Easy Kohler Illumination Method

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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.

1 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.

2 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).

3 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.

4 References Leica Microsystems [Online]. Richmond Hill, Ontario. Cited September 15, Available online: Misseri, A., Microscopy. Kingston, Ontario: St. Lawrence College. Appendix A: Operational Parts of a Microscope

5 The Academic Skills Centre

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 1: THE LIGHT MICROSCOPE

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 1: THE LIGHT MICROSCOPE Prior to lab: 1) Read these instructions (p 1-6) 2) Go through the online tutorial, the microscopy pre-lab