The immersion method of microscopic observation involves the introduction of a special fluid between the objective of the device and the studied object. It provides increased brightness and expansion of the boundaries of the magnification of the image. Thus, the object can be significantly approximated and considered its smallest elements without changing equipment. Accordingly, the fluid is called immersion. As it can be 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 (until hardening). Accordingly, the refractive index also changed . In the 20th century, synthetic immersion oil began to be produced. This liquid did not have the above drawback.
Immersion oil: standards
The key parameters of the liquid are established 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 considered to be 20 degrees. There are also international standards. In accordance with ISO 8036/1, the refractive index is 1.518 + 0.0005, and the transmittance at a layer of 10 mm for the spectral range from 500 to 760 nm is 95%, and at 400 nm - 60%.
The specified parameters correspond to oil immersion non-fluorescent . ISO 8036-1 / 2 defines luminescence fluids. The transmittance at a spectral range from 500 to 700 nm in a 10 mm layer is 95%, from 365 to 400 nm - 60%.
Difficulties of parameter discrepancies
The detected difference in the above standards may lead to a deterioration in the performance of a particular lens when using inappropriate liquid. As a result:
- Contrast is reduced due to spherical aberration.
- The field at the object of study is painted.
- Illumination in the plane of the studied subject and in the area of formation of its image becomes uneven.
- The picture becomes blurry.
Nuances
Optical microscopes have an upper limit of resolution a little more than 100 times. At this level of increase, the illumination of the subject under study should be of high quality. Otherwise, the resulting image will be so dark that it will be simply impossible to examine the object. The fact is that the refraction and scattering of light occurs between the coverslip and the lens. Immersion oil contributes to its greater capture. As a result, the image becomes sharper.
Features of light refraction
How to get a clear picture? In different environments, light refraction 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 can reduce the refractive index of the rays that pass through the studied object. The fact is that liquid has the same parameter as 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 device. This produces a clear image.
Technical points
As a rule, on the cases of lenses intended for immersion, there is an engraving 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 liquid. In this regard, they are completely tight. This provides high protection against damage to the lens oil.
Classification
In practice, two viscosity oils are used: 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 airspace, and with high - together with condensers.
Terms of use
To get a clear image of the investigated object, you need to follow fairly simple recommendations:
- Find the studied subject on the slider in the center of the field at a small magnification. For this, a low-magnification lens is used.
- Rotate the turret.
- Enter the lens 100 times into the working position.
- Place a drop of oil on the glass slide, the second - on the lens.
- Adjust the working distance by precise focusing until a clear picture of the object appears.
When working, be careful. It is important to prevent air from entering between the coverslip and the lens.
Oil immersion "Minimed"
The liquid is used when working with achromatic and apochromatic lenses of any type of device, except for fluorescent ones. As experts who used this immersion oil note, it has several beneficial properties. The liquid significantly improves the visibility of the object, minimizes glare, loss of light and aberration of optics. The use of oil significantly expands the range of equipment capabilities.
Equipment cleaning
After working with immersion oil, the device must be tidied up. Cleaning must be done before the lens dries. Clean lens paper is used to remove oil residues. Rolled sheet clean all glass surfaces. The lens paper should be moistened with a special solution and the remaining oil removed.
Historical reference
The first scientist to explain the mechanism of immersion was Robert Hooke. In 1678, his book Microscopium was published, in which all 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 manufactured. Their creator was DB Amici. Initially, researchers used anise oils as an immersion fluid. The refractive index was close to that of glass.