Immersion (microscopy)

Immersion ( Latin immersio , 'immersion', 'embedding') describes in light microscopy a process in which an immersion liquid (embedding liquid) is placed between the objective and the specimen, namely immersion oil ( cedar oil , fatty oil, turpentine oil , petroleum, 1- Bromonaphthalene , water or glycerine , paraffin or silicone oil , as well as benzene and xylene ). Sometimes a condenser with immersion is also used.

Immersion objective in use

Immersion is used with different objectives.

• To increase the achievable resolution . High-resolution oil immersion objectives, for example with magnifications between 60 and 100 times and a high numerical aperture, are used for this purpose .
• To observe living cells or tissues surrounded by aqueous solutions. In this case, water immersion with immersed objectives can be used so that the specimen does not dry out.
• To suppress contrast-reducing reflections by avoiding changes in the refractive index at air-water or air-material boundaries. This aspect plays a role in reflected light microscopy both when observing living objects with immersion objectives and when examining ores and coals . Oil immersion objectives with low magnification such as 2.5x are also used for the latter.

history

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

In 1858 Robert Tolles (1822–1883) built an objective with an exchangeable front lens: one for use with water immersion and another for dry observations. In 1873 he built a well-known “1/10 lens”. In 1859 Edmund Hartnack presented his first water immersion objective, in which he built a correction ring for the first time. His immersion lenses were considered the best of his time, he was able to sell about 400 copies in the following five years. Ernst Gundlach (1834–1908) presented the first glycerine immersion objective at the World Exhibition in Paris in 1867 , in order to be able to use an immersion medium with a higher refractive index than water.

Tolles introduced Canada balsam as an immersion medium in 1871 after discovering that it had the same index of refraction as crown glass , which was used for lenses. In 1873 he built a three-lens objective for "homogeneous immersion" with balm, which reached a numerical aperture of 1.25. Homogeneous immersion means that the refractive index between the condenser , preparation and objective does not change, i.e. that it is the same for the glasses used and for the immersion liquid. Almost at the same time, he also presented a glycerine lens that achieved a numerical aperture of 1.27.

From 1877 onwards, Carl Zeiss manufactured lenses for homogeneous immersion, which were designed by Ernst Abbe . In 1878 Abbe published an article in the journal of the Royal Microscopical Society in which he described the corresponding optics and pointed out that homogeneous immersion enabled the maximum theoretically achievable aperture.

Increase in resolution

Working principle

Principle of increasing the resolution with immersion medium. Left side beam path with immersion medium (yellow), right without. Rays of light (black) coming from the object (red) and passing through the cover slip (orange, like the slide below) are only picked up by the objective (dark blue) in a certain angular range if an immersion medium causes a refraction at the transition from the cover slip to air is prevented or at least reduced.

The achievable resolution of an objective and thus of the entire microscopic system depends on its effective opening angle : the more light that can be captured that has passed through the specimen from different directions, the greater the total information content and the better the achievable resolution. For an objective, this is specified as the numerical aperture (NA). The NA is defined by the opening angle of the objective and the refractive index (also: refractive index) n _{i of} the medium between the objective and the specimen.

${\displaystyle {\text{NA}}=n\cdot \sin \alpha }$

${\displaystyle n}$ : Refractive index of the immersion medium or of air

${\displaystyle \alpha }$ : half the opening angle

Air has a low refractive index of approximately 1. When light from aqueous or embedded biological specimens passes into air, it is therefore deflected away from the optical axis by the refraction that occurs . When using a cover slip , the same undesirable effect occurs at the transition from the cover slip to air. That part of the light that has been so strongly deflected that it can no longer be captured by the objective is lost for microscopy and with it its informational content.

When using the microscope, the object is usually covered with a cover glass. The light from the object is first refracted when it passes into the cover slip and then again when it passes into the space between the cover slip and the objective. At the second transition, total reflection can occur if the space is filled with an optically thinner medium than glass. The amount of light that enters the optical system is then reduced. Total reflection can be avoided by using an immersion oil that has roughly the same refractive index as glass.

