Immersion oils improve the resolution and brightness of images viewed through a microscope, owing to their optical and viscosity characteristics.
Microscopy wouldn’t have advanced to where it is today without the use of immersion oil. Without it, the resolving power of light microscopes in revealing the details of cells would have hit a brick wall.
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What is the Function of Immersion Oils?
Immersion oils are used to increase the resolving power of light microscopes. These oils are transparent and have a high refractive index, meaning they can significantly increase the magnification and contrast of a specimen under a microscope.
The optical and viscosity characteristics of immersion oils are crucial in light microscopy because of the way they effectively focus light without blurring any details.
Typically, immersion oils have a refractive index of 1.1515. This is unitless because it’s a ratio. The refractive index is typically measured in terms of the ratio between the angle of refraction in a medium, and the angle of incidence of light from the source in relation to an imaginary line that’s perpendicular to the medium, as shown in the illustration below:
Refractive index is also measured in terms of the ratio between the speed of light in a vacuum and the speed of light in a medium. This definition is more accurate, but harder to measure experimentally. Higher refractive index means light is slower and can be bent more in the medium.
Specialised microscope objective lenses are used with immersion oils. Ordinary objective lenses can still work, but not as effectively. Optimal resolution is also achieved with the help of condenser lenses immersed in oil.
Oil immersion is a special technique used in light microscopy that involves the immersion of both the specimen and the objective lens in a transparent oil that has a high refractive index. This has the effect of increasing the numerical aperture of the objective lens, meaning that the lens can gather more light despite its small size. In order for a lens to have a large aperture, it has to have a larger diameter.
This also means that the lens has to be thicker in order to have greater magnification. But this would negatively affect the amount of light that could pass through the lens. Therefore, using immersion oil is a better solution to the inevitable compromises between aperture and magnification, as well as between the thickness of the lens and its light permissibility.
Light scattering is more pronounced in the absence of immersion oils. Without immersion oil, light waves are reflected off the slide specimen, through the glass covered slip, through the air, and finally into the lens of the microscope. The light waves are refracted based on the angle of incidence. The refractive indices of the glass and air are very different, causing some distortions of the image as light passes through different media with different refractive indices. This has a detrimental effect on the quality of the magnified image. Fewer details are preserved in the process. Meanwhile, immersion oil has similar refraction properties to glass. This preserves image quality because there are very few distortions in the light rays.
As shown in the illustration below, light rays aren’t distorted and are more focused when they pass through the oil medium, represented by the yellow colour. The blue-coloured shape represents the objective lens of a microscope. As a result, more light rays enter the objective lens, translating into better magnifications and greater details.
What Are the Uses of Immersion Oil?
Immersion oils are used for various types of microscopy. Each type of microscopy typically requires a different type of immersion oil, which primarily depends on the details that need to be revealed:
- Normal light microscopy: This is the most common type of microscopy that uses normal light sources, such as ambient sunlight or light from an electric lamp. It uses two types of immersion oils, with one being more viscous than the other. These are general purpose immersion oils that can be combined with each other to produce intermediate viscosities.
- High viscosity oil microscopy: The oils used for this purpose have a viscosity of 21,000 centistokes. This type of immersion oil is used for inverted, horizontal, and inclined specimen mounts. It’s also used for long working distance objectives and specimens that use condensers with wide gaps.
- Fluorescence microscopy (type DF): The immersion oils that are used for this type of microscopy provide the highest resolution, and produce a pale green background colour. In some setups, laser light is used with a toluene-water interface.
- Fluorescence Microscopy (Type FF): For this application, an improved oil is used without background fluorescence. The oil is crystal clear and non-hygroscopic. This oil can also be used in other types of fluorescence microscopy making it a universal oil.
What Are the Types of Immersion Oil?
Immersion oils are classified based on their clarity, applications, and viscosity, which is measured in centistokes. A wide range of synthetic oils are commercially available for various microscopy applications.
Different countries have their own standards of quality, but the most recognised standard for immersion oil is specified by the International Standards Organization in its ISO 8036 standard for immersion oils intended for general purposes. The specifications are the following:
- ne = 1.5180 ± 0.0005 (nD = 1.515)
- Ve = 44 ± 3 (Abbe Number – a measure of dispersion)
- Temperature = 23 ± 0.1 degrees Centigrade
Here are some of the common types of immersion oils based on viscosity and applications:
- Types A and B: These have a 150 to 1,250 centistokes viscosity. You can mix types A and B to get intermediate viscosities.
- Type NVH: These are high viscosity immersion oils with viscosity levels of 21,000 centistokes.
- Type OVH: These immersion oils have very high viscosity at 46,000 centistokes. They’re ideal for inverted, inclined, and long focus instruments.
- Fluorescence microscopy types: The immersion oils used for fluorescence microscopy are types LDF, HF, and FF. They have viscosities of 500 centistokes, 700 centistokes, and 170 centistokes, respectively.
- Type 37: This has a refractive index of 1.515 and a viscosity of 1,250 centistokes, similar to type B. However, it’s stable above the standard calibration temperature of 23°C, and is designed to remain stable at 37°C. Image quality is preserved even at this temperature.
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