What is Oil Immersion?
- Oil immersion is a technique, used to increase the resolving power or microscopic resolution of a light microscope.
- This is done by immersing the objective lens and specimen into a transparent oil containing a high refractive index, as a result, it increases the numerical aperture of the objective lens.
- The immersion oils have the index of refraction around 1.515.
- The objective lens, which is immersed in the immersion oil is called an oil immersion objective.
What is Microscopic Resolution?
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Microscopic Resolution Definition
The microscopic resolution also is known as resolving power. Resolution is defined as the ability of a lens to separate or distinguish between small objects that are close together or the ability to distinguish two adjacent points as distinct and separate.
Equation of Microscopic resolution
The microscopic resolution can be calculated by using Abbe Equation. Resolution is described mathematically by an equation developed in the 1870s by Ernst Abbe, a German physicist responsible for much of the optical theory underlying microscope Design.
The Abbe equation is;
d = Distance between two objects.
𝞴 = Wavelength of light.
nsin𝝧 = Numerical Aperture.
When d becomes smaller, the resolution increases, therefore the resolution (r );
Why is Oil Immersion used?
When light rays pass through two different mediums of different refractive index, it is bent.
For example, in a light microscope, the light rays pass through two different mediums which containing different refractive indices, such as the glass (objective lens and slide) and air.
During the passes of light from the specimen slide to the objective lens, it also travels through the air space between the objective lens and slide. During this time some of the light rays are scattered and lost, because of the variation in the refractive index of air and glass.
The air has the refractive index is approximately 1.0, whereas the glass has approximately 1.5. During the passes of light through both glass and air, it is refracted. The light rays of different wavelengths bend at different angles, so as the objects are magnified more, and images become less distinct.
This means if you increase the magnification power of the objective lens the image will be blur or dark.
The light refraction or this problem is highly noticeable in the 100x objective lens it is not noticeable during the uses of lower magnification microscope objective lenses such as 4x, 10x, 40x.
If somehow we reduce the amount of light refraction, more light will be pass through the objective lens from the specimen slide and as a result, the image will be clear and crisp.
The amount of light refraction can be reduced by placing a drop of immersion oil between the objective lens and slide, which has an equal refractive index of the glass slide and the space filled with air. As a result more light will pass through the objective lens and it will form a clear image.
Immersion oil Objectives
- The objective lenses which are specifically designed for this purpose is known as the Oil immersion objectives.
- The objective lens improves the resolving power by a factor 1/n.
- They are used for very large magnifications that require high resolving power.
- They have short focal lengths, facilitating the use of oil.
- In the conventional microscope, the immersion oil is applied over the specimen slide and the stage is raised to immerse the objective lens in oil.
- In inverted microscopes, the immersion oil is applied to the objective.
Immersion oil Types
- There are mainly present two types of oil which are used as immersion oil such as; Cedar tree oil and synthetic oil.
- In the 1940s, synthetic immersion oils were first developed before that cedar tree oil was used as immersion oil.
- Cedar tree oil has a refractive index of about 1.516.
- Cedar tree oil objectives also has a numerical aperture of around 1.3.
- There are present several disadvantages of Cedar tree oil such as;
- Absorbs blue and ultraviolet light.
- Become yellows with age.
- It potentially damage objectives by attacking the cement used to join lenses, due to presence of sufficient acidity.
- It needs to remove immediately after use before it can harden.
- Due to these disadvantages now synthetic oils are used as an alternative of Cedar tree oil, because it eliminates most of these problems of Cedar tree oil.
- The synthetic oil has a numerical aperture around 1.6, which can be achieved with different oils
Immersion oils are categories into different classes such as Type A and Type B, based on their properties and the type of microscopy will be performing. Type F immersion oil is used for fluorescent imaging and type N used to visualize living cells.
Immersion oil Technique
- After observing the sample in 40x rotate the objective lens partway between the 40x and 100x lens.
- The place a drop of type A immersion oil at the top of the coverslip.
- Now carefully set the 100x objective lens on the drop of immersion oil.
- Then adjust the focus by controlling the fine focus knob until a clear and crisp image appear.
- After observing the image wipe the oil from all glass surfaces using a piece of lens cleaning paper.
- Again clean the lens using a small amount of alcohol or lens cleaning solution.