Do you know why immersion oil and objectives are used for high power magnification? Do you know how to use an immersion objective correctly? Then review with me the why and how of immersion objectives.
The quality of your image depends on your Numerical Aperture (NA) and resolution. To very briefly recap, 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 an immersion lens and oil can improve both your resolution and NA. Now let us take a look at why this is.
Refraction and the Refractive Index
The exact physical property of the medium/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. And as you know, 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 travelling 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 mis-match. Most of the commercial immersion oils have refractive indexes of around 1.51 – similar to glass. This 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/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 millimetres 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 ?m. 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 and 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. And 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 which holds the objective front lens in place. 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 colour stable over time as well as being 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 that require temperature controlled chambers around the cells and stage, then you need to choose your immersion oil carefully. 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, use commercial oil designed to work at 37°C.
Beware of Autofluorescence
Another point to bear in mind is that general use oils 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.
Steps for Oil Immersion
- 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 centre of view when you look down the eyepieces. To set up the microscope correctly for Koehler illumination read my 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 becoming trapped in the concave recess.
- Now 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 focussing 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 solutions 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 and you now know why and how to use an immersion objective correctly. To learn more, read Cynthia Barber’s article on NA, “That Other Number –The Meaning of Numerical Aperture in Microscopy” and Erin Bank’s article 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!