Oil Immersion Microscopy
Applications, Advantages/Disadvantages and Cleaning
Oil Immersion Microscopy increases the refractive index of a specimen when used properly. With limited disadvantages, slides prepared with oil immersion techniques work best under higher magnification where oils increase refraction despite short focal lengths.
It is best to use an oil-immersed objective at high magnification as the oil compensates for short focal lengths associated with larger magnifications. The oil has a similar refractive value to the glass slides and slipcovers.
Each time light-waves pass through objectives with similar reflective indexes, images are not reduced or distorted. Ideal for short focused, large magnifications oil immersion microscopy yields bright images of fine resolution ranging from 40x – 120x. Depending on the sample, oil immersion can provide the most powerful lens potential on a light microscope.
Oil is an ideal conduit in the preparation of slides because the refractive index is the same or similar as glass. Although color can increase or decrease in oil immersion microscopy, color in this circumstance is characterized through the loss of light via absorption.
Researchers should consider whether color is important to a particular sample analysis in relation to the high resolution achieved via oil-immersed slides.
The most important aspect of an oil-immersed specimen is image quality; the lines and features may retain full integrity even though the value of color may be reduced.
Oil immersion microscopy utilizes one or more oils on an oil appropriate lens. Oil immersion slides are best suited for inanimate or dead subject matter. This commonly includes bacterium, muscle tissue with pronounced striations and specimens with small definable structures.
The specimens should not include items that can be destroyed by oils – as would be the case of acidic oil, where high Ph might cause deformities in samples containing metal.
In oil immersion microscopy diffraction is minimized as light bends the same as it passes through the layers of glass and oil. This is different from other mediums, such as water or glycol, where refractive indexes change each time light hits the glass and water, potentially decreasing image integrity.
Oil immersion at its most ideal includes a similar Ph between glass and oil; one or more synthetic oils are preferred over the former use of natural oils commonly derived from cedar-wood or sandalwood.
In addition, researchers, scientists, school labs, and hobbyists can order a mixture of different oils to achieve a specific viscosity. Advantages include:
- Offered by most manufacturers
- Reusable for second slides (under certain circumstances)
- Appropriate with wide condensers
- Objectives of long focus at low power
Can use on inclined stages, including horizontal and a range of slanted angles
Synthetic oils alone or in combination with others contain viscosity properties superior to natural oils such as cedar wood, which was the most commonly used natural source of oil. Synthetics keep the Ph at a more desirable level.
A low Ph, indicative of an acidic environment, can lead to the deterioration or degradation of samples and specimens. If unaware, viewers may mistake these for artifacts.
Users need to be careful to avoid inadvertently getting oil directly onto the microscope, especially the lens. If the slides are not prepared correctly, the cements used to seal the slides may be ineffective or possibly adhere to other slides or the microscope.
In addition to the caution required in making and preparing slides, disadvantages may include:
- If not maintained through application and cleaning, damage can occur to the lens
- If the cements or adhesives used to contain the oils underneath the slide are not placed on properly, they may allow outside particles such as dust to enter
If the cement dries on the microscope, it will be difficult to remove and may cause damage to the lens or other parts of the microscope
Certain solvents can be purchased to remove cement from unwanted areas. However, there is still a slight risk of scratches and abrasions.
Also note that oil is incompatible with dry lenses; using oil inappropriately can distort images.
As far as objective cleaning protocols are concerned there is an excellent book -"The Microscope" by Wredden.JH, from back to the year 1948 which sums up the best cleaning technique. At page 67 it reads...
"The best method of cleaning an immersion objective is to commence by wiping off the majority of the oil with a piece of dry lens tissue. The front of the lens may then be polished with the aid of the tongue and a clean soft handkerchief."
Basically, just wipe off the oil with lens paper for microscope lenses. If necessary and for the least harsh approach to cleaning, mix a solution of 5-10 drops dish washing liquid (no ammonia) in 10 ml of distilled water.
Excessive use of chemicals can ruin your lens so avoid them, especially if your lens is basically clean and there is no dried oil.
Lenses certainly don't improve from repeated cleaning.
For more information continue reading the best practices in cleaning your microscope. And check out Cell Staining to add to your microscopy talents.
Here is a MicroscopeMaster pick for the perfect oil for oil immersion microscopy
Oil Immersion Microscopy is an essential tool in examining specimens under a compound microscope. Although its few disadvantages are significant, careful technique will minimize problems such as cement drying on a lens.
Similar refractive indexes allow for large bright images, especially useful to the study of inanimate objects, striated tissue and bacteria; a mixture of synthetic oils can create the most suitable viscosity to achieve high resolute quality images.
Check out other relevant and interesting microscopy imaging techniques.
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