The immersion method of microscopic observation involves the introduction of a special liquid between the lens of the device and the object under study. It enhances the brightness and expands the scope of the image magnification. Thus, the object can be significantly zoomed in and its smallest elements can be examined without changing the equipment. Accordingly, the liquid is called immersion. It can serve as a variety of compositions. The most popular is immersion oil. Consider its features in more detail.
General information
The first immersion oil for microscopy was cedar. However, it had one significant drawback. Over time, its properties changed, and it did not allow to obtain the desired results. In the open air, the liquid began to gradually condense (up to hardening). Accordingly, the refractive index also changed. In the 20th century, synthetic immersion oil began to be produced. This fluid did not have the above deficiency.
Immersion Oil Standards
Keyfluid parameters are set in GOST 13739-78. According to the standard, immersion oil has:
- refractive index nd=1.515±0.001;
- transmittance in the spectral range from 500 to 700 nm with a layer thickness of 1 mm - 95%, from 400 to 480 nm - 92%;
The optimum temperature at which immersion oil can be used is 20 degrees. There are also international standards. According to ISO 8036/1, the refractive index is 1.518 + 0.0005, and the transmittance at 10 mm layer for the spectral range from 500 to 760 nm is 95%, and at 400 nm it is 60%.
The indicated parameters correspond to non-fluorescent immersion oil. The ISO 8036-1/2 standard defines the characteristics of liquid for luminescence. The transmittance in the spectral range from 500 to 700 nm in a layer of 10 mm is 95%, from 365 to 400 nm - 60%.
Difficulties of parameter discrepancies
Identifiable difference in the above standards may result in deterioration of the performance of a particular lens when using an inappropriate fluid. Result:
- Contrast is reduced due to spherical aberration.
- The field on the research object is colored.
- Illumination in the plane of the object under study and in the area of its image formation becomes uneven.
- The picture becomes blurry.
Nuances
Optical microscopes have an upper limit of resolutiona little more than 100 times. At this level of magnification, the illumination of the object under study should be of high quality. Otherwise, the resulting image will be so dark that it will be impossible to see the object. The fact is that the refraction and scattering of light occurs between the cover glass and the objective. Immersion oil contributes to its greater capture. As a result, the image becomes clearer.
Features of light refraction
How do you get a clear picture? In different media, the refraction of light occurs in different ways. For example, the angles of refraction of rays in air and glass are different. In the first case, the indicator is 1.0, in the second - 1.5. This is the main problem.
The use of oil allows you to reduce the refractive index of the rays that pass through the object under study. The fact is that the liquid has the same parameter as the glass. As a result, a homogeneous medium is formed between the slide and the lens, and most of the light passing through the object enters the instrument. This results in a clear image.
Technical points
As a rule, immersion lens barrels are engraved with Oil. The element itself is used when an aperture of 1.0 or more is needed. Such "immersion" lenses are used for direct immersion in a liquid. In this regard, they are completely sealed. This provides high protection against oil damage to the lenses.
Classification
Oils are used in practicetwo viscosities: high (type B) and low (A). Often on the packaging you can find information about the refractive index. For example, they produce immersion oil (100 ml), the refractive index of which is 1.515. Liquids with low viscosity are applied to the air space, and with high viscosity - together with condensers.
Terms of Use
To get a clear image of the object under study, you need to follow fairly simple recommendations:
- Find the object under study on the slider in the center of the field at a small increase. For this, a low magnification lens is used.
- Turn the turret.
- Introduce lens 100x into working position.
- Place a drop of oil on the slide glass, the second one on the lens.
- Adjust the working distance with fine focus until the subject is clearly visible.
Care must be taken when working. It is important to prevent air from getting between the coverslip and objective.
Immersion oil "Minimed"
Liquid is used when working with achromatic and apochromatic lenses of any type of devices, except for luminescent ones. According to experts who have used this immersion oil, it has several useful properties. The liquid significantly improves the visibility of the object, minimizing glare, light loss and optical aberrations. The use of oil significantly expands the range of equipment capabilities.
Cleaningequipment
After working with immersion oil, it is necessary to put the device in order. Cleaning should be done before the lens dries. Clean lens paper is used to remove oil residue. A rolled sheet is used to clean all glass surfaces. The lens paper should be moistened with a lens solution and any remaining oil should be removed.
Historical background
The first scientist to explain the mechanism of immersion was Robert Hooke. In 1678, his book Microscopium was published, in which all the explanations were given. In 1812, immersion was proposed as a means of correcting lens aberrations. The author of the idea was David Buster. Around 1840, the first immersion lenses were made. Their creator was D. B. Amici. Initially, the researchers used anise oils as the immersion liquid. The refractive index was close to that of glass.
