Microscope advice

Setting up and using your microscope

Setting up Köhler illumination

Most blurry, washed-out, or unevenly lit pictures are not a problem with the lenses. They are a problem with the light. Köhler illumination (named after August Köhler, who worked it out in 1893) is a quick way to set up the light so that the whole view is lit evenly and you get the best contrast and sharpness your microscope can give. It takes a couple of minutes, and it is well worth it for pale, nearly transparent cells like dinoflagellates.

You need a microscope with a field diaphragm (an adjustable iris near the light, in the base) and a condenser that moves up and down. If yours lacks these, jump down to "If your microscope is simpler".

1. Put a slide on the stage, with something easy to focus on (a prepared slide, or the crayon-line trick below), and focus with the 10X objective.
2. Close the field diaphragm until you see a small polygon of light in your view. The edges will probably be fuzzy and it may be off to one side.
3. Move the condenser up or down with its focus knob until the edges of the polygon are sharp.
4. If the polygon is not in the middle of the view, use the two centering screws on the condenser to move it there.
5. Open the field diaphragm until the polygon just disappears past the edge of the field of view. Do not open it further, because extra stray light lowers the contrast.
6. Now set the condenser aperture diaphragm, which controls contrast. Closing it makes pale cells stand out, but close it too far and the image gets dim and develops dark fringes and loses detail. A good starting point is about two-thirds to three-quarters open. If you remove an eyepiece and look down the tube, you will see the diaphragm in the objective lens, and a setting of about 70 to 80 percent of that circle is the usual target.
7. When you switch to a different objective, check both diaphragms again. The field diaphragm usually needs a small adjustment, and the aperture setting changes more.

If your microscope is simpler

Many simple student scopes have no field diaphragm and a condenser that cannot be centered. You can still get most of the benefit: keep the condenser raised close to the slide, use the aperture diaphragm (or the rotating disc of holes under the stage) to adjust contrast, and change brightness with the lamp. This is also one more reason to look for a scope with a full condenser when you shop for a used one.

Focusing

The two focus knobs

Most microscopes have a large coarse focus knob and a smaller fine focus knob. Use the coarse knob only with the lowest-power objective, to get close. Once you move up to 10X and 40X, use only the fine focus knob, so that you cannot drive the objective into the slide. Start at the lowest power, find your cells, and move them to the center of the view before you switch to a higher objective.

Finding focus when there is nothing to lock onto

Focusing is hardest when the view looks empty. Two tricks help:

• A crayon line. Draw a line on the slide with a crayon, put your algae next to the line, add a drop of water, and cover it with a cover slip. Focus on the line first, then move to the algae. A crayon leaves a rough, grainy surface, which makes it much easier to tell when you are in focus. A marker works too, but it is harder.
• The edge of the cover slip. If you do not have a crayon, focus on the spot where the cover slip ends, then move over to your sample.

Focus through the cell

Many of these cells are big enough that you can focus through them. Once you are looking at a cell, use the fine focus to move slowly up and down through it. Your brain will make much better sense of the three-dimensional shape that way than from looking at just one slice of the cell.

Keep the lenses clean

Student microscopes are often dirty. Clean the lenses well, with lens cleaner and lens paper, not a shirt.

Next: collecting samples, making slides, and taking pictures.