Oil immersion

Immersion (Latin Immersio, immersion ',' embedding ') referred to in the light microscopy, a method in which, between the lens and the preparation an immersion liquid, namely, immersion oil, water or glycerin, is introduced. Sometimes, in addition, a condenser with immersion is used.

Immersion is used with different objectives.

• To increase the achievable resolution. Serve this purpose very high resolution oil - immersion objectives, for example, with magnifications between 60 - and 100-fold and a high numerical aperture.
• To observe living cells or tissues, which are surrounded by aqueous solutions. Here water immersion can be used with the immersed lens, so that a drying of the preparation is avoided.
• For the suppression of contrast -reducing reflections by avoiding refractive index changes at air -water or air - material boundaries. This aspect plays in the reflected-light microscopy in both the observation of living objects with immersion lenses as well as a role in the study of ores and coals. In the latter, see also oil - immersion objectives with low magnification as 2.5 -fold application.

History

Already published in 1678 book " Microscopium " by Robert Hooke was the use of an immersion liquid discussed to unify the refractive index of the optical path and thereby achieve clearer and brighter images. Other mentions of the immersion microscopy can be found at 1812 David Brewster and around 1840 in Giovanni Battista Amici. Amici built lenses for use with anise oil, which has a refractive index similar to that of glass. He did this in order to reduce the chromatic aberration of his system. Since slides were very expensive at that time, to oil immersion continued, however, at first not by Amici and switched to water immersion. In 1853 he built a first water lens, which he presented in Paris in 1855.

1858 built Robert Great ( 1822-1883 ), a lens with interchangeable front lens: One for use with water immersion and another for dry observations. In 1873 he built a very well known become " 1/10 lens ". 1859 Edmund Hartnack presented before his first water-immersion objective, in which he included a correction ring for the first time. His immersion objectives were considered the best of his time, he was able to sell over the next five years, about 400 copies. Ernst Gundlach (1834-1908) presented in 1867 at the World Exhibition in Paris in a first glycerol - immersion objective before to use an immersion medium with a higher refractive index than water can.

Great led a 1871 Canada balsam as an immersion medium, since he discovered that it has the same refractive index as crown glass, which was used for lenses. In 1873 he built a three-lens objective for " homogeneous immersion " with balsam, which reached a numerical aperture of 1.25. Homogeneous Immersion means that the refractive index between condenser, specimen and objective does not change, so that it is equal to the used glasses and for the immersion liquid. Almost simultaneously, he also presented a glycerol lens, which reached a numerical aperture of 1.27.

From In 1877 Carl Zeiss lenses for homogeneous immersion ago, designed by Ernst Abbe. 1878 Abbe published an article in the Journal of the Royal Microscopical Society, in which he described the appropriate optics, pointing out the homogeneous immersion enabled the maximum theoretically achievable aperture.

Increase the resolution

Principle of operation

The achievable resolution of a lens and thus of the entire microscope system depends on its effective aperture angle from the more light can be collected, which has traversed from different directions, the preparation, the greater the summed information content and the better the achievable resolution. This is indicated for a lens as the numerical aperture (NA). NA is determined by the opening angle of the lens and the refractive index (also: refractive index) of the medium defined ni between objective and specimen.

Air has a low refractive index of approximately 1 When light from aqueous or embedded biological specimens is excreted in air, it is therefore deflected away by the apparent refraction of the optical axis. With the use of a cover glass of the same adverse effect on the transition from the cover glass to the air occurs. The part of the light which was so distracted that he can no longer be absorbed by the lens, is lost for microscopy and with him his information content.

At microscopy, the object is usually covered with a cover glass. The light of the object is first broken during the transition to the cover glass and then again during the transition into the space between the cover glass and lens. The second transition may occur, for the total reflection when the intermediate space is filled with an optically thinner medium than glass. The amount of light that enters the optical system, is then reduced. By the use of an immersion oil, which is about the same refractive index as does glass, total internal reflection can be prevented.

Immersion media have a much higher refractive index than air, so that the described undesired refraction at least does not occur or less away from the optical axis. More light and therefore more information can be picked up by the lens. The resolution improves. The resolution limit, ie, the minimum resolvable feature, for example, can be determined according to the Rayleigh criterion:

The resolution depends on the numerical aperture of the refractive index of the immersion medium. Since the incident light microscopy, the lens is also used as a condenser, is considered here in the denominator 2 · NA. This is the case for example with the usual form of fluorescence microscopy. The given formula is valid only if both points lie in the focal plane (xy plane ). Along the optical axis (z- direction), the resolution is worse.

Example

Dry lenses, ie those without immersion achieve because of the refractive index of air is maximum theoretical NA 1 ( with sin α = 1, corresponding to an opening angle of 2α of 180 ° ), and virtually has an NA of 0.95 ( angle 144 degrees). With an oil -immersion objective with a numerical aperture NA = 1.4 is valid for immersion oil with a refractive index of 1.518: 1.518 = 1.4 * sin α. From the opening angle 2α followed by 134 °.

For the above dry objective with NA = 0.95 ( or fluorescence microscopy ) results at 500 nm wavelength and using a good condenser as a maximum resolution of 1.22 × 500 nm / (0.95 0.95) = 321 nm. described for the oil immersion objective NA = 1.4 is obtained, however, 1.22 × 500 nm / (1.4 1.4 ) = 217 nm, as mentioned is selected in this example, NA = 0.95, the maximum possible for dry objectives. With an NA of 0.65 and 0.3 of the lens of the condenser would be obtained: 1.22 × 500 nm / (0.65 0.3 ) = 642 nm

Immersion oils

From the 19th century cedar oil was used for oil immersion. On thickening higher refractive indices can be achieved. In air, however, it gummy.

Today, largely synthetic oils are used, which are not hard. Standard oils have a refractive index of 1.5180 ( at 546.1 nm wavelength) and therefore lie close to the refractive index of glass plates ( 1.5255 ). Oil immersion objectives are calculated so that they achieve the maximum resolution with such oils and a cover glass of the correct thickness.

For certain applications, however, there is also immersion oils with different refractive indices, for example, from 1.30 to 2.11. In specific cases oils can be mixed with high and low refractive index in order to achieve a necessary refractive index exactly.

In fluorescence microscopy, it must be ensured that the oil used does not have any intrinsic fluorescence. An "F " in the model name indicates that it is suitable for fluorescence microscopy.