Immersion media have a significantly higher refractive index than air, so that the described undesired refraction away from the optical axis does not occur or at least less strongly. More light and thus more information can be captured by the lens. The resolution improves. The resolution limit , i.e. the smallest resolvable structure, can be determined, for example, according to the Rayleigh criterion , which is used, for example, in fluorescence microscopy (see also resolution (microscopy) ):

${\displaystyle d_{\mathrm {min} }={\frac {0{,}61\cdot \lambda }{\text{NA}}}}$

${\displaystyle \lambda }$ : Wavelength of the light used.

The resolution depends on the numerical aperture of the objective used on the refractive index of the immersion medium. In the formula given, d corresponds to the distance that two punctiform, fluorescent structures, both of which lie in the plane of focus ( xy plane ), must at least have in order to be able to be resolved as separate structures. The resolution is worse along the optical axis (z-direction).

example

Due to the refractive index of air, dry objectives, i.e. those without immersion, achieve a maximum theoretical NA of 1 (with sin α = 1, corresponding to an aperture angle 2α of 180 °) and practically an NA of 0.95 (aperture angle 144 °). In the case of an oil immersion objective with a numerical aperture NA = 1.4 for immersion oil with a refractive index 1.518, the following applies: 1.4 = 1.518 · sin α. This results in the opening angle 2α of 134 °.

For the stated dry objective with NA = 0.95, a maximum resolution of 0.61 * 500 nm / 0.95 = 321 nm results at 500 nm wavelength according to the Rayleigh criterion in fluorescence microscopy. For the oil immersion objective described NA = 1.4 In contrast, the result is 0.61 * 500 nm / 1.4 = 217 nm. As mentioned, the NA = 0.95 selected in this example is the maximum possible for dry objectives. With an NA of 0.5 for the objective, the result would be: 0.61 x 500 nm / 0.5 = 610 nm.

Immersion oils

From the 19th century cedar oil was used for oil immersion . Higher refractive indices can be achieved through thickening. However, it becomes resinous in the air .

Nowadays, synthetic oils that do not harden are largely used. Standard oils have a refractive index of 1.5180 (at 546.1 nm wavelength ) and are therefore close to the refractive index of cover glasses (1.5255). Oil immersion objectives are designed to achieve the maximum resolution with such oils and a cover slip of the correct thickness.

For certain applications, however, there are also immersion oils with different refractive indices, for example from 1.30-2.11. In special cases, oils with high and low refractive indexes can be mixed to precisely achieve a required refractive index.

With fluorescence microscopy , care must be taken that the oil used does not have any inherent fluorescence . An "F" in the type designation indicates that it is suitable for fluorescence microscopy.

See also

• Immersion lithography
• Immersion method (mineralogy)

literature

• ISO 8036: 2006 (E): Optics and photonics - Microscopes - Immersion liquids for light microscopy .

Footnotes and individual references

1. ^ Paul Heermann, Alois Herzog: Microscopic and mechanical-technical textile investigations. 3rd edition, Springer, 1931, ISBN 978-3-642-98596-6 , p. 87.
2. ↑ ^{a b c d e} Highlights from the History of Immersion Objectives . In: Carl Zeiss AG (Ed.): Innovation . tape 15 , 2005, pp. 16–17 ( zeiss.de [PDF; accessed October 6, 2013]).
3. ↑ Since materials usually have a wavelength-dependent refractive index ( dispersion ), for the sake of simplicity, mostly only the refractive index at a certain wavelength is given. These are typically n _e , i.e. the value at 546.1 nm (mercury e-line), or n _D , at 589.3 nm ( sodium D-line ). Examples (for this article):
   • Air: n _D  = 1,000293.
   • Water: n _e  = 1.33.
   • Glycerin: n = 1.47.
   •  Cover slip glass: n _e = 1.5255, n _D  = 1.5230.
   • Standard immersion oil from Zeiss: n _e  = 1.5180, n _D  = 1.5151.
4. ↑ 's website Cargille , accessed on 3 July of 2009.