What is a Water Immersion Objective - Uses and Advantages
In some of our other articles, you might’ve seen us refer to an oil immersion, but today we’re going to talk about a similar technique: a Water Immersion.
You’ll learn when to use this technique, how it compares to the oil immersions you might be familiar with, and the situations where you would choose it over other immersive solutions.
In addition, you will also get a brief overview of the history of immersion lenses to understand how the technique itself was invented.
Who Invented Immersion Lenses?
The concept for immersion lenses began in 1678 by the first person to visualize a micro-organism, Robert Hooke.
As the Southwest Museum of Engineering, Communication and Computation describes, the first actual immersion lenses were made in 1840 by Professor Giovanni Battista Amici. However, this wasn’t done to increase resolution, which immersions are commonly used for today, but instead to correct reflective aberrations seen when looking through glass sample surfaces.
The First Immersion Lenses
The first water immersion objective was completed in 1853 and subsequently introduced to the world at the 1855 Paris Exposition. In 1858 Robert Bruce Tolles produced microscope objectives that had two exchangeable front elements, one corrected for dry work and the other for immersion work.
This work was then adapted by Charles Stodder, who made a famous microscope in August of 1873 called the Homogenous Immersion 1/10th. Before that, though, Hugh Powell and Peter Lealand introduced this technology into England, creating a 1/25” high-quality immersion lens and then a 1/50” the subsequent year.
Scientists continued to experiment with and iterate on this unique approach to microscopy and worked to improve it. In 1867, Ernst Gundlach exhibited “microscopes et loupes” at the 1967 Paris International Exposition, where he introduced the idea of using immersion fluids with higher refractive indexes than water.
That year Tolles would also experiment using glycerin as an immersion fluid, and in 1871 he also used Cana balsam as an immersion medium. His 1/10th inch objective lens had an aperture of 100 degrees or a numerical aperture of 1.25 on a three-lens system.
This is regarded as the first publicly announced homogeneous immersion system within microscopy.
What Is a Water Immersion Objective?
Water immersion is a less commonly used form of immersion. It is used for research-grade microscopes. Here, we introduce a bit of water between the highly magnified objective lens and our sample, bridging the gap between the two surfaces and expelling any air from the area.
As with oil immersion, the objective is to increase the resolution of the microscopy image by using a solution with a higher refractive index than seen in the air. This results in the light being more directly channeled into the objective lens and is most commonly seen in those with the highest magnification levels, between 40x and 100x.
You can quantify this as the immersion increases the microscope lens’s numerical aperture, which is imperative for the deepest levels of microscopy. Being unable to discern details properly leads to inaccuracies in data and should be avoided at all costs.
Water Immersion Benefits
As Agar Scientific points out, this technique greatly benefits the imaging of live cells, as these are contained within a chamber and spaced further from the lens. Water helps to bridge this distance in ways that oil cannot due to the working focal distances of the substances.
Oil has a higher refractive index than water does, 1.51 for the former and 1.33 for the latter, but this results in a shorter focal distance. This shorter distance means it is harder for an oil immersion to focus on more distant objects, such as a contained specimen.
Because of this, the oil immersion does not complete its stated goal of offering better imaging clarity, but water can. In addition, the refraction of the plastic container holding specimens or cells should also be acknowledged, as this container can introduce another layer of refractions, and oil immersions can lead to aberrations.
While it is true that oil has a higher refractive index and can ultimately achieve a larger numerical aperture than water can, it should also be known this is not the case in every situation. Even when paired with research-grade objective lenses, these immersions can introduce refractions and other artifacts that compromise visual quality and can also lack the focal distance required for live specimens.
To that end, it is important to understand how your microscope lens is corrected, as different immersion substances require different corrections due to the levels of refraction present.
What Is The Difference Between Water and Oil Immersions?
Comparatively, both water and oil immersions have something distinct to add, but water immersions are often looked over and disregarded for one basic fact. The purpose of a fluid immersion is to increase the numerical aperture for an objective lens by bridging the gap between a sample’s cover lens and the lens’s glass with fluid, which is more refractive than water.
Oil and water differ because oil is more refractive than water, leading to a higher numerical aperture and simply better results. Not only that, but oil has similar refractive properties to a sample’s cover glass, meaning that water is evenly channeled from the sample to the lens.
Because these refractions are different in water, the microscope’s objective lens will require corrections to accommodate for this. Microscopy-UK explains this property to be known as spherical aberration, which can also occur with non-immersion lenses used without a cover glass they are designed for.
While oil immersions were rather unpopular for most of the 20th century, this changed in the 1990s, as life sciences began to examine fresh biological specimens that needed to be mounted within the water.
As earlier explained, the refractive properties for oil and water vary, so using these two substances in conjunction would damage data collection. Using water on an oil immersion lens will degrade the quality of the information collected and lead to inaccuracies.
In addition, oil immersions often have a very low depth of field. With these applications requiring cover glass to be used, oil immersions are simply unable to reach the necessary depth to focus on a sample properly.
To that end, water immersions gained popularity as life sciences advanced and found samples with needs to accommodate them. While it can be easy to overlook this method in favor of one with slightly better statistics, microscopy applications are often more difficult than this, requiring customized solutions.
Which Is Best for You?
In designing your solution, it is important to understand that the correction of your objective lens can directly impact its compatibility with immersion methods.
The oil or water is intended to help channel light through a path, and the lens does this as well, but it can damage results if these two are misaligned. This would lead to chromatic aberrations in your image or other artifacts that obscure data or your subject.
So, lenses are designed to compensate for this, but no one can accommodate both perfectly with different refractive indexes for water and oil. As a result, most homogenous solutions simply implement multiple objective lenses into the same microscopy solution, with the ability to rotate a mechanism and switch lenses with ease.
In acquiring lenses, you should be sure which ones are intended for which purpose and use them correctly.
There is a lengthy history to immersion microscopy, which was born out of the want to explore deeper and clearer with the power of lenses. They date as far back as the 1670s but took centuries to develop and evolve into the technology we understand today properly.
This technology aids microscopes in producing clear images at incredible magnification levels by channeling the refractive indexes in liquids to help boost clarity. With this innovative technique, specially corrected lenses can drive incredible results and help push the medium of magnification forward.
In designing your microscopy solution, be sure to acknowledge which form of immersion may be for you. If you are viewing a live specimen or one through an extra layer of glass, you may want to opt for water immersion, as it can reach the depth needed to capture these.
Or, if you are looking for the maximum magnification and resolution possible on more shallow samples, then you could opt for an oil immersion instead. These are both compelling options with their own use-cases, so picking what works best for you is the real answer.
SMECC - History of Oil Immersion Lenses | SMECC
Agar Scientific - Looking Down and Through: Microscope Optics 4: Water Immersion Objectives | Agarscientific.com
Microscopy-UK - Advanced Notes On Water Immersion Lenses | Microscopy-uk.org.uk