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Kohler illumination

Please note that we have two different kinds of microscopes that we will be using during this semester: the compound microscopes and the dissecting microscopes.  Pay attention to the type of microscope called for in the instructions!

THE BASICS OF LIGHT MICROSCOPY

 The Olympus BH-2 microscope.
 

Adjusting the Olympus BH-2 microscope Kohler illumination.

Kohler illumination is required whenever maximum resolution of detail in opaque or colored objects is sought. It might be regarded as the "standard" illumination for a compound microscope.

Although most students have previous experience with finding and focusing on specimens with a compound microscope, few have experience with the specific light adjustments necessary to use the Olympus BH-2 microscope.  It is imperative that you learn to properly adjust the light on these microscopes.  Failure to do will result in loss of image quality and if the light is too far out of adjustment, you may be unable to see anything at all clearly. Although you can still obtain an image if you skip the adjustment or do it incorrectly, for some experiments the image quality will be too poor to achieve the objectives of the lab.

Procedure:

A. Initial setup

Important!  The microscope adjustments must be done with a slide present on the stage.  As you practice making the adjustments, use the prepared slide that will be provided.  Because you will have a different slide than the one used to prepare the instruction illustrations, the item you see under your microscope will be different from what is shown in the illustrations, although the characteristics of the image (fuzzy edges, size, etc.) will be similar.


1. Plug the microscope into a 120 V outlet. Turn on the lamp using the power on/off switch located on the front of the base and adjust voltage to a low value where the bulb emits light, but at an intensity level that can be observed directly without harm to the eyes.  Do NOT turn on the preset light intensity toggle switch on the right side of the base.  This switch overrides the voltage control and makes it impossible to adjust the brightness of the lamp.  Always use the voltage control to adjust the light intensity - not the field or condenser diaphrams.


2. Rotate the turret until the lowest power objective lens (10x) clicks into position.  Make sure that the phase contrast selection wheel is clicked into the zero position (turned off).


3. Turn the field diaphragm adjustment dial all the way clockwise to open the field diaphram to its maximum aperture.


4. Rotate the condenser focusing knob until the condenser is at its top position. Do not confuse the one condenser focusing knob found under the stage on the left side with the two sets of coarse and fine focusing knobs located on both sides of the lower arm.

B. Finding and focusing on the specimen


5. Place a microscope slide (with specimen) on the stage, and position it so the specimen is over the light beam. Use the two knobs hanging below the right side of the stage to move the slide around.


6. Focus the microscope on the specimen.  The high power objectives (20x and 40x) can easily be smashed into the slide, resulting in the destruction of the slide and possible damage to the objective as well.  For this reason, always begin focusing by looking at the side of the objective (NOT in the eyepiece) and turning the coarse focus knob so that the slide is moved as close as possible to the slide without touching it.  Then look into the eyepiece and rotate the coarse focus knob so that the slide moves away from the objective lens until the image comes into clear focus (as in the image above).  If the light adjustment is terrible, the image you see may be obscured.  In this case, proceed with the Koehler illumination adjustments and then try focusing again afterwards.

C. Focusing and centering the condenser.

The adjustments in steps C, and D if examining an opaque specimen should be repeated each time you switch objectives (change to a different power).  It may be possible to skip the readjustment, but if the exceptional image quality is required, you should repeat those steps.


7. Close the field diaphragm by turning its adjustment dial all the way counterclockwise. If you are re-doing the light adjustment because you have changed objectives, raise the condenser all the way to the top again as described in step 4.


8. Close the condenser diaphragm by moving its lever (located below the stage) all the way to the right.


9. The edges of the image seen through the eyepiece should now look fuzzy like this.  (The object that you are viewing on the slide will be different.)  If you see nothing but darkness, the condenser could be so far off the side that the light beam is missing the objective.  You may need a TA's help with centering the condenser (described below) before you can proceed with focusing the condenser.  
 
Lower the condenser slowly by turning the condenser focusing knob (NOT the coarse and fine focusing knobs!!!) until an image of the edges of the field diaphragm (which form a decagon) is brought into sharp focus as seen in the two figures above. (As you lower the condenser the beam of light will become smaller and sharper around the edges. If the beam of light starts to increase in size, you have gone beyond the point of focus).


10. Slowly open the field diaphragm by turning its dial clockwise and the decagon representing the edge of the field will increase in size as seen to the left.  (Compare with the image above.)  Continue opening the field diaphram until the edges of the decagon expand just beyond the field of view.  If the decagon is off-center and touches one side of the field before the rest, you may need to center the condenser as described in the next step.  Otherwise proceed to step 12.


11. To center the condenser, use the 2 condenser centering screws that come out of the condenser toward you at an angle. Do NOT turn the screw that comes straight out to the right.  Doing so will cause the condenser to fall off of the microscope.  Do not use the two screws that angle out from the condenser away from you (these are used in phase contrast adjustment).  While looking through the eyepiece, turn the screws until the decagon is centered.  Because the image is inverted, the direction that the decagon moves when you turn the screws may seem strange.)  After centering the decagon, open the field diaphram until the edges of the decagon expand just beyond the field of view.

12. If necessary, re-adjust the focus of the specimen (using the fine focus knob, NOT the condenser focusing knob).

D. Filling the field


13.  Carefully remove an ocular lens as shown above.
 
14. Make sure that the condenser diaphragm is closed completely (the lever is all the way to the right). While looking through the tube where you removed the ocular lens, slowly open the condenser diaphragm until the entering light almost coincides with the edges of the field (tube). This operation is called "filling the field".

15. Adjust the light intensity to a comfortable level using the sliding brightness control.  If you are looking at a stained or opaque sample, stop here.