An oil-immersion objective is a high-powered microscope objective that uses a drop of oil between the objective lens and the specimen to reduce the refractive index between them. This allows for greater resolution, since the oil has a higher refractive index than air, and a greater numerical aperture (NA) than air.

Oil immersion

Principle of immersion microscopy. Path of rays with medium immersion (yellow) (left half) and without (right half). Rays (black) coming from the object (red) at a certain angle and passing through the coverslip (orange, as is the slide at the bottom) can enter the objective (dark blue) only when immersion is used. Otherwise, refraction at the coverslip-air interface causes the ray to miss the target and its information is lost.

Two Leica oil immersion objective lenses. Oil immersion objective lenses appear superficially identical to non-oil immersion lenses.

At the microscope light, oil immersion It is a technique used to increase the resolving power on one microscope. This is achieved by dipping both the objective lenses and the sample in a transparent oil of high refractive indexthus increasing the numerical aperture of the objective lens.

Without oil, light waves are reflected from the specimen on the slide through the glass coverslip, through the air, and into the microscope lens (see color figure at right). Unless a wave exits at a 90-degree angle, it bends when it hits a new substance, and the amount of bending depends on the angle. This distorts the image. Air has a very different refractive index than glass, resulting in a greater curvature compared to oil, which has a more similar index to glass. Specially manufactured oil can have almost exactly the same refractive index as glass, making an oil-immersed lens almost as effective as having all glass in the sample (which would be impractical).

immersion oils are transparent oils that have optical and viscosity characteristics necessary for use in microscopy. Typical used oils have a refractive index from about 1515. One oil immersion objective it is an objective lens specially designed to be used in this way. Several capacitors they also provide optimal resolution when the condenser lens is immersed in oil.

Theoretical basis

Lenses reconstruct light scattered by an object. To successfully achieve this goal, ideally, all diffraction orders should be collected. This is related to the aperture angle of the lens and its index of refraction. The resolution of a microscope is defined as the minimum separation required between two objects under examination for the microscope to differentiate them as separate objects. This minimum distance is labeled δ. If two objects are separated by a distance less than δ, they will appear as a single object under the microscope.

A measure of the resolving power, RP, of a lens is given by its numerical apertureN/A:




δ
=


λ

two
No
AN





\displaystyle \delta =\frac \lambda \mathrm 2NA

where λ is the Wave-length of light. From this it is clear that a good resolution (small δ) is connected to a high numerical aperture.

The numerical aperture of a lens is defined as





No
AN

=
n
sin


α

0





\displaystyle \mathrm NA =n\sin \alpha _0\;

where α0 is half the angle measured by the objective lens seen from the sample, and n is the index of refraction of the medium between the lens and the sample (≈1 for air).

High-end objectives can have a numerical aperture of up to 0.95. because the sin0 is always less than or equal to unity (the number “1”), numerical aperture can never be greater than unity for an objective lens in air. However, if the space between the objective lens and the sample is filled with oil, the numerical aperture can get values ​​greater than unity. This is because oil has a refractive index greater than 1.

Oil immersion goals

Oil immersion objective in use

From the above, it is understood that the oil between the sample and the objective lens improves the resolving power by a factor 1/n. Objectives specifically designed for this purpose are known as oil immersion objectives.

Oil immersion objectives are only used at very high magnifications that require high resolving power. Objectives with high power magnification have a short focal lengths, facilitating the use of the oil. Oil is applied to the specimen (conventional microscope) and the stage is lifted by immersing the objective in the oil. (At the inverted microscopes oil is applied to the target).

The refractive indices of the oil and the glass in the first lens element are almost the same, which means that the refraction of light will be small as it enters the lens (oil and glass are optically very similar). The correct immersion oil for an objective lens must be used to ensure that the refractive indices match closely. Using an oil immersion lens with the wrong immersion oil, or without immersion oil altogether, will suffer from spherical aberration. The strength of this effect depends on the size of the refractive index mismatch.

Oil immersion can generally only be used on rigidly mounted samples, otherwise the superficial tension of oil can move the coverslip and thus move the specimen underneath. This can also happen in inverted microscopes because the coverslip is below the slide.

immersion oil

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this section needs expansion. you can help by adding to that. (January 2008)

Prior to the development of synthetic immersion oils in the 1940s, cedar oil was widely used. Cedarwood oil has a refractive index of approximately 1.516. The numerical aperture of cedar oil targets is usually around 1.3. However, cedar oil has a number of disadvantages: it absorbs blue and ultraviolet light, yellows with age, has enough acidity to potentially damage Goals with repeated use (attacking the cement used to bond lenses), and diluting it with solvent changes its viscosity (and refractive index and dispersal). Cedar oil must be removed from the lens immediately after use before it hardens, as removing hardened cedar oil can damage the lens.

In modern microscopy, synthetic immersion oils are most commonly used as they eliminate most of these problems. NA values ​​of 1.6 can be achieved with different oils. Unlike natural oils, synthetics do not harden on the lens and can usually be left on the objective for months at a time, although to best maintain a microscope it is best to remove the oil daily. Over time, oil can enter the front lens of the objective or the objective barrel and damage the objective. There are different types of immersion oils with different properties based on the type of microscopy you will be performing. Type A and Type B are general purpose immersion oils with different viscosities. Type F immersion oil is best used for fluorescent imaging at room temperature (23 °C), while type N oil is designed to be used at body temperature (37 °C) for live cell imaging applications. everyone has oneD from 1515, quite similar to the original cedar oil.

See too

References

  • practical microscopy by LC Martin and BK Johnson, Glasgow (1966).
  • microscope light by JK Solberg, Tapir Trykk (2000).

External Links


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