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Immersion Media and Microscope Objectives

Most high numerical aperture and high magnification objectives need immersion media in order to properly illuminate a sample and to capture the emitted photons. The numerical aperture (NA) of the objective helps to define its resolving power (how small of an object can the objective accurately resolve):

Resolving Power = Lambda/(2*NA)

As can be seen, the greater the NA, the smaller the Resolving Power (the smallest size the objective can resolve).

The NA is defined by the following equation:

NA = n*sina0

Where a0 is the half angle of the objective lens and n is the refractive index of the medium between the lens and the sample. As a result, air objectives have a maximum NA of 0.95 (n = 1, and there needs to be some space between the sample and the objective (arcsin(0.95) = 71.8 degrees)). To get beyond this NA limit you need to increase the refractive index of the medium, n. Here are several standard immersion media used for microscope objectives:

Immersion MediumRefractive Index
Air 1.00
Water 1.33
Glycerin 1.47
Oil 1.515

Nikon has a pretty good overview that you should look at (click here).  Olympus and others do as well. Warning: increasing the NA of the objective greatly reduces the the working distance of the objective. In other words, your sample will need to be closer to the objective with higher NA.

 

So, now that you are infatuated with the idea of using immersion oil, here is how you should use it:

  1. Manually lower the objective to a position well below the bottom of your sample.
  2. Place ONE small drop of your immersion medium onto the correct objective (oil for oil objectives, DI water for water objectives, air for air objectives).
  3. Place your sample onto the microscope stage. The coverslip goes towards the objective (down for inverted microscopes).
  4. Slowly raise the objective until the oil comes into contact with your sample. You should see the oil spread as contact is made. You are near the focal plane once this occurs.
  5. Find the sample as you normally would.

As you change samples or move around a lot (like from well to well of a multiwell plate), you may not have enough immersion medium to image appropriately. An indicator for this is that your sample will appear to be out of focus or blurry. If you need to add more immersion medium, please follow these simple instructions:

  1. Remove your sample from the microscope stage.
  2. Remove the oil from your sample using a kimwipe. You do not need to remove all of it, but more is better.
  3. Remove the oil from the objective using lens paper, moving the lens paper in one continuous direction across the top of the objective. DO NOT WIPE BACK AND FORTH OR SCRUB!!!!
  4. Repeat around the cone of the objective using a clean section or a new piece of lens paper.
  5. Repeat steps 4 and 5 until no more immersion medium is found on the lens paper.
  6. Once the objective is clean you can add new immersion medium and start again.

So, some of you may be asking yourself, "Why should I use only ONE drop of immersion medium?".  I will tell you why.  IMMERSION MEDIUM (ESPECIALLY OIL) GETS EVERYWHERE!!!  Don't believe me? Put one drop on your finger and then see how many fingerprints you leave on beaker.  Need more proof?  Look at the two images below:

Oil in PFS Case 2

Oil in PFS Base 2

 

What you are looking at here is the inside of the Nikon NSTORM Perfect Focus System case. That shiny stuff you see is IMMERSION OIL! The oil is also on the PFS filter and dichroic. For those of you that cannot picture where the PFS sits on the microscope, it sits directly below the objective. That means, for the oil to reach the PFS, it need to first go from the lens of the objective, down the cone, fill up the containment ring, spill down the side of the objective, cover the objective turret, before dripping down onto the PFS housing. Then, it has to accumulate in significant enough volume to enter into the PFS and spread all over. That is a ridiculous amount of oil. More importantly, this is an extreme amount of negligence. Left unchecked, this amount of oil will probably ruin the objective, neighboring objectives, and the PFS. Oil objectives can range from $5000 - $15000 each, the PFS is on the order of $25000. Oil costs about $100 per bottle (50 mL or so).

Please be careful using immersion media.

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