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    • CommentAuthorcpgruber
    • CommentTimeNov 27th 2011
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    Oil immersion protocol revised

    This protocol explains how to: use the 100x objetive lens immersed in oil.

    Protocol revised: 11/27/2011

    Protocol written by: Chase Gruber

    To acquire a high-resolution image using the 100x objective, light collected by the objective must pass through immersion oil, which has a greater refractive index than air, to increase numerical aperture. The following steps outline the procedure used to acquire an image using the 100x objective lens immersed in oil and the proper technique to clean the 100x lens after oil immersion.

    1. View the slide with a low power objective before moving up to the dry 100x objective. At this magnification, find the field of view you would like to image. If the image is brightfield, set up Kohler illumination before continuing with this protocol (see Kohler illumination protocol if necessary).

    2. With the desired image in view, rotate the nosepiece so that no objective is pointing directly towards the slide. You can turn the nosepiece counterclockwise, so that the specimen is between the 40x and 100x objectives, or clockwise, so that only the 100x objective is near the slide. The latter is the safest way to go about this, as you decrease the chance of inadvertently passing other objectives through oil.




    3. Place 1-2 drops of immersion oil (located on the shelves above the microscope) directly onto the slide where the objective will be located. You can use the column of light transmitted through the slide as a guide for oil placement. You may also remove the light shield to make the slide more accessible.



    4. Rotate the nosepiece back to the 100x objective without passing any other objectives through the oil. The 100x objective lens should now be immersed in oil.

    5. The stage can be moved, but doing so may spread the oil too thin. Repeat steps 2-4 if oil reapplication is necessary. It is best to move the stage as little as possible after applying oil.

    6. Once finished with the 100x objective, rotate the nosepiece so that there is no objective pointing toward the slide, like in step 2.

    7. Lower the stage and remove the slide. Wipe up oil with a kimwipe or any absorbent paper. Be sure to remove all oil from the slide, as any remnants will make future imaging of the slide difficult.

    8. ONLY use lens cleaning tissues, found in packets on the shelves above the microscope, to clean the objective lens. DO NOT use a kimwipe, as it will scratch the lens. Lower the stage to give you more room to adequately clean the lens. 


    9. Place a lens cleaning tissue between your finger and the lens. Pull the paper without moving your finger so as to drag the lens wipe across the objective lens. Wiping in circles smears oil in the lens without removing it. Repeat 3-4 times using a clean section of tissue for each pull.


    10. If any other objective accidentally passed through the oil, clean the lens using the same technique described in the previous step.

    *If the objective still looks foggy or unclean for any reason, see the lens cleaning protocol for more thorough lens cleaning instruction.