How do you suppose I found the depth of focus for each of the objective lenses on my microscope? Such practical information is hard to find in textbooks.It is tricky to measure distances in the vertical dimension, but it can be done.

The microscope I use has fine focus controls, each marked with 100 divisions. With the stage lowered, I measured the distance from top of the stage to a point on the microscope stand directly above the stage. I then brought the stage up by rotating the fine focus twenty turns and re-measured the distance. I found that with each full rotation, the stage traveled 0.2 mm, or 200 µm. That explains why the divisions on the fine control were marked from zero to 200! If I had looked in the manual, I might have found that the instrument was designed with a calibrated fine focus.

To measure a vertical dimension using the fine focus, one must be able to focus separately on the top, and on the bottom of a specimen. For example, to measure the depth of liquid under a cover slip, it is necessary to measure the distance from the top of the slide to the bottom of the cover slip. To do so while lowering the focus requires one to focus on the bottom of the cover slip (or on a small object attached to it). While keeping track of the distance traveled, one then focuses on the top of the slide or on a small object on top of the slide.

It is easiest to measure the depth of an object that provides distinctly different cross sectional views from top to bottom. For example, the amoeboid protists known as Difflugia develop a shell, called a test, that encloses the cell. The test is usually textured and curved, and it is semi-transparent. When the organism is active, pseudopodia protrude from an aperture in the base of the test.

Depending on the type of microscope you are using, you will either raise the stage or lower the nosepiece until the object comes into view. When the central part of the test comes into focus, you are looking at the very top. When the aperture appears in focus, you are viewing the bottom of the test. The tips of the pseudopodia are attached to the surface of the slide. When you measure the distance traveled in the vertical direction from the top of the test to the tip of the pseudopodia, you have measured the height of the organism.

I would not rely too heavily on the precision of measurements using a fine focus control. For one thing, depth of focus limits the accuracy with which one can estimate a position. There also is the matter of hysteresis. We have hysteresis when the direction of change has a significant effect on a result. If I focus precisely on a specimen, then de-focus by moving the stage down a precise number of turns, the specimen is not in focus when I move the stage back up the exact number of turns. No machinery is perfect. Hysteresis results from slippage, for example, as a knob is turned. To minimize the effect, one should approach calibrated positions by moving a control in the same direction each time.

Guide students as they observe the cells. Ask questions that will encourage them to look at relative sizes and shapes of cells. Help students to identify visible structures: cell walls in both kinds of cells; chloroplasts in Elodea leaf; and nuclei (one per cell) in onion epidermis.

Cell nuclei usually are easy to observe in the onion samples, but they will not be visible in all cells. Students also may notice one or more vacuoles within the onion cell cytoplasm, and tiny perforations, or pits, in the onion cell walls that connect the cytoplasm of adjacent cells. Nuclei are much less visible in the Elodea cells, which have abundant green chloroplasts.

Encourage students to make detailed drawings of their observations. Also, have students use the fine focus knob of the microscope to focus down through the layers of cells. Ask students to think about whether cells are flat or three-dimensional structures.

The cell membrane in Elodea cells can be observed if a sample is prepared in a drop of salt water. The movement of water out of the cell in the presence of saline solution will cause the cell membrane (also called plasma membrane or plasmalemma) to pull away slightly from the cell wall.

Viewing this presentation fulfills part of the requirements for completing the short course on The Science of Microbes, offered on BioEd Online for professional development contact hours. The Science of Microbes Teacher's Guide may be obtained in its entirety from the Center for Educational Outreach, Baylor College of Medicine (1-800-798-8244).

You can download a PDF of this lesson, including the pre-assessment, from BioEd Online or K8 Science.

The Science of Microbes and accompanying online professional development were supported, in part, by Science Education Partnership Award number 5R25RR018605 from the National Center for Research Resources of the National Institutes of Health (NIH) to Baylor College of Medicine. The unit was developed in partnership with the Baylor-UT Houston Center for AIDS Research, an NIH-funded program (AI036211). The opinions, findings, and conclusions expressed in this presentation are solely those of the authors and do not necessarily reflect the views of Baylor College of Medicine or the sponsoring agencies.

