Setting up Koehler Illumination
adapted from Foster
This should be done first thing whenever you sit down to use a light microscope. If the person before you used a different type of sample, it will be set wrong for your sample. Many things on a microscope do not need routine adjustment, but Koehler Illumination does! You must do this each time you use the microscope! If you are doing fluorescence imaging, this does not apply.
Koehler Illumination can be done with any objective, but it is easier with lower magnification objectives. If your objectives are parfocal (all objectives are in perfect focus as you switch objectives), you don’t need further adjustment. However, if they are not parfocal (and ours are not), you need to adjust each of them separately.
Reminder: If you are using phase contrast, be sure to select the correct phase ring in the condenser first, otherwise you may have trouble finding your cells and the correct focal plane.
Step 1. Focusing the Objective on the Sample
1. Place a dish or slide (with the coverslip facing the objective) on the stage of the microscope.
2. Use the coarse and fine focus to bring the image of your sample into focus.
IMPORTANT REMINDERS:
A. When using an upright microscope it is a good idea to focus away from the specimen. For instance, if you are not sure where to be, bring the lens close to your sample and then move it away from the sample to find the correct focal plane. This protects you from smashing the objective into the sample and breaking your slide or the objective.
B. For our inverted microscopes, because the sample is not fixed, overfocusing will simply lift the slide or dish off the surface. Lens elements are recessed, so it is hard to damage the objective this way. Therefore, it is okay in that case to slowly and carefully focus toward the specimen.
C. It is a good idea to look at the spacing for each objective when in focus so you can estimate the distance where correct focus should be. As the magnification increases, the working distance increases, so the 100X oil immersion objective must be very close to the sample while the 10X air objective will need to be further from your sample to be in focus.
Step 2. Adjusting the Condenser
1. While looking at the image with the microscope, close the field iris (circle of light) so that only about one-quarter of the field is illuminated. The field iris ring is located above the condenser.
2. Using the condenser focus knob, move the condenser up and down to sharpen the focus of the image of the field iris so that the dark/light transition zone is sharp.
3. If the circle of light is way off center, bring the image of the field iris (circle of light) into the middle of the field of view using the centering screws to crudely adjust the beam onto the center of the objective.
4. Open the field iris so that the lighted portion is just outside the field of view.
Step 3. Adjusting the Eyepiece
1. Using the binocular or slider adjustment on the eyepieces, adjust the interpupillary distance (distance between the eyepieces) so that you see one clear, round field of view.
2. Using your dominant eyeª, focus on the specimen with the non-adjusting eyepiece. Next, close that eye and use the focusing eyepiece to make the focus sharp with your other eye. Check the focus of the objective and, if necessary, fine-tune with the fine-focus control. Close your dominant eye and, using the focus control ring on the other eyepiece, bring the image into focus for your non-dominant eye.
NOTE: Some microscopes have no focusing eyepieces while others have only one. Check your microscope. If the focusing eyepiece is determined by microscope design and not by your choice, use the fixed eyepiece as your “dominant eye” and the focusing eyepiece as the other eye.
To determine which is your dominant eye, focus on a target 20-30 feet away through a cardboard tube. Without moving the cardboard tube, close one eye. If the tube is in front of the open eye, that is the dominant eye. If the tube is in front of the other eye, then the closed eye is your dominant eye.
http://www.microscopyu.com/tutorials/java/kohler/index.html - good java
Köhler Microscope Illumination
http://micro.magnet.fsu.edu/primer/anatomy/kohler.html
Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography. Köhler illumination was first introduced in 1893 by August Köhler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination.
This technique is recommended by all manufacturers of modern laboratory microscopes because it can produce specimen illumination that is uniformly bright and free from glare, thus allowing the user to realize the microscope's full potential.
The manufacturers have designed modern microscopes so that the collector lens and any other optical components built into the base of the microscope will project an enlarged and focused image of the lamp filament onto the plane of the aperture diaphragm of a properly positioned substage condenser. Closing or opening the condenser diaphragm controls the angle of the light rays emerging from the condenser and reaching the specimen from all azimuths. Because the light source is not focused at the level of the specimen, the light at specimen level is essentially grainless and extended and does not suffer deterioration from dust and imperfections on the glass surfaces of the condenser. Opening and closing of the condenser aperture diaphragm controls the angle of the light cone reaching the specimen. The setting of the condenser's aperture diaphragm, along with the aperture of the objective, determines the realized numerical aperture of the microscope "system". As the condenser diaphragm is opened, the working numerical aperture of the microscope increases, resulting in greater resolving power and light transmittance. Parallel light rays that pass through and illuminate the specimen are brought to focus at the back focal plane of the objective, where the image of the variable condenser aperture diaphragm and the image of the light source will be seen in focus.