How does immersion oil affect the image

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 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.

Leave them in the comments section below! Has this helped you? Then please share with your network. You must be logged in to post a comment. This site uses Akismet to reduce spam. Learn how your comment data is processed.

Facebook Twitter LinkedIn More. Written by Martin Wilson. Leave a Comment Cancel Reply You must be logged in to post a comment.

Share via. Copy Link. The refractive index of air is 1, while the refractive index of immersion oil is 1. Therefore, allowing all the light to pass to the lens. Below is an example. The images were captured using a microscope with a 63x objective lens. The left image was dry no oil and the right image was with microscope immersion oil.

Notice the difference in image quality and the Resolution between the image captured dry versus that captured with immersion oil. Not every lens is suitable for immersion oil use. You will need immersion oil when using a higher magnification lens, but be sure that the lens is an oil lens. If you are not sure, check with your manual or supplier. Use immersion oil on the non-oil objective lens could damage the lens.

Make sure you read the manual of your microscope first.