Using a Bright Field Light Microscope
Focusing with an Oil Immersion Lens
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.
- Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell (4th ed.). New York: Garland Science.
- Caprette, D. (2005). Light microscopy. Retrieved 09-12-2005 from http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html
- Lodish, H., Berk, A., Zipursky, L., Matsudaira, P., Baltimore, D., & Darnell, J. (2000). Molecular cell biology (4th ed.). New York: W.H. Freeman and Co.
- Nave, C.R. (2005). Hyperphysics (light and vision). Retrieved 09-12-2005 from http://hyperphysics.phy-astr.gsu.edu/hbase/hframe.html
- Wolfe, S.L. (1993). Molecular and cellular biology. Belmont, CA: Wadsworth Publishing Company.
- Caprette, D. (2005). Microscope images.
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