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Oil Immersion Microscopy

11/7/16


Oil Immersion Microscopy

When light passes through different media, such as from glass to air, it is refracted, and different wavelengths of light bend at different angles. Therefore, as white light passes across several refractive surfaces the waves spread out and the edges of objects appear blurred. Resolution is defined as the minimum distance between objects that can be visually detected. With dry lenses resolution is unsatisfactory at final magnifications above 400x.

A drop of oil with the same refractive index as glass, placed between the specimen and the objective lens, eliminates two refractive surfaces. Final magnifications of 1000x or greater are attainable, with satisfactory resolution.

Caution: Oil will ruin other objective lenses. Use it only on lenses that are clearly labeled for oil immersion. If you inadvertently get oil on another lens, such as the high dry lens (40x), clean it immediately. Before you put the microscope away, always check the high dry lens for oil contamination if you have been using oil. The lens has a very narrow focal plane, so that it is easy to focus right through the specimen and hit the slide with the lens. Be extremely careful when focusing. The oil lens must be very close to the specimen to bring it into focus. Therefore it is used only with a proper (thin) single cover slip, or without a cover slip in the case of a smear. The lens will not focus through the thickness of a glass slide.

Focus and center the specimen at the highest dry magnification, using the FINE focus control. Center a small object that is very easily seen in the field. Using the coarse focus control, lower the stage sufficiently to apply a drop of oil to the slide. Keep the oil confined to the illuminated area of the specimen. Looking from the side and OBSERVING CAREFULLY, raise the stage until the objective just touches the oil. You will see a change in the quality of light when the objective makes contact. Lower the stage a little without breaking contact with the oil.

Attempt to focus using the FINE focus control. This is one situation in which you may have to focus by moving the stage toward the objective lens. Go slowly because the focal plane is extremely narrow. It is so easy to drive the specimen into the objective lens that most 100x lenses have a telescoping “shock absorber” built into them for protection. Keep checking that you are not pushing the cover slip against the lens.

If you cannot focus, do not attempt to go back to using the 40x objective. You will get oil all over it, and the oil will blur the image anyway. Think about why you cannot focus. Low illumination and a lower degree of contrast are typical of high magnifications. You may wish to temporarily close down the aperture diaphragm to enhance details for focusing, then readjust it once you are there. Move the slide back and forth just a little, and see if you can detect movement. For wet mounts the surface tension of the oil may pull up the cover slip. Petroleum jelly can be used to secure the edges of the cover slip to the slide. If you simply cannot get a satisfactory image, get help.

Even a thinly cut specimen is three-dimensional. If the specimen will not focus, but it defocuses in both directions, either the lens is contaminated or the specimen is too thick. Try the lens on a thin specimen such as a blood smear or very thin prepared slide before you try to clean it. You may just have to prepare a thinner specimen or work at a lower magnification.




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