Immersion Oil Alternatives: Which Oils Can Replace Traditional Microscopy Immersion Oils?

Microscopes use immersion oils to improve the resolution and contrast of specimens under high magnification objectives. The key requirements for a suitable immersion oil are appropriate viscosity, refractive index close to the glass, lack of autofluorescence, and minimal volatility.

With rising costs and supply chain issues with traditional immersion oils, some have proposed alternatives like coconut oil and glycerine. In this article, we evaluate the suitability of these alternatives.

Requirements for Immersion Oil

Refractive Index

The refractive index of immersion oil needs to be close to that of glass (around 1.5) to allow light to travel from the glass slide to the oil and into the objective lens without refraction. This minimizes spherical aberrations and improves resolution.

Most standard immersion oils have refractive indices between 1.5 and 1.58. The refractive index is temperature dependent so it should match glass at working temperatures.


Viscosity affects the working distance between the specimen slide and the objective lens. Oils with too low viscosity have poor adherence while highly viscous oils limit working distance. Standard immersion oils have a viscosity of 30-100cSt.


The immersion oil should be free of autofluorescence which can obscure dim signals from fluorescent specimens. Good quality fluorescence immersion oils are purified to remove fluorescent contaminants.


Immersion oils need to be stable under prolonged illumination without bleaching or volatilization. Oils that evaporate or oxidize can compromise image quality and require frequent reapplication.

Glycerine as Immersion Oil Alternative

Glycerine is a colorless, odorless liquid produced from fats and plant oils. Here are its pros and cons as an immersion oil:


Refractive Index

Glycerine has a refractive index of 1.47 at room temperature, lower than glass. This can introduce refraction artifacts and loss of resolution. However, glycerine’s refractive index increases to 1.49 at 50°C, closer to glass.


Pure glycerine has a very high viscosity of 934cSt at room temperature. This limits working distance for high magnification objectives unless diluted with water. A 30:70 glycerine: water mixture gives a viscosity in the ideal 30-100cSt range.


Glycerine itself has very low autofluorescence but improper purification can leave fluorescent contaminants. High-purity glycerine is comparable to the fluorescence of standard immersion oils and can be obtained through vacuum distillation.


Glycerine has good optical stability. It does not oxidize or volatilize under prolonged illumination. No significant bleaching or evaporation occurs compared to some plant-based immersion oils.


Food-grade glycerine is inexpensive but vacuum-distilled high-purity glycerine suitable for microscopy costs as much as traditional immersion oils.

Coconut Oil as Immersion Oil Alternative

Coconut oil is comprised mainly of saturated fats. Here are its characteristics:

Refractive Index

The refractive index of coconut oil ranges from 1.44-1.47, significantly lower than glass. This can reduce resolution compared to standard immersion oils. The refractive index also varies with coconut oil composition.


Coconut oil has a viscosity of around 30cSt at room temperature, ideal for immersion oil. However, being fat, the viscosity varies greatly with temperature – too low when warm and too high when cool.


Virgin or crude coconut oil has very high autofluorescence due to the presence of plant phenols and antioxidants. Extensive processing and bleaching is required to remove fluorescent contaminants.


Coconut oil is prone to oxidation and volatility under prolonged illumination and heating from microscope lamps. This requires frequent reapplication compared to standard synthetic oils.


Refined low-fluorescence coconut oils suitable for microscopy are often as costly as standard immersion oils. Unrefined oils are ineffective due to autofluorescence.

Verdict: Coconut Oil and Glycerine Work in a Pinch But Not Ideal

While alternatives like glycerine and coconut oil can technically function as microscope immersion oils when optimally processed and used under controlled conditions, they do not match traditional oils in resolving power, working conditions, and stability. Their optical properties vary with composition, purity, and temperature. This can compromise image quality.

For critical high-resolution microscopy, standard synthetic immersion oils remain the best choice. They deliver consistent performance and are engineered for optimal light transmission.

Coconut oil and glycerine may be suitable for basic microscopy if the cost and availability of commercial oils are a concern. With care, usable images can be obtained, but these alternatives come with caveats and limitations.

Potential Alternatives That Partially Fulfill the Requirements

Here are some other potential alternatives that have been examined for use as microscope immersion oils:

  1. Mineral oils – Refined mineral oil can achieve refractive indices close to glass but still suffers from temperature variability and autofluorescence issues.
  2. Silicone oils – Silicones have excellent optical stability and can be formulated to a precise refractive index. However, they are expensive and can be difficult to clean.
  3. Plant oils – Aside from coconut oil, other plant oils like clove, cedarwood, and castor oil have been suggested. However, extensive processing is needed to remove autofluorescence and achieve ideal viscosity.
  4. Synthetic esters – Esters like dioctyl sebacate can mimic the properties of natural oils at a lower cost. However, refractive index and fluorescence still vary.
  5. Ionic liquids – Water-stable liquids like imidazolium compounds have potential but their viscosity is often too high. Toxicity may also be a concern.
  6. Solid immersion lenses – These exotic lenses eliminate the need for oil altogether. However, they are very expensive specialized equipment with limited working distances.


Can I use olive oil or other cooking oils as immersion oil?

No, common cooking oils like olive oil have very low refractive indices (around 1.4) and high autofluorescence, making them poorly suited for microscopy. Their properties will degrade image quality compared to standard oils.

Do I need a special immersion oil for each objective lens?

No, a single immersion oil with a moderate refractive index of around 1.5 is suitable for most objectives from 40X to 100X. Specialized oils are only needed for particular techniques like phase contrast or ultra-high objectives above 100X.

Can I reuse immersion oil multiple times?

It’s generally advised to use fresh immersion oil each time to prevent dust or impurities from degrading image quality. However, oil can be reused for a few sessions as long as it remains optically clear and free of debris. Over time, oxidation and evaporation will degrade reused oil.

Is castor oil a good immersion oil substitute?

No, castor oil has very high autofluorescence that will obscure microscopy images. It also has a lower refractive index (around 1.48) than ideal. Extensive purification is required to make castor oil suitable as an immersion medium.

Can I use water instead of oil for high-magnification objectives?

Water’s refractive index is too low (1.33) for high-power immersion objectives designed for oil. It will drastically degrade resolution. Water immersion lenses are special low-magnification objectives designed just for water-based samples.

Leave a Reply

Your email address will not be published.

This field is required.

You may use these <abbr title="HyperText Markup Language">html</abbr> tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <s> <strike> <strong>

*This field is required.