Oil Immersion Technique - Objectives, Procedure, Types

Oil immersion technique is a light microscopic technique used for increasing the resolving power of microscope at high magnification.

In this technique, a special transparent oil is used between the cover glass and the objective lens. This oil is called immersion oil. It is mostly used with oil immersion objective lens.

Normally, when light passes from glass slide to air, the light bends due to difference in refractive index. Some light rays are scattered and some rays do not enter into the objective lens. So the image becomes less bright and less clear.

Immersion oil has almost same refractive index like glass. So when oil is placed between cover glass and lens, it removes the air space. The light rays can pass through a continuous medium.

Due to this, more light rays enter into the objective lens. It increases the numerical aperture and improves the resolving power. Thus a bright, clear and sharp image is formed.

Oil immersion technique is used to observe very small microscopic structures. It is useful for observing bacteria and other minute details under high power microscope.

What is Microscopic Resolution?

Microscopic resolution is the ability of microscope to distinguish two close points as separate points.

It is also known as resolving power. It helps to see the fine details of a microscopic object clearly. If the two points are very close than the resolving limit, then they appear as one single blurred point.

Resolution mainly depends on the wavelength of light and numerical aperture of the objective lens. Shorter wavelength of light gives better resolution. Higher numerical aperture also gives better resolving power.

The numerical aperture means the capacity of lens to collect light rays from the specimen. When more light rays enter into the objective lens, the image becomes more clear and detailed.

Oil immersion is used to increase the numerical aperture. It removes the air gap between cover glass and objective lens and helps more light rays to enter into the lens.

Thus microscopic resolution is important for observing small structures. It helps to identify minute details of cells, bacteria and other microscopic particles.

Objectives of Immersion oil

To increase the numerical aperture of the objective lens.
To improve the resolving power of microscope.
To observe very small structures which cannot be seen clearly by dry objective.
To increase the brightness of the microscopic image.
To collect more light rays from the specimen into the objective lens.

To reduce refraction of light between cover glass and objective lens.
To prevent scattering and reflection of light rays.
To make a continuous optical path between the specimen and the lens.
To reduce optical aberration and image distortion.
To produce a clear, bright and sharp image at high magnification.

Why Oil Immersion used?

Oil immersion is used to reduce the bending of light rays.
When light passes from glass slide to air, the light rays are refracted. Some rays are scattered and some rays are reflected away. So less light reaches into the objective lens.
Immersion oil has nearly same refractive index as glass. For this reason, it fills the air gap between the cover glass and the objective lens.

It forms a continuous optical path. The light rays pass easily from the specimen slide to the objective lens.
It increases the numerical aperture of the objective lens. More oblique light rays are collected by the lens.
It also increases the resolving power of microscope. Fine and minute structures can be seen separately and clearly.

The image becomes brighter because more light enters into the lens.
It reduces hazy image, spherical aberration and chromatic aberration. So the final image becomes sharp, bright and less distorted.

Why oil immersion is used in microscope 100x?

Oil immersion is used in 100x objective because at this high power more light is required for seeing the object clearly.

In 100x objective, the lens remains very close to the slide. The space between cover glass and lens contains air. When light passes from glass to air, the light rays are bent and many rays are lost.

So the image under 100x becomes dim and not clear. The small details also cannot be separated properly.

Immersion oil is used in this space because its refractive index is nearly same as glass. It makes the passage of light almost continuous from slide to objective lens.

Due to this, the bending of light is reduced. More rays enter into the objective lens and image becomes brighter.

It also increases the numerical aperture and resolving power of the objective lens. So bacteria and other very small structures can be seen more clearly under 100x objective.

Immersion oil Types

The following are the important types of immersion oil-

Type A

It is a low viscosity immersion oil. It is thin and spreads easily between the cover glass and objective lens. It is used in routine brightfield microscopy and also useful for beginners.

Type B

It is a high viscosity immersion oil. It is thicker than Type A oil. It remains in place for longer time and is used when many slides are examined one after another.

Type C

It is a synthetic immersion oil. It is used as a substitute of natural cedarwood oil. It does not become hard and does not turn yellow easily with time.

Type 300

It is a special type of immersion oil. It is made for automated hematology instruments. It has controlled consistency which is needed for automatic microscopic system.

Type NVH

It is a very high viscosity immersion oil. It is used in inverted, inclined, projection and long-focus microscopes. It is useful where normal oil may flow away from the lens area.

