Oil Immersion, Refractive Index & Objective Front Lens Design 

This article explains how to use immersion liquids, like oil or water, between the front lens of a microscope objective and the sample to improve resolution and also eliminate false light. Compared to dry objectives, immersion objectives usually have higher numerical apertures. Water immersion objectives are preferred for living specimens in aqueous media.

Key Learnings:

• Immersion liquids can improve the resolving power of a microscope and eliminate false light.
  
• The numerical aperture (NA) is an important factor in determining the resolution of an objective. Generally, immersion objectives are made with higher NAs than dry objectives of the same magnification.
  
• Compared to dry objectives, immersion objectives can correct many image aberrations better.
  

The resolving power of the microscope is often increased by using an immersion liquid between the front lens of a dedicated immersion objective and the cover glass/sample.
Immersion objectives have been devised several times: The modern immersion objective was most probably invented by the Italian instrument maker Giovanni Battista Amici in 1847. Its function principle was fully understood scientifically in 1873, when Prof. Ernst Abbe published the first and still valid theory of image formation in the light microscope. Since then, many objectives in the magnification range between 40x and 100x and beyond have been designed for use with a suitable immersion liquid.
Most often, a synthetic immersion oil is applied. In former times, also cedar wood oil was popular, which still has excellent optical properties, but will harden out quickly during usage. Good results have been obtained with an oil with a refractive index of n = 1.518, which is close to the refractive index of glass. As living objects are embedded in aqueous media, water immersion objectives are becoming increasingly popular.

Another advantage of immersion objectives: they efficiently eliminate the otherwise unavoidable amount of false light, commonly caused by reflections on the path from the specimen to the objective.

This tutorial explores how changes in the refractive index of the medium between the objective’s front lens and the specimen (sometimes called the “imaging medium”) can affect how light rays are captured by the objective. It also clarifies the advantages of the immersion objective principle for the calculation and optical design of highly aberration-free objectives in general.

In this tutorial, the space between the objective front lens and a cover glass preparation is displayed. The objective has an arbitrarily fixed angular aperture of 65 degrees. The illuminating light rays are depicted either entering the front lens or being refracted outwards into the air surrounding the front lens or being totally reflected into the cover glass. To operate the tutorial, move the Refractive Index (n) slider and adjust the effective refractive index (n) of the optical matter within the object space. The ten virtual light rays emanating from the specimen pass through the cover glass, but only half (five rays) are refracted into the objective front lens at the lowest refractive index value. The other five light rays are either stopped by the objective front lens housing, refracted into the air surrounding the objective, or reflected into the cover glass. These light rays cannot contribute to the formation of the microscopic image.

One of the most important factors in determining the resolution of an objective is the angular aperture, which for a dry objective has a practical upper limit of about 72 degrees (with a sine value of 0.95). When taking the refractive index of the optical matter between sample and front lens into account, the following product is obtained:

n(sin(θ))(1)

It is consistent for all types of objectives from dry to immersion. The product n(sin(θ))(1) is known as the numerical aperture (NA). The NA is a convenient indicator of the resolution for any objective. It is generally the most important optical property (other than magnification) to consider when selecting a microscope objective. Values range from 0.025 for objectives with a very low magnification (1x to 4x) to as much as 1.6 for high-performance objectives (63x to 100x), utilizing special immersion oils and cover glasses. As the 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. Nevertheless, objectives with high numerical apertures require exact preparation (e.g. cover glass thickness) and samples close to the cover glass underside.

The usual way to increase the objective’s numerical aperture is to design 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 cover glass, image degradation due to thickness variations of the cover glass is 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 1.518 and a dispersion matching to that of the cover glass.

The illustration depicts the aplanatic refractions that occur at the first two lens elements in a typical oil immersion objective. The specimen is sandwiched between the microscope slide and the cover glass. It is in focus at point P, the aplanatic point of the hemispherical lens element. The front lens is followed by one or more meniscus-shaped lenses. The light rays refracted at the rear of the hemispherical front lens appear to proceed from point P(1), which is also the center of curvature of the first surface of the meniscus lens. The refracted light rays enter the meniscus lens along the radius of its first surface and are not refracted at this surface. At the rear surface of the meniscus lens, the light rays are refracted aplanatically, so that they appear to originate/diverge from point P(2). Such rays can be more easily combined to one focal plane, which is done by another meniscus-shaped lens element inside the objective. The result is an image that is free of spherical aberration.

Color aberrations are mainly corrected in lens groups containing optical elements with anomalous dispersion (e.g. CaF₂). An objective that is aplanatic for two remote colors, such as blue and red, has no visible longitudinal chromatic aberration for three main colors (blue, green, and red). It has been called an apochromat since its invention by Prof. Ernst Abbe in Jena in 1886.

If the wrong immersion liquids are used, e.g. oils from suppliers that do not exactly match the refractive index and dispersion values set by the original microscope objective manufacturer, the image will suffer from chromatic and spherical aberration.

For living specimens, usually embedded in aqueous media, water immersion objectives are favored. Although they have a maximum NA of only 1.2, the water immersion objectives allow to optically penetrate such samples up to ~400 µm without considerable spherical aberration.