Using oil immersion microscopy can help increase your resolution in light microscopy. This, in turn, can lead to better images- and who doesn’t want nicer microscopy images?
But, do you know why immersion oil and objectives are used for high power magnification?
And do you know how to use an immersion objective correctly?
The quality of your image depends on your Numerical Aperture (NA) and resolution. NA relates to the light gathering properties of the optical components of your microscope, whereas resolution is your ability to distinguish details within your specimen.
Using oil immersion microscopy can improve both your resolution and NA!
Now let us take a look at why this is.
Reasons Why You Should Use Oil Immersion Microscopy
The Role of Refraction and the Refractive Index
The exact physical property of the medium or material through which light passes determines the amount of diffraction of the light. This is known as the ‘Refractive Index’ and is without units, as is NA.
With non-immersion (or ‘dry’ as they are called) objectives, there is an air gap between the front lens of the objective and the top surface of the coverslip. Most microscope slides and coverslips will have refractive indexes of 1.5, whereas air has a refractive index of 1.0.
When light passes from one medium to another, say from glass to air, it ‘refracts’ or bends and scatters. Therefore, if you use a dry objective, the light rays from your specimen will undergo refraction when traveling from the glass coverslip into the air. Refracted rays are not usually collected by the objective front lens and are lost to the final image.
Now, if you use an immersion medium to replace the air gap, you can correct this mismatch. Most of the commercial immersion oils have refractive indexes of around 1.51 – similar to glass.
This is how oil immersion microscopy improves the resolution of your high-power immersion objective and increases your NA by lowering refraction.
The Working Distances of Objectives
When you focus an objective and move it closer to the slide or coverslip, the focal plane moves further into your specimen. However, there is a physical limitation to how far you can go before the nose tip of the objective hits the coverslip. Go any further than this distance, and you will not be popular! Cleaning glass shards out of a condenser is a time-consuming and expensive business. Not to mention you can damage the objective front lens!
When a specimen is in sharp focus, the actual distance (in millimeters or microns) between the front lens of an objective and the surface of the coverslip is known as the ‘Working Distance’. In general, there is an inverse relationship between magnification and working distance. An objective with 10x magnification has an approximate working distance of 4 mm, whereas a 100x oil immersion objective has a working distance of approximately 130 µm. You can find your objectives Working Distance or ‘WD’ engraved on the barrel of your objective (just don’t confuse this with ‘Water Dipping’).
Natural vs. Synthetic Immersion Oils
Cedar Wood Oil
Cedar Wood oil was the immersion oil of choice for many years before the large-scale manufacture of synthetic alternatives. You can still buy Cedar Wood oil today. However, this oil can have many disadvantages.
If not correctly cleaned up after use, it can penetrate and damage the cement that holds the objective front lens. Cedar wood oil can also ‘yellow’ with age and has a tendency to absorb light in the ultraviolet and blue range of the spectrum.
Modern synthetic oils are designed to remain color stable over time as well as be relatively inert. Although this isn’t an excuse to avoid correct cleaning after use! If you are carrying out long-term, live-cell imaging experiments requiring temperature-controlled chambers around the cells and stage, you must carefully choose your immersion oil.
Most oils are designed to work at room temperature (i.e., 23°C). A change in temperature causes a change in the refractive index of the oil. In fact, a temperature difference of only 1°C can cause a change in the refractive index of the oil by a factor of 0.0004. This might not seem much, but if you are capturing images over many hours, these subtle differences will be present in your images and data collected. So if you are planning on such long-term experiments using oil immersion microscopy, use commercial oil designed to work at 37°C.
Beware of Autofluorescence
Another point to bear in mind is that general use oils can auto-fluoresce. When planning and carrying out fluorescence microscopy, you should use a non-fluorescent oil. These oils have a letter ‘F’ before or after the oil code or name. Finally, no matter what imaging you are carrying out, you should always use an oil that is recommended by the manufacturer of your microscope and objectives.
6 Easy Steps for Awesome Oil Immersion Microscopy
- Find your slide’s area of interest using a low magnification objective.
- Move the nosepiece around from the low power up to the 40x objective, stopping at each one to focus and make sure your area of interest is still in the center of view when you look down the eyepieces. To set up the microscope correctly for Koehler illumination, our other article, “How to Transform Your Images from Mediocre to Publication Quality with Kohler Illumination”.
- Once you have aligned the microscope, carefully turn the nosepiece around to between the 40x and the oil immersion objective, but don’t fully engage the oil immersion objective yet.
- Turn your attention from “down the eyepieces” to “the side of your microscope”. Carefully place one drop of immersion oil directly onto your coverslip. That’s one drop—you’re not oiling a bike chain!
- Now, turn the nosepiece around to fully engage the oil immersion objective and bring the nose of your immersion objective into contact with the drop of oil. Some immersion objectives have concave front lenses. If you are using one of these, you also need to add one drop of immersion oil to the front lens to prevent air bubbles from becoming trapped in the concave recess.
- Next, look back down the eyepieces and, using only the fine focus control, very slowly bring your specimen back into focus. Although immersion objectives have spring-loaded nose cones, this is not an invitation to rack up the focus. Quick focusing at this stage can easily result in the objective cracking the coverslip and the slide. A costly accident, as doing so can cause slivers of glass (and oil) to fall into the substage condenser (or over the objectives and nosepiece on an inverted microscope) and can even damage the front lens of the immersion objective—one of the most expensive components of microscopes. So be patient and focus slowly.
After you have finished imaging with your immersion objective, clean up the oil. Even if you are planning to use this objective again in the same session, clean up! Immersion oil can (and will) penetrate the microscope components and can damage ‘dry’ objectives, as immersion oil can corrode the cement used to hold objective front lenses in place.
To clean your immersion objective, use a lens cleaning tissue to sweep across the surface of the objective front lens in one direction only. Continue cleaning in the same manner (using a clean section of lens tissue for each sweep) until no oil is seen on the tissue. You can also use a commercial immersion oil removal solution or a small amount of xylene for the final cleaning. As always, check the manufacturer’s recommendations before using any solutions to clean your objectives.
Hopefully, that helps with your oil immersion microscopy, and you now know why and how to use an immersion objective correctly. Using oil immersion microscopy will help increase your resolution and in turn, lead to better images!
To learn more, check out our other articles on NA, “That Other Number –The Meaning of Numerical Aperture in Microscopy” and on resolution, “Rubbing your Microscope’s Eyes: A Guide to Optical Resolution”.
Have any tips on using an immersion objective? Leave them in the comments section below!
Originally published on 31 March 2015; updated and republished June 2022.