Why immersion oil is used

To examine specimens at high magnifications using the microscope, there are a number of factors which need to be taken into consideration. These include resolution, numerical aperture NA , the working distance of objectives and the refractive index of the medium through which the image is collected by the front lens of an objective.

In this article, we will briefly look at how using an immersion medium between the coverslip and the objective front lens helps to increase the NA and resolution. In addition, we will consider the refractive index of air and the glass with which slides and coverslips are composed and how an immersion medium is used to partially reduce the mismatch when light travels from one medium to another.

There are also practical tips for using oil immersion systems as well as the benefits of using water immersion objectives, especially when imaging live cells.

One of the main problems in light microscopy is to overcome some of the limits of optical resolution and to increase the NA of the system. In brief, the NA of an objective is the ability to gather light from a specimen whereas resolution is the ability of an objective to distinguish details in the specimen. Resolution and NA will be covered in other articles , but we will now examine immersion techniques available to microscopists which allow the imaging of specimens at high magnification whilst overcoming some of the limits of resolution.

Having an immersion liquid in place of the air gap between the front lens of an objective and the cover glass of a specimen increases the resolution of an objective. When light passes from one medium to another for example, through glass to air it refracts - in other words, it bends and scatters. Any light rays which are refracted into the air, reflected by the cover glass or actually blocked by the metal housing of the objective front lens do not contribute to the image formation.

The purpose of the immersion liquid is to decrease the amount of refraction and reflection of light from the specimen and increase the ability of the objective to capture this otherwise deviated light s.

Figure 1. The physical properties of the medium through which light rays travel determines the degree to which the light will be refracted. Air has a refractive index of 1. Taking this difference into account, the purpose of the immersion liquid is to match as closely as possible the refractive index of the glass in which the specimen is mounted, therefore increasing the amount of light rays which will form the final image.

Subsequently, most immersion oils have a refractive index of 1. For common refractive indices, see Table 1. Using this system, it is possible to achieve the maximum resolution and NA. Figure 2 and Table 1. Placing immersion liquid on the lens of the condenser is usually not necessary.

If the microscope is correctly set up and aligned to achieve optimal contrast and illumination across the specimen see the article on Koehler Illumination , then the position and settings of the condenser will be optimised so as to contribute to the overall NA of the microscope system. This is simply the actual distance between the objective front lens and the surface of the cover glass when the specimen is in sharp focus s.

Figure 3. When the objective is moved to be closer to the slide, the focal plane moves further into the specimen. However, this is physically limited by the fact that the objective can only be moved until it is in contact with the cover glass.

There is an inverse relationship between working distances and the magnification of each objective. The disadvantageous properties of cedarwood oil are: high absorption of blue and UV light, yellowing with age, a tendency to harden on lenses due to uneven volatility, acidity, and changing viscosity diluting with solvent changes the index and dispersion.

What would you observe if you forgot to use oil with the oil-immersion lens? The purpose of the oil is to contain the light rays and direct them upwards. If oil was not used, the rays would scatter, and the image would also be less magnified and lower clarity. Never go back to the 10x or 40x objectives after you have applied oil to the specimen since oil can ruin the lower power objectives.

An eyepiece reticle is a small piece of glass with a ruler or grid imposed on it that fits into the microscope eyepiece. When looking through the microscope, the reticle image is imposed upon your specimen image. Most often the reticle is used to make measurements or count particles. Begin typing your search term above and press enter to search.

Press ESC to cancel. Objective Selector. Overview Specifications Immersion oil increases the resolving power of the microscope by replacing the air gap between the immersion objective lens and cover glass with a high refractive index medium and reducing light refraction.

Type N Type N is a general purpose oil recommended for transmitted light applications, including brightfield, darkfield, DIC and phase contrast. Type F Type F is designed to ensure the highest image quality for fluorescence applications, with maximal signal-to-noise ratio and minimal autofluorescence.

Silicone Silicone immersion oil is used for deep observations of live specimens. These oils are transparent and have a high refractive index, meaning they can significantly increase the magnification and contrast of a specimen under a microscope.

The optical and viscosity characteristics of immersion oils are crucial in light microscopy because of the way they effectively focus light without blurring any details. Typically, immersion oils have a refractive index of 1. Refractive index is also measured in terms of the ratio between the speed of light in a vacuum and the speed of light in a medium.

This definition is more accurate, but harder to measure experimentally. Higher refractive index means light is slower and can be bent more in the medium. Specialised microscope objective lenses are used with immersion oils. Ordinary objective lenses can still work, but not as effectively. Optimal resolution is also achieved with the help of condenser lenses immersed in oil. Oil immersion is a special technique used in light microscopy that involves the immersion of both the specimen and the objective lens in a transparent oil that has a high refractive index.

This has the effect of increasing the numerical aperture of the objective lens, meaning that the lens can gather more light despite its small size. In order for a lens to have a large aperture, it has to have a larger diameter. This also means that the lens has to be thicker in order to have greater magnification.