Immersion Oil and Refractive Index

The refractive index of the imaging medium is critical in determining the working numerical aperture of a microscope objective. A dramatic increase in numerical aperture is observed when the objective is designed to operate with an immersion medium such as oil, glycerin, or water between the front lens and the specimen cover glass.

This tutorial explores how changes in the refractive index of the imaging medium can affect how light rays are captured by the objective, which has an arbitrarily fixed angular aperture of 65 degrees. To operate the tutorial, use the mouse cursor to translate the Refractive Index slider and adjust the effective refractive index (n) of the imaging medium in the object space. The 12 hypothetical light rays emanating from the specimen pass through the cover glass, but only four are refracted into the objective at the lowest refractive index (n) value. The other eight light rays are either stopped by the objective front lens housing, refracted into the air surrounding the objective, or reflected back into the cover glass. These light rays and do not contribute to formation of the image. As the refractive index value of the imaging medium is increased by moving the slider to the right, successively more light rays are able to refract into the objective front lens until, at the highest n value, all 12 rays enter the objective.

One of the most important factors in determining the resolution of an objective is the angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of 0.95). When combined with refractive index, the product:

Formula 1 - Numerical Aperture
$$n(sin(θ)) $$

is known as the numerical aperture (abbreviated NA), and provides a convenient indicator of the resolution for any particular objective. Numerical aperture is generally the most important design criteria (other than magnification) to consider when selecting a microscope objective. Values range from 0.1 for very low magnification objectives (1x to 4x) to as much as 1.6 for high-performance objectives utilizing specialized immersion oils. As numerical aperture values increase for a series of objectives of the same magnification, we generally observe a greater light-gathering ability and increase in resolution.

Objective numerical aperture can be dramatically increased by designing the objective to be used with an immersion medium, such as oil, glycerin, or water. By using an immersion medium with a refractive index similar to that of the glass coverslip, image degradation due to thickness variations of the cover glass are practically eliminated whereby rays of wide obliquity no longer undergo refraction and are more readily grasped by the objective. Typical immersion oils have a refractive index of 1.51 and a dispersion similar to that of glass coverslips. Light rays passing through the specimen encounter a homogeneous medium between the coverslip and immersion oil and are not refracted as they enter the lens, but only as they leave its upper surface. It follows that if the specimen is placed at the aplanatic point of the first objective lens, imaging by this portion of the lens system is totally free of spherical aberration.

Figure 1 - Oil-Immersion Microscope Objective

Contributing Authors

Matthew Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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Immersion Oil and Refractive Index