragm ring to the
point where the circle of light is just visible. Adjust the condenser screws as
necessary to centre the circle of light. Close the field diaphragm ring again and
proceed to step 8.
8) Adjust the condenser focus knob until the image of the circle of light is sharp.
NOTE: Completion of steps 6, 7 and 8 are important for a) centring the light path
and b) focussing light onto the specimen.
9) Take out the removable eyepiece (Figure 2). Look down the eyepiece cylinder.
Observe a circle of light located in the centre of a dark field.
Figure 2: Removable left eyepiece
Open the condenser aperture diaphragm ring slowly until the circle of light fills ¾
of the field. Replace the removable eyepiece. Record the setting for the condenser
aperture diaphragm for the 10X objective (Figure 3).
Figure 3: Settings for the condenser aperture diaphragm
10) The Kohler illumination procedure is now complete. Note: a Kohler illumination
is required on a daily basis and every time the objective lens is changed. Once the
condenser aperture diaphragm setting is established for each objective lens for the
day, it is easy to dial the setting to switch between the objectives rather than
repeat the entire procedure when an higher or lower magnification is necessary.
References
Leica Microsystems [Online]. Richmond Hill, Ontario. Cited September 15, 2010.
Available online: http://www.leica-microsystems.com/
Misseri, A., 2008. Microscopy. Kingston, Ontario: St. Lawrence College.
Appendix A: Operational Parts of a Microscope
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