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iLabs: What Is In Immersion Oil for Microscopes?

June 24, 2012

Last Saturday we had a visitor in the lab who was fascinated by our compound microscopes. While he has worked with the usual 4x, 10x, and 40x objectives, he was really interested in the 100x objective. This is a special objective that combined with the 10x power of the ocular lens, gives a total magnification of 1000x to any object being viewed. It is also a lens that needs to have a special oil called “immersion oil” placed between the slide specimen and the 100x lens.

This man was very interested and asked all kinds of questions, though his final one, I could not answer: What is in immersion oil, what is the base product it is made from?

We welcome questions from our visitors, and especially love it when you ask us something we have to go and research. Then we both learn something new. I did some digging on his question and without further delay, here are some results about immersion oil:

What is immersion oil and what does it do?

It is an oil placed on a slide of material to be examined using a special, 100x objective lens on a compound microscope. The lens is moved down toward the slide and into the oil, thus there is no air gap between the slide and the lens.  It is meant to have the refractive index of glass so as not to refract the beams of light being focused on the item being examined. Basically this means that all light beams are focused onto the object to be examined, as opposed to light beams going through air and being scattered, thus losing some of their power. Using oil results in higher brightness at high magnification (1000x), and high image resolution. Examples of things best viewed under oil immersion are bacteria, blood cells, various blood, fecal and tissue parasites, various cell types from biopsy specimens, etc.

Neutrophil in a Wright's Stain of a blood smear

Here is an image of a stained blood smear showing red cell appearance and a white cell called a neutrophil. This type of image is best seen at 1000x magnification using immersion oil.

A good summary of immersion oil comes from the Cargille Labs Website:

Immersion Oil contributes to two characteristics of the image viewed through the microscope: finer resolution and brightness. These characteristics are most critical under high magnification; so it is only the higher power, short focus, objectives that are usually designed for oil immersion.”

Now, getting back to the original question, “What is in it?,” well, that seems to vary depending on manufacturer:

From a 2002 Flinn Material Safety Data Sheet

“Clear liquid with slight yellow color. Odorless.
Solubility: Not soluble in water. Material is a proprietary
mineral oil mixture.”

From a 1998 New England Journal of Medicine article:

“According to Leica, modified epoxy resins were contained in the new oil,”

From a 2012 entry at Wikipedia on Oil Immersion:

“Before the development of synthetic immersion oils in the 1940s Cedar tree oil was widely used. …Cedar oil has a number of disadvantages however: it absorbs blue and ultraviolet light, yellows with age, has sufficient acidity to potentially damage objectives with repeated use (by attacking the cement used to join lenses), and diluting it with solvent changes its viscosity (and refraction index and dispersion). Cedar oil must be removed from the objective immediately after use before it can harden, since removing hardened cedar oil can damage the lens. In modern microscopy synthetic immersion oils are more commonly used, as they eliminate most of these problems.[2] NA values of 1.6 can be achieved with different oils. Unlike natural oils synthetic ones do not harden on the lens and can typically be left on the objective for months at a time, though dust accumulation can be a problem. Xylene is often used to remove oil from the objective when desired.”

From Microscopy UK;

These lenses are designed to be used with cedar wood oil, a naturally occurring product from the tree. Its refractive index of approximately 1.52, is almost identical to that of the glass used for slides and coverslips. Synthetic oils have been formulated which are more stable, and can be obtained in various viscosities. Other fluids such as glycerine, and also mineral and vegetable oils work , but their refractive indices and dispersive powers vary somewhat from that of glass, and hence cannot be expected to elicit the best imagery from the specimen.

I think it is safe to say that immersion oils used now are synthetic or variations of mineral oil with added ingredients to make sure they do not damage lenses, they maintain image clarity, and do not turn yellow with age. The point is to get an oil mixture that is very close to the refractive index of glass so as not to interfere with the appearance of the object being examined, while at the same time intensifying the brightness of the light and the resolution of the image.

If you absolutely loved all these details, I suggest you go to the Cargille Labs website, which seems to have every last detail you’d ever want to know, and more, about oil immersion microscopy theory, and their specifications sheet on their oil.

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2 Comments leave one →
  1. Joe permalink
    July 20, 2012 3:03 pm

    Great info! What brand and model microscope were those? I remember they looked pretty cool.

  2. July 22, 2012 2:33 pm

    We have Leica microscopes. The model we use for our classes is: DM500. Here’s a link to the Leica website: http://www.leica-microsystems.com/products/light-microscopes/education/life-science/details/product/leica-dm500/

    The other microscope we have in the lab, also capable of oil immersion microscopy, is the EVOS XL. It is an inverted microscope, meaning that the objectives are BELOW the stage, vs above on standard microscopes. A link for that is: http://www.amgmicro.com/products/evos_xl.php

    Thanks for your comment and hope to see you in the lab again!

    Deb

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