Type OVH

It is an ultra high viscosity immersion oil. It is very thick oil. It is mainly used in projection system and long-focus instruments where the gap between lens and cover glass is more.

Type 37

It is an immersion oil used at 37°C temperature. It gives proper refractive index at body temperature. It is mainly used for live cell observation in heated chamber.

Tropical Grade

It is made for laboratories present in warm climate. It gives proper refractive index at higher room temperature. It is useful in tropical areas.

Low fluorescence oil

These are special immersion oils used in fluorescence microscopy. They do not produce much background fluorescence. So the specimen fluorescence can be seen more clearly.

Some examples are Type LDF, HF, FF and DF. Type FF is almost fluorescence free. Type HF is low fluorescence and halogen free. Type LDF gives very low fluorescence background. Type DF is medium viscosity oil and gives good resolution with pale green background.

Procedure of Immersion oil Technique

The following are the procedure of immersion oil technique-

Step 1

The microscope is kept on clean working table. The objective lens and eyepiece are checked before use.

The slide is cleaned properly. Dust and finger mark are removed from the slide.

Step 2

The slide is placed on the stage and fixed with stage clips.

The specimen is first focused under low power objective. The required field is brought to the centre.

Step 3

The specimen is then focused under high dry objective.

The light and condenser are adjusted for clear viewing. After proper focusing, coarse adjustment knob is not used.

Step 4

The nosepiece is rotated slowly between high dry objective and oil immersion objective.

One small drop of immersion oil is placed on the cover glass over the focused area.

Step 5

The oil immersion objective is slowly brought over the oil drop.

The lens should touch the oil drop, but it should not press the slide. This forms oil bridge between cover glass and objective lens.

Step 6

The specimen is observed through the eyepiece.

Fine adjustment knob is used to focus the image. Coarse adjustment is not used because the working distance is very short.

Step 7

The light is adjusted again if needed.

The condenser is raised and diaphragm is adjusted to get bright image. In some cases, oil may be placed between condenser and slide also.

Step 8

The specimen is examined under oil immersion objective.

The bacteria and other small structures are observed clearly due to better resolving power.

Step 9

After observation, the objective lens is cleaned immediately.

Lens paper is used to remove the oil from the lens. The slide is also cleaned. Oil should not be kept on the lens for long time because it may become sticky and damage the lens.

Advantages of Immersion oil Technique

The following are the advantages of immersion oil technique-

It increases the resolving power of microscope.
It helps to see the fine details of small microscopic structures.
It increases the numerical aperture of the objective lens.
More oblique light rays are collected by the lens due to use of oil.

It increases the brightness of image.
The image appears more clear because more light enters into the objective lens.
It reduces the refraction of light.
Immersion oil has almost same refractive index as glass. So the light rays do not bend much between cover glass and objective lens.
It prevents scattering and loss of light rays.
The light passes through a continuous medium from slide to lens.

It reduces optical aberration.
Spherical and chromatic defects are decreased and the image becomes less distorted.
It gives sharp and high contrast image.
It is useful for observing bacteria, blood cells and other minute structures under high power microscope.

Limitations of Immersion oil Technique

The following are the limitations of immersion oil technique-

It is not much suitable for live and watery samples.

When the specimen is present in water, the refractive index becomes different. So deeper part of the sample may appear blurred and dim.
The oil may move the cover slip.
Due to thick nature of oil, the cover glass can shift slightly. This may disturb the position of the specimen.
It may damage the objective lens if not cleaned properly.

Immersion oil collects dust and dirt easily. If the oil is dried on lens, it becomes sticky and hard film.
It may contaminate the dry objective lens.
If 40x or other dry objective touches the oil, the image becomes unclear. The oil may also enter into the lens part.
Air bubbles may be formed in the oil.
These bubbles scatter the light rays. So the image becomes less bright and contrast is reduced.

The working distance is very short.
The oil immersion objective stays very close to the slide. If focusing is not done carefully, the lens may strike the cover glass.
It depends on proper cover slip thickness.
If the cover slip is too thick or too thin, the image may not be sharp. Spherical aberration may occur.
It needs careful handling and cleaning.
After use, the lens and slide must be cleaned properly with lens paper. Otherwise the microscope may be damaged.

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Oil Immersion Technique – Objectives, Procedure, Types