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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.

Key Takeaways

  • Immersion oils increase magnification and contrast in light microscopy

  • They help preserve image clarity and detail by reducing light distortion

  • Oils with a high refractive index work best for focusing light without blurring

How Immersion Oils Work

Immersion oils are used in light microscopy to boost 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. This helps achieve more clear and detailed images at higher magnification than without this medium. Typically, immersion oils have a refractive index sitting at a ratio of 1.1515, 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:

Illustration showing how the refractive index is measured

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. Generally speaking, a higher refractive index means light is slower and can be bent more in the medium.

The refractive index explained, with an equation

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 is 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.

Benefits of Immersion Oil

Perhaps the most important benefit of immersion oil is its ability to enhance both image quality and magnification. By reducing the scattering of light, immersion oils allow microscopes to capture more light from the specimen they are studying. This is vital when viewing very small structures like bacteria or cells, where clarity can be the difference between diagnoses or discoveries being spotted or missed. Beyond that, some other key benefits include:

  • Increasing the numerical aperture of the lens, boosting magnification for clearer views at high power
  • Matching the refractive index of glass, preventing distortion and preserving image accuracy
  • Oil enhances contrast, helping finer details stand out against the background
  • Immersion oil helps achieve higher resolution by focusing light more effectively
  • At high magnifications, immersion oil ensures maximum image quality by enabling better light gathering

What Are the Uses of Immersion Oil?

Immersion oils are used for various types of microscopy. They have a high refractive index, which lets them be more effective at bending light. This helps in achieving clearer, more detailed images, especially at high magnifications. Without immersion oil, light waves can scatter when they pass through the specimen and the glass slide, leading to blurring and a loss of detail. The oil fills the gap between the slide and the lens, ensuring that light continues through without distortion, leading to a clearer and more precise magnified image.  This process also helps prevent the light from being refracted at unwanted angles, which could otherwise degrade the image quality. 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.

The oil’s viscosity and refractive index are essential for its effectiveness. With a refractive index of around 1.515, immersion oils help focus light more precisely, which in turn improves the image quality. This is why they are often put into droppers as part of the chemical bottling process, to allow for easier application.

Distribution of immersion oil under light microscope objective lens

Using Dry Objectives Versus Immersion Oil

There are times when using a dry objective is more appropriate than using an immersion oil. These include lower power viewings, where objects don’t require such precise levels of enhancement to make an assessment. Some other pros of using dry objectives include:

  • There’s no need for additional oil or special preparation, making them quick and easy to use
  • Dry objectives can be used with any slide without worrying about oil compatibility or cleaning
  • They are generally less expensive than immersion oils
  • There’s no need to worry about oil getting on the lens or slide, which requires cleaning

Meanwhile, these are the cons of dry objectives:

  • Dry objectives gather less light, resulting in lower resolution and less detail at high magnifications
  • High-magnification dry objectives may not provide the clarity needed for viewing fine details
  • They are less effective in enhancing contrast, especially for high-resolution imaging

While we’ve discussed many of the positives of immersion oils so far, it’s important to note that they do have some drawbacks, including:

  • Extra preparation: Using immersion oil requires applying and cleaning the oil after use
  • They can get messy: Oil can get on the lens or slide, requiring thorough cleaning to avoid contamination or damage
  • Cost: Immersion oil and the necessary objectives are typically more expensive than dry objectives

Conclusion

Immersion oil plays a vital role in modern microscopy by improving image quality, enhancing contrast, and increasing magnification. Its ability to focus light effectively and reduce distortion ensures that scientists can observe even the most intricate details of specimens. Choosing the right immersion oil for the task at hand is crucial, as it directly impacts the quality of the results, and helps minimise the negatives of using this medium.

About the author

Jessica Clifton

Chief Strategy Officer

Jessica is Chief Strategy Officer at ReAgent and leads a variety of growth projects. She has an extensive background in marketing, and has worked in the chemical industry since 2019. Outside of work, Jessica can be found on a run, building LEGO, or watching Star Wars.
View all posts by Jessica Clifton

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