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How to add Oil or Water to an objective

Objectives with a large numerical aperture (NA) require the application of immersion media (oil, water, glycerin) to function properly. In our facility, primarily only oil and water objectives are present (in addition to air objectives.A very nice overview of immersion media and its applications can be found here. If you are not sure what immersion medium is necessary, please ask for help. Here is a general guide to help you determine which objectives require immersion medium (and which type) for our facility:

Objective Numerical ApertureObjective MagnificationImmersion Medium
0.2 - 0.95 4x, 10x, 20x, 40x Air
1.1 - 1.3 60x Water
1.4 - 1.49 40x, 60x, 100x, 100x TIRF Oil

You may have noticed that some of the objective appear twice. This is not a typo. For example, in our facility we have 40x Air and 40x Oil objectives (on the A1R, for example). You need to be careful with these objectives to make sure you use the correct immersion medium with the correct objective. Often, if you hover your mouse over the objective within the software (in Elements, for example), a tooltip appears giving you the details of that objective. These include the magnification, the numerical aperture (NA) and the immersion medium. If you see a W or WI, then you need to add distilled water. If you see Oil, you need to add oil. if you do not see either, then it is most likely an air objective and you do not need to add anything (except for air but that is all around us anyways).

One must take care when applying immersion media as too much can cause thousands of dollars of damage to the objective and the microscope. Further, tacky immersion media such as oil can get all over everything (stage, joystick, mouse, keyboard,...) and your hands.

Here is a very simple protocol to follow when adding oil or water to an objective:

  1. Lower the objective down well below the anticipated focal plane
  2. Add the appropriate stage insert to the microscope
  3. Adjust the stage to the appropriate XY position for your sample
  4. Using the Oil or Water dropper, carefully add 1 SMALL DROP to the top of the objective. It is not necessary to touch the dropper to the objective. Let gravity be your friend.
  5. If you add too much oil or water, please remove all of it using lens paper before repeating step 4. See below for instructions on how to clean the objective.
  6. Place your sample into the stage insert. If you are using a fixed sample be sure to put the coverslip towards the objective (down in our facility).
  7. Slowly raise the objective until you see the oil or water come into contact with the sample. You will see the oil spread out. At this point you are very near your focal plane
  8. If you accidentally add immersion medium to an air objective, please remove the immersion medium using lens paper (see below) and notify me via email or text. I will come by and appropriately clean the objective.

Click here to watch an brief and incredibly awesome video on how to add immersion medium to an objective.

Want to know how to remove oil or water from an objective? Click here.

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