Oil immersion objective
In light microscopy, an oil immersion objective is a specially designed objective lens used to increase the resolution of the microscope. This is achieved by immersing both the lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.
The resolution of a microscope is defined as the minimum separation needed between two objects under examination in order for the microscope to discern them as separate objects. This minimum distance is labelled δ. If two objects are separated by a distance shorter than δ, they will appear as a single object in the microscope.
A measure of the resolving power of a lens is given by its numerical aperture, NA:
where λ is the wavelength of light. From this it is clear that a good resolution (small δ) is connected with a high numerical aperture.
The numerical aperture of a lens is defined as
where α0 is the angle spanned by the objective lens seen from the sample, and n is the refractive index of the medium between the lens and specimen (≈1 for air).
State of the art objectives can have a numerical aperture of up to 0.95. Because sin α0 is always less than or equal to unity, the numerical aperture can never be greater than unity for an objective lens in air. If the space between the objective lens and the specimen is filled with oil however, the numerical aperture can obtain values greater than unity. This is because oil has a refractive index greater than 1.
From the above it is understood that oil between the specimen and the objective lens improves the resolving power by a factor 1/n. Objectives specifically designed for this purpose are known as oil immersion objectives. Cedar tree oil is the most utilized oil for this purpose, as it has an index of refraction of 1.5. The numerical aperture of cedar tree objectives are generally around 1.3, but NA values of 1.6 can be achieved with different oils.
Oil immersion objectives are only used at very large magnifications that require high resolving power. Objectives with high power magnification have short focal lengths, facilitating the use of oil. The oil is applied to the specimen (conventional microscope), and the stage is raised, immersing the objective in oil. (In inverted microscopes the oil is applied to the objective).
The refractive indices of the oil and the lens are of the same order of magnitude, which means that the refraction of light will be small upon entering the lens (The oil and glass are optically similar). The oil immersion objectives are constructed with this in mind, and can not be used without oil, because in this case there will be considerable refraction of light at the glass/air interface, making it impossible to achieve focus. Using the oil immersion objective with oil can only be done on fixed speciments.
In addition to improving resolution, the use of oil is also advantageous in that it reduces the reflective losses as light enters the lens (again because the oil and glass are optically alike).
- Practical Microscopy by L.C. Martin and B.K. Johnson , Glasgow (1966).
- Light Microscopy by J.K. Solberg, Tapir Trykk (2000).