Using a Bright Field Light Microscope
Placing an Oil Immersion Lens
When a lens is marked with the word "oil," the tip of the lens itself is made to be dipped into a transparent oil drop placed in the light path directly on a coverslip or dried smear. Light passes through the specimen and/or coverslip, into oil, and then into the objective without passing through air. The result is a dramatic improvement in resolution.
A typical oil immersion lens magnifies between 95x and 100x. The lens must be brought extremely close to the specimen and the depth of focus is very shallow. Unless one is very experienced and is working with familiar specimens, it is essential to start at low magnification and work up as described previously. The specimen should be viewed, centered, and focused using the high dry lens before adding the oil and moving the oil immersion lens into position.
To place the oil immersion lens, move the high dry lens out of the way and place a generous drop of oil directly on top of the slide. You should have a convex drop in the center of the light path. Carefully rotate the oil immersion lens into place so that the end of the lens contacts the oil. You should be looking directly at the lens and specimen at this point, not through the eyepieces.
It probably will be necessary to increase the illumination to maximum, because the illuminated surface area is now very small. The light path must be well aligned. If the elements of your microscope are adjustable, your manual should indicate how to optimize alignment for maximum brightness. If the images are still too dim, be sure that the lamp is putting out enough light and replace if necessary.
Because the depth of focus is so shallow with a 100x lens, there probably will be no sign of the specimen at first, even with parfocal lenses. Even knowing the correct direction to focus, if you go too fast you might move right through the focal plane and not even recognize the specimen. Students in microbiology frequently encounter this problem when trying to view Gram negative bacteria.
You may use the stage translational controls with an oil immersion lens, just as you would any other lens. But if you move too far from the center of the drop, the lens may lose contact with the oil. If that happens you can carefully move the lens out of the way, add more oil, and replace the lens without losing your focus. The hard part is finding the focal plane in the first place.
- 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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