Using a Bright Field Light Microscope
Focusing on Multiple Surfaces
Even with parfocal objectives, each time you change lenses, you will have to make some adjustment to focus. When you go to high dry magnification, and especially to an oil immersion lens, the depth of focus is so narrow that the specimen may be faint or invisible. If you lose track of a specimen completely, it usually is fruitless to remain at high power and search. You are searching in three dimensions, and your quarry occupies only a small volume in the three dimensional search area. It is best to enlarge your field of view in all three dimensions by going back to a lower magnification, re-focusing and re-centering a specimen before returning to high magnification.
A trick for spotting your target when it is out of focus is to jiggle the slide with the mechanical control and focus on any object that appears to be moving. You should be aware, though, that your slide consists of multiple surfaces, and that you naturally must focus on the surface that bears your specimen.
With the objective lens well away from the specimen so that no part of the slide can be in focus, suppose you begin moving the stage toward the objective. The first surface that comes into focus is the top of the coverslip (A), although in bright field you may not see anything without stopping down the aperture diaphragm in the condenser. You may see scratches, dust, and fibers on the surface. The next surface is where the coverslip contacts the specimen. In a wet mount, the bottom of the coverslip (B) might be above the target material. To focus on a specimen at the surface of the slide (C) you would need to raise the stage. The next surface is the bottom of the slide (D). If you raise the stage too far, you actually may focus on the surface of the condenser lens (E). You can tell if you are focused on the condenser by jiggling the slide. Anything on the slide will be seen to move, but the condenser image will remain still.
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- 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
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- Caprette, D. (2005). Microscope images.
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