Unless you are so familiar with a type of specimen that you can go straight to an appropriate magnification and find your target immediately, it is best to take the same approach to finding specimens each time you observe. The most consistently effective strategy is to start at low magnification, find the target, adjust illumination, resolution and contrast, focus and center the object, and then raise magnification. Most sets of objective lenses are parfocal, meaning that the objectives are matched, so that if a specimen is in focus using one objective, it will be very nearly in focus when you raise the magnification using the next objective lens. Thus, if you re-focus, using only the fine focus control, and center the target each time you change magnification, you should have no trouble obtaining the image you seek at the desired final magnification.

After reaching 100x magnification, it is a good to re-adjust the microscope for binocular viewing, if you have a binocular eyepiece tube. You can see more detail now, and the better the oculars are adjusted to match your eyes, the more satisfactory the viewing.

At low magnifications (up to 100x or so total magnification), you should use the coarse focus control. Not only does it take too long to move a distance with the fine control, but the limit of travel with the fine focus may be less than with the coarse. Trying to focus past the limit of travel can damage a focusing mechanism.

When you bring in a high dry objective (a high power lens which is used without oil, usually a 35x or 40x lens) with the specimen in focus, the end of the objective will approach the specimen closely. It is unwise to use the coarse objective with such a lens, because it is too easy to ram the lens into the slide. In this case, use the fine control only.

Suppose you mount your slide upside-down. You will be able to focus at 40x total magnification, and again when you go to 100x magnification by swinging in the 10x objective. However, the thickness of the slide may exceed the depth of focus with the high dry objective (35x or 40x). If so, you won't be able to focus at all. If you don't pay attention, you probably will bump the slide with the end of the objective. Good high power lenses will telescope so as to buffer such shocks, but if you reach the limit, further movement will damage the slide and also may scratch the objective, and even the exit lens of the condenser. Such damage cannot be repaired.

Because high magnification lenses come so close to the specimen, to reduce the risk of a disaster, you might want to take your eyes from the eyepieces and instead watch the lens as you rotate it carefully into place. Until you are used to your microscope, you should check the position of the lens frequently while focusing, or (better) have someone else watch the objective and warn you if it contacts the slide.

The first time you use an oil immersion lens, it might help to have someone watch the objective lens while you attempt to focus. As with any high power lens, use the FINE FOCUS CONTROL ONLY. It may take many turns to bring the specimen into focus, so be patient. The lens will nearly touch the specimen before you reach focus. If it contacts the slide, it will begin to telescope, and your observer should warn you that you have gone too far. Moving in the other direction will bring the specimen into focus. However, if the gap between the lens and specimen reaches a couple of millimeters, you are too far above the specimen, and have missed the focal plane completely.

Now what? Here's what not to do: do NOT go back to the high dry lens. It will contact the oil, fouling the surface of the lens, which is meant to be used only in air, and not in oil.

Check that your specimen is indeed on top.  Every time I teach microbiology, someone, sooner or later, puts a bacterial smear on a stage upside down and I spend quite a bit of time trying to help him or her focus before I realize what happened. If you cannot find the focal plane, you may go back to a low magnification objective, such as 4x or 10x, to refocus and re-center the specimen. The direction needed to bring the specimen into focus at low magnification may give you a clue about how far out you were with the high power lens, and in what direction. To protect your high dry objective, you will have to "jump" directly to back to the oil immersion lens.

When you go back to the oil lens, make sure your oil drop is big enough to accept it; you may need another drop. This time, with illumination turned way up, try stopping down the aperture diaphragm in your condenser. The image will be distorted, but if there is anything to be seen, it will have greater contrast and you are more likely to find it. Slowly rotate the fine focus control. It may help to move the mechanical stage slightly back and forth. The eye can detect movement more readily than it can see a stationary image. When you identify your specimen, you can readjust the aperture to optimize resolution and contrast.

One more strategy is to place the lens as close to the surface as you dare, and then slowly move the stage away from the objective. This method requires patience because you may begin far out of focus. But at least you know you are moving in the right direction.

When you finish using an oil immersion lens, you must dab off the oil with good quality lens tissue. Dried oil will interfere with viewing and can be difficult to remove.