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What is immersion oil used for and what is the proper technique for using it?

Compound microscope objectives in the 90X to 100X range require immersion oil.

Background

The upper limit of the resolving power of light microscopes is slightly above 1000X. Objectives of 90 to 100X, when coupled with a 10X eyepiece, approach that upper limit. Even in the range of 900 to 1000X, a clear image is only possible if every bit of available light is directed through the microscope optics to the viewer's eye. Immersion oils play an essential role in maximizing the amount of light producing the image the viewer sees.

In the airspace between the slide and the objective lens, light is refracted, scattered, and effectively lost. This happens because the refractive index of air (approximately 1.0) is very different from that of glass (approximately 1.5), and light passing through a glass/air interface is refracted (bent) to a large degree. By reducing the amount of refraction at this point, more of the light passing through the slide will be directed to the very narrow diameter lens of the high-power objective. The more light, the clearer the image. Placing a material with a refractive index equal to that of glass in the airspace between slide and objective directs more light through the objective and produces a clearer image. Immersion oils are formulated for just this purpose.

High-power objectives of 90X or higher are almost invariably intended for use with oil and will be engraved with the words, "oil", or "immersion", or "HI" (homogeneous immersion). These objectives are assembled with special sealants that prevent penetration of oil into the lens system. Applying oil to an objective not designed for immersion will ruin the objective.

Immersion oils are commonly available in two viscosities-low viscosity (Type A), and high viscosity (Type B)-and should be labeled with a refractive index of 1.515. The low viscosity oil is applied to the airspace between slide and objective, the high viscosity oil is (less commonly) applied between the condenser and the slide.

Procedure

Low viscosity oil between slide and objective:

1. With low- or medium-power objective, locate a point or area of interest on the slide and center it in the image field.

2. Rotate the objective turret so that the high power objective is just to one side of the slide. Place a single drop of immersion oil (low viscosity, Type A) on the slide (using the circle of light from below as a guide) and place a drop directly on the objective lens. Failure to apply oil to the objective will likely result in trapped air and reduced image quality.

3. Slowly rotate the high power objective into place and adjust the fine focus to fully resolve the image.

High viscosity oil between condenser and slide (optional).

Condensers with a numerical aperture (N.A.) of 1.0 and greater (usually engraved directly on the condenser) are also sealed to prevent oil penetration. Do not immerse condensers with an N.A. less than 1.0.

1. Before placing the slide on the microscope stage, rack the condenser down (using the condenser focusing mechanism) and apply a drop of oil (high viscosity, Type B) to the condenser lens.

2. Apply a drop of oil to the bottom of the slide directly below the specimen, and place the slide on the stage so that the drops will meet when the condenser is raised.

3. Raise the condenser until the drops converge. Follow the steps detailed above to oil the slide to the objective.

Cleanup

Immersion oil should be cleaned from lens and slide surfaces when observations are complete. Oil left on lens surfaces will eventually dry and be very difficult to remove.

1. Carefully wipe oil from all glass surfaces with a folded piece of clean lens paper.

2. With a second piece of lens paper, moistened with a small amount of alcohol (ethyl or isopropyl), wipe glass surfaces to remove any streaks of residual oil.

3. To remove oil that has been allowed to dry on lens surfaces, moisten a folded piece of clean lens paper with a small amount of xylene. Gently wipe lens sufaces, giving the xylene a few moments to work. Xylene may soften cements used to assemble the objective-so wipe the surfaces again with clean lens paper moistened with dilute alcohol or distilled water.

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