Oil immersion
Encyclopedia
Optical microscope
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the...
, oil immersion is a technique used to increase the resolution
Optical resolution
Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged.An imaging system may have many individual components including a lens and recording and display components...
of a microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
. This is achieved by immersing both the objective lens
Objective (optics)
In an optical instrument, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be single lenses or mirrors, or combinations of several optical elements. They are used in microscopes, telescopes,...
and the specimen in a transparent oil of high refractive index
Refractive index
In optics the refractive index or index of refraction of a substance or medium is a measure of the speed of light in that medium. It is expressed as a ratio of the speed of light in vacuum relative to that in the considered medium....
, thereby increasing the numerical aperture
Numerical aperture
In optics, the numerical aperture of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the property that it is constant for a beam as it goes from one...
of the objective lens.
Immersion oils are transparent oils that have specific optical and viscosity
Viscosity
Viscosity is a measure of the resistance of a fluid which is being deformed by either shear or tensile stress. In everyday terms , viscosity is "thickness" or "internal friction". Thus, water is "thin", having a lower viscosity, while honey is "thick", having a higher viscosity...
characteristics necessary for use in microscopy. Typical oils used have an index of refraction around 1.515. An oil immersion objective is an objective lens specially designed to be used in this way. Many condensers also give optimal resolution when the condenser lens is immersed in oil.
Theoretical background
The resolution of a microscope is defined as the minimum separation needed between two objects under examination in order for the microscope to discern them as separate objects. This minimum distance is labelled δ. If two objects are separated by a distance shorter than δ, they will appear as a single object in the microscope.A measure of the resolving power of a lens is given by its numerical aperture
Numerical aperture
In optics, the numerical aperture of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the property that it is constant for a beam as it goes from one...
, NA:
where λ is the wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...
of light. From this it is clear that a good resolution (small δ) is connected with a high numerical aperture.
The numerical aperture of a lens is defined as
where α0 is the angle spanned by the objective lens seen from the sample, and n is the refractive index of the medium between the lens and specimen (≈1 for air).
State of the art objectives can have a numerical aperture of up to 0.95. Because sin α0 is always less than or equal to unity, the numerical aperture can never be greater than unity for an objective lens in air. If the space between the objective lens and the specimen is filled with oil however, the numerical aperture can obtain values greater than unity. This is because oil has a refractive index
Refractive index
In optics the refractive index or index of refraction of a substance or medium is a measure of the speed of light in that medium. It is expressed as a ratio of the speed of light in vacuum relative to that in the considered medium....
greater than 1.
Oil immersion objectives
From the above it is understood that oil between the specimen 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 used only at very large magnifications that require high resolving power. Objectives with high power magnification have short focal length
Focal length
The focal length of an optical system is a measure of how strongly the system converges or diverges light. For an optical system in air, it is the distance over which initially collimated rays are brought to a focus...
s, facilitating the use of oil. The oil is applied to the specimen (conventional microscope), and the stage is raised, immersing the objective in oil. (In inverted microscope
Inverted microscope
An inverted microscope is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up. It was invented in 1850 by J. Lawrence Smith, a faculty member of Tulane University...
s the oil is applied to the objective).
The refractive indices of the oil and of the glass in the first lens element are nearly the same, which means that the refraction of light will be small upon entering the lens (the oil and glass are optically very similar). The correct immersion oil for an objective lens has to be used to ensure that the refractive indices are correctly matched. Use of an oil immersion lens with the incorrect 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 specimens otherwise the surface tension
Surface tension
Surface tension is a property of the surface of a liquid that allows it to resist an external force. It is revealed, for example, in floating of some objects on the surface of water, even though they are denser than water, and in the ability of some insects to run on the water surface...
of the oil can move the coverslip and so move the sample underneath. This also happen on inverted microscopes because the coverslip is below the slide.
Immersion oil
Before the development of synthetic immersion oils in the 1940s Cedar tree oilCedar oil
Cedar oil, also known as cedarwood oil, is an essential oil derived from the foliage, and sometimes the wood and roots, of various types of conifers, most in the pine or cypress botanical families...
was widely used. Cedar oil has an index of refraction of approximately 1.516. The numerical aperture of cedar tree oil objectives is generally around 1.3. Cedar oil has a number of disadvantages however: it absorbs blue and ultraviolet light, yellows with age, has sufficient acidity to potentially damage objectives
Objective (optics)
In an optical instrument, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be single lenses or mirrors, or combinations of several optical elements. They are used in microscopes, telescopes,...
with repeated use (by attacking the cement used to join lenses
Lens (optics)
A lens is an optical device with perfect or approximate axial symmetry which transmits and refracts light, converging or diverging the beam. A simple lens consists of a single optical element...
), and diluting it with solvent changes its viscosity (and refraction index and dispersion
Dispersion (optics)
In optics, dispersion is the phenomenon in which the phase velocity of a wave depends on its frequency, or alternatively when the group velocity depends on the frequency.Media having such a property are termed dispersive media...
). Cedar oil must be removed from the objective immediately after use before it can harden, since removing hardened cedar oil can damage the lens. In modern microscopy synthetic immersion oils are more commonly used, as they eliminate most of these problems. NA values of 1.6 can be achieved with different oils. Unlike natural oils synthetic ones do not harden on the lens and can typically be left on the objective for months at a time, though dust accumulation can be a problem. Xylene
Xylene
Xylene encompasses three isomers of dimethylbenzene. The isomers are distinguished by the designations ortho- , meta- , and para- , which specify to which carbon atoms the two methyl groups are attached...
is often used to remove oil from the objective when desired.
External links
- "Microscope Objectives: Immersion Media" by Mortimer Abramowitz and Michael W. Davidson, OlympusOlympus Corporationis a Japan-based manufacturