Water & Oil Immersion Lens - Everything You Need To Know
When searching for a high-quality microscope, you may see the terms ‘Water Immersion’ and ‘Oil Immersion’ thrown around when describing lenses. But what do these terms mean, and should you be looking for a lens like these?
The answer requires a bit of extended explanation about the different ways microscopy is implemented and how the technology developed, so let’s go over some of the basics of water and oil immersion techniques.
In this article, you will learn what immersion is, how to perform one, and when you might actually need to do that in your microscopy application.
What Is An Immersion Lens?
As microscopy technology developed, researchers worked to develop microscope objectives with more powerful objective lenses that could offer higher levels of magnification and further detail on a specimen.
But they ran into a problem. The images produced by their 40x objective and 100x objective lenses suffered from a spherical aberration that caused them to blur and significantly impacted the quality of data collected. As they learned, this was because the light rays themselves were not being narrowly channeled between the objective lens and the area of interest, causing refractions.
To solve this problem, they developed both the oil immersion objective and water immersion objectives, the latter of which can also be referred to as aqueous dipping. As they discovered, light passes through liquids differently due to the refractive index of the medium.
They also found differing results, depending on what fluid they used. Beyond just water and oil, other immersion media include ethyl cinnamate, TDE, Mineral oil, silicone oil, FocusClear, Scale, and even some organic media.
However, this technique does not function properly on standard lenses and instead requires the objective to be specifically corrected to utilize this effect correctly.
To that end, an immersion lens is one manufactured with several internal correction collars that can be adjusted within the microscope, allowing a single lens device to alternate between immersion mediums.
Why Use Microscope Immersion Oil?
Microscope immersion oil helps produce high-magnification images with fine focus while using objective lenses that have a high numerical aperture. With the increase in magnification comes a decrease in the objective lens’s working distance and focal length. This decrease means that, with very little space to pass between the glass coverslip and the front lens of the objective, the light often has reflections that distort the image.
But, liquids have a higher refractive index than air, so immersion oils help to narrowly channel the rays of light into your light microscope. Some examples of the differences in image quality with and without immersion oil are available at Microscope World.
When To Use Microscope Immersion Oil?
According to Rice University’s Experimental Bioscience, the best time to use microscope oil is for viewing a fixed subject that is no thicker than a few micrometers in thickness. As they explain, immersion oil is best used for magnification of 400x or above, as this is the point where the image quality through light rays starts to deteriorate severely. At lower levels of magnification, the oil immersion simply isn’t necessary.
At this immense magnification depth, the working area seen is immensely tiny, leading to their stipulation on specimen size and movement. The specimen must be fixed, dead or un-moving, to properly be seen, making oil immersion best for viewing individual bacteria or the striations of skeletal muscle.
How Immersion Microscopes Work
Immersion microscopes work in the same basic ways that traditional microscopes do, with light passing through a specimen, up into an objective lens, and through a second optical lens to a viewer’s eyes.
The difference here lies in the specific tuning of that objective lens, which differs for immersion objectives.
Typically in microscopy, light is lost between the specimen and the objective as it is reflected outward beyond the objective lens. For visual learners, R’s Science has an excellent diagram to help explain how to use microscope immersion oil.
However, this light is channeled directly into the lens in immersion microscopy to provide the image’s additional detail and ‘resolution.’ Immersion objectives must be specifically tuned to receive extra light and still produce a discernible image.
This tuning is accomplished through the spacing between the layers of glass within an objective lens. As these lenses are brought closer together, they can better accept this wider spectrum of light and better view images filtered through oil.
In certain cases, lenses can be configured with moving layers of glass, enabling them to function in both immersion and ‘dry’ settings to switch between them easily. Immersion lenses are also constructed with a special coating that prevents the oil from sticking to the glass and leaving lasting marks or smudges.
For this reason, you mustn’t use immersion oil on microscope lenses that aren’t designed for it, as it can permanently damage them.
Microscope Immersion Oil
Microscope immersion oil is a specially concocted substance designed to offer very specific light reflections. It is completely unlike conventional oils you would find in a retail store. Instead, this oil is meant to be used in just a few drops at one time, gently applied to the top of a sample so a lens can be placed directly over top of them.
Importantly, there is not just one variety of immersion oil, but instead, there are four. Type N is made for transmitted light applications and is the most typical one used. Type B is engineered to help with time-lapse imaging experiments, most commonly light-based transmitted ones.
Type F is designed to work with fluorescent microscopy by absorbing as little of the light as possible and optimizing the signal-to-noise ratio. Finally, silicone immersion oil is most useful for deeper field-of-view imaging on live specimens. It matches the refractive index of cells and produces the most accurately scaled images.
Oil Immersion Microscope Uses
Oil immersion is used on microscopy applications requiring above a 400x magnification, as this is where light refraction is most detrimental to image quality. One commonly known use for this level of magnification is life sciences, where researchers study specimens as small as individual cells to learn more about the natural world or work to develop medical solutions for human illness.
However, when viewing a live science submerged within water, a different aqueous immersion medium like water is used instead of oil. Water is used because the fluid refractions need to be kept consistent between the immersion and the specimen.
The primary difference between water and oil immersions is that the numerical aperture of oil is higher, but water enables a better depth of field for examinations. This means that when using the highest levels of magnification, like 1000x, oil should be utilized for the clearest results at those immense depths.
But, in situations where the depth of field should be a priority (live specimens, as explained above), water should be utilized to properly view the subject, even if this occurs at a slightly diminished resolution.
Recommended Oil Immersion Microscope
When searching for an immersion microscope that works best for your application, look for these specifications.
Primarily, you want a lens that handles immersion media with a refractive index of 1.33 to 1.56, as this includes all commonly used types. In addition, you want a working distance of between 12 and 10 millimeters, with a field of view of 1.2 or 1 millimeters and no correction collar. These will allow you to work at an optimal viewing distance unobstructed.
Other important factors are the numerical aperture, flat sample depth, spherical correction, and parfocal length. All of these microscope elements are essential to its use for immersion techniques, so ensure that what you’re purchasing fits your application.
Quality lens makers list these facts and figures alongside their products.
Water and oil immersions are very specifically developed methods for increasing the image quality at the deepest levels of magnification, from about 400x to 1000x. At these levels, the light is reflected between the specimen and lens surfaces, and enough of it is lost to degrade image quality. These immersion techniques were developed to use liquids to channel the light directly To prevent light from leaving the microscopy area. However, this requires specially tuned lenses to get the optimal effect, crucial for scientific data collection.
For more information on water and oil immersion lenses, visit Navitar.
Microscope World Resources - Using Microscope Immersion Oil | MicroscopeWorld.com
Experimental Biosciences - Microscopy with oil immersion | rice.edu
Rs' Science - How to Use Microscope Immersion Oil to Get Higher Resolution Images | rsscience.com