If you have used a high-powered microscope, you must have across various oil immersions. This might be your very first project with such a high scaled magnification.
Oil immersion forms the fundamentals of high-quality microscopy. To understand the concept behind oil immersion, you must first understand several other principal factors.
The Refraction and the Refractive Index
The diffraction of light passing through a medium is mostly dependent on its physical properties. This phenomenon is known as Refractive Index.
In this context, the objective lens can be classified into two types. The dry objective or the non-immersion types and the immersion objectives.
There is a definitive air gap between the end of your objective lens and the coverslip’s summit. The air gap is present when you are using a non-immersive objective.
A typical coverslip has a refractive index on 1.5, while that of air is 1. When light passes from one medium to another, it bends. This bending of light rays is what we called refraction.
Hence, with a non-immersion objective, the incoming light rays of the specimen will inevitably undergo refraction. As these rays scatter right in front of the objective, a grade of the final image is lost.
You can eliminate this air gap with a very straightforward solution. You need to use an immersion objective.
Typically, all immersion mediums (oils) have a refractive index of 1.51, close to that of glass. Due to this similarity of the refractive index of the two mediums, the resolution improves by default.
The working distance
The actual distance between the front of the objective lens and the coverslip is the working distance. The magnification and the working distance are inversely proportional to each other.
While focusing on an objective, you invariably bring it close to the specimen slide or the coverslip. This causes the focal plane to move deeper into the specimen.
Due to the placement of different structures in a microscope, there is a physical limitation. There is a certain distance to which your objective’s nose tip can move.
Focusing the objective any further can not only damage its front end but also destroy the slide. This is an expensive and time-consuming disparity.
So how do you overcome it? The answer is straightforward. If you are using a high-powered microscope, it is best to use an immersion lens.
What are the applications of immersion oils?
The importance of immersion oil can be appreciated mainly in high-powered microscopy. Immersion microscopy becomes essential for viewing microscope dead matter.
The specimen viewed via immersion microscopy include bacteria, biological tissues, and other smaller inanimate structures. Suitable samples are not affected by the immersion medium.
If your specimen is sensitive, it can get destroyed if you are using an acidic medium. The Ph of the immersion oil is what causes deformities in your specimens.
Types of immersion oils
There are several types of immersion oils present in the market today. We have mentioned the most used immersion oils for microscopy.
The Cedar Wood Oil
The cedarwood oil has been in use for microscopy for much longer than any other type. This is still considered as the best alternative for synthetic oils.
But this type of oil has several disadvantages. The biggest issue with cedar oil is the cleaning.
When you use the oil avidly, it penetrates inside of the cement. This damage the summit of the objective lens.
Synthetic Immersion Oils
Modern synthetic oils are well known for their colour stability. They remain relatively inert over more extensive periods.
If your goal is to increase the longevity of your equipment, synthetic oils are your best bet. Most immersion oils are safe to use at room temperatures.
We recommend doing that you maintain the temperature while carrying out the experiments. Typically, any temperature fluctuation causes a refractive index to alter.
An increase or decrease of temperature by even a degree changes the refractive index by 0.0004 units. This change might seem significant at first.
But when your goal is microscopic imaging, even a minor difference can seem apparent in the final image. The calculations and inference, which then entered the database, cause discrepancies.
What is Autofluorescence?
When you are using immersion oils, auto-fluorescence is a critical consideration. It will greatly help your cause if you use non-fluorescent immersion oils.
To find whether the oil is non-fluorescent or not, you can check if the packaging has a letter ‘F’ on it. The trick is to use the type of oil that the manufacture of your microscope recommends