Oil immersion microscopy is a good way of increasing resolution when examining a specimen. When the process is used correctly, oil immersion methods can help to increase the refractive index of a sample.
This all helps to produce more detailed microscopy pictures.
Planes that have been prepared with oil don’t have many disadvantages, but immersion methods work better under greater magnification.
This helps the oils to increase refraction, even though the focal lengths are shorter.
The details within your microscopy images rely on the resolution and numerical aperture. Resolution is how well you can see distinct details within the sample you’re examining.
The numerical aperture tells you how well the microscope’s optical parts can collect light.
Oil immersion microscopy can improve the numerical aperture and the resolution to produce better quality images.
We’ll cover more about how immersion oil techniques do this in this article, including the pros and cons of this method, and cleaning tips to help you clean your objectives.
Keep reading to learn more about oil immersion microscopy!
The Significance Of The Refractive Index And Refraction
The physical properties of the material that light passes through will determine how much the light diffracts. These properties are known widely as the ‘Refractive Index’.
Like numerical aperture, the refractive index isn’t measured in units.
Dry objectives, also known as non-immersion objectives, have an air space between the coverslip’s surface and the objective’s front lens.
Air has a refractive index of 1, but coverslips and microscope slides tend to have a refractive index of 1.5.
When light moves from one material to another, like through glass, then through the air, it will bend, or ‘refract’.
If you use a non-immersion objective, the light rays from your sample will refract as they move to the air from a glass coverslip.
The objective front lens generally won’t gather the refracted rays, losing them to the final picture instead.
You can correct this issue by substituting the air gap for an immersion medium. Commercial immersion oils tend to have refractive indexes that are like glass, which is roughly 1.51.
Through this, oil immersion methods will lower refraction to increase the numerical aperture and improve the immersion objective’s resolution.
Comparable Refraction Index
Greater magnification levels have shorter focal lengths, which is why it’s a good idea to use oil-immersed objectives at higher magnification levels.
Oil will have a refractive index that’s like slipcovers and glass slides.
The image quality won’t be distorted or reduced when light waves pass through objectives with comparable refractive indexes.
Oil immersion microscopy produces clear, contrasted pictures with an impressive resolution, ranging between 40x to 120x.
This is ideal for higher magnifications with short focal lengths.
Oil immersion can bring out the best in powerful lenses when using light microscopes, though this will depend on the specimen being examined.
Oil is a good conduit to prepare slides with, as its refractive index is like glass.
Color can decrease and increase in oil immersion microscopy, but in this case, the color will be defined through light loss that occurs during absorption.
Before using oil immersion techniques, researchers should decide if color is significant when analyzing a sample, in comparison to the greater resolution that oil immersion produces.
Image quality is the most significant element of oil-immersed samples. Despite any reduction in color values, the details and lines may keep their bold tones.
Oil Immersion Applications
Oil immersion microscopy uses one or several oils on a lens suitable for oil immersion techniques. Oil immersion slides are often used to look at dead or non-moving specimens.
These include muscle tissue with distinct grooves, microbes, and samples with smaller identifiable components.
Oil immersion slides shouldn’t be used to look at samples that can decompose in contact with oil. One example is acidic oil, as its high pH can break down any specimens that contain metal.
Synthetic And Natural Immersion Oils
Cedarwood oil was a popular immersion oil choice before commercial manufacturing processes created synthetic substitutes.
You can purchase cedar oil today, but it comes with many drawbacks.
If cedar oil isn’t cleaned up properly, the oil can break down the cement used to hold the objective front lens. This natural oil can also change color, becoming yellow over time.
As its form changes, cedarwood oil can take in light from the blue and UV range of the spectrum.
Modern synthetic oils were designed to stay inert and maintain their color with age. Despite this, you should still make sure you clean the oil up correctly.
It’s important to choose your immersion oil with care, particularly if you are carrying out lengthy experiments that require temperature-controlled settings.
The majority of oils were made to work well at room temperature, roughly 23°C. Temperature changes can make an oil change its refractive index.
Just a variance of 1 degree can make the oil’s refractive index change by a factor of 0.0004.
This may not seem like a huge amount, but if you are carrying out lengthy experiments and examining specimens over several hours, these variances can affect the images and information that are collected.
If you are planning to carry out longer experiments with oil immersion techniques, it’s a good idea to use commercial, synthetic oils made to work their best at 37°C.
Pros And Cons
Oil immersion microscopy reduces diffraction, as light passes through glass layers and oil layers in the same way.
Light travels through other materials, like glycol and water differently. The refractive index changes as it passes, lowering the image quality in the process.
At its best, oil immersion should involve a pH level between oil and glass. It’s better to use one or several synthetic oils over any natural oils that come from sandalwood or cedar.
Scientists, researchers, and school laboratories can order several oils to reach a particular thickness.
The advantages of this are:
- Available at most manufacturers
- Can be used for secondary slides (depending on circumstances)
- Suitable for broader condensers
- Objectives of extended focus at low energy
- Suitable for several inclines, including slants and even horizontally
Cedarwood oil was a popular natural oil source in the past. However, using synthetic oils on their own, or with a mix of others, delivers thickness properties that are better than natural oils.
Synthetic substances maintain a desirable pH level compared to natural ones. Lower pH levels indicate that an environment is acidic.
Acidic settings are bad for specimens as they can make samples break down.
Microscope users need to take care when applying oil, as they need to avoid the substance coming into contact with the microscope, particularly the lens.
If the slides are prepared incorrectly, the cement used to lock the panels might not work properly. The glue might stick to the microscope or other panels.
Other than the care needed to prepare and produce the slides, some disadvantages of this are:
- Lens damage may occur if caution isn’t taken during application and cleanup
- If glue or cement used to contain oils beneath the slide isn’t applied correctly, outside particles like the dust may penetrate it
- Cement may dry on the microscope itself. Removing this will be hard and may damage the microscope and lens in the process
You can buy particular solvents that can remove dried cement from microscope components, but removal still comes with a risk of damage and scratches.
Take note that oil is also unsuitable for dry lenses, as using oil incorrectly can lead to lower quality or deformed pictures.
One of the best ways of cleaning immersion objective lenses is by using dry lens tissue to wipe off the oil.
After you’ve cleaned off most of the oil, you can use a soft tissue to clean the front of the objective.
If you’d prefer to clean your objectives with a solution, create a mixture of water and dish soap to do so.
Mix five to ten drops of the soap with ten milliliters of distilled water. Make sure that the dish soap doesn’t contain any ammonia.
It’s important to avoid harsh cleaners when cleaning your lenses, as harsh chemicals can damage them.
Take particular care if your lens is already clean and free from dry oil, as this can make it prone to damage.
You don’t need to clean your lenses over and over again. As long as they are free from debris and residue, once is more than enough.
Now you know why oil immersion microscopy is so important!
Oil immersion microscopy is a significant tool when using a microscope to view samples.
The method does come with its drawbacks, but remaining cautious and handling components with care, can prevent problems occurring, like adhesive drying on the slides or lens.
Comparable refractive indexes can produce bigger, clearer images. This is ideal when examining inanimate specimens, muscle tissue, and bacterium.
Using a blend of synthetic oils can produce an oil with the optimum thickness to produce detailed, high-quality pictures.
Make sure that you always clean up oil after you’ve used your immersion objective lens, even if you’re using the lens again in the same period.
Immersion oil can enter the microscope’s parts, breaking down the cement that fixes the front lenses in place.
Oil immersion microscopy can help enhance resolution, producing better quality images as a result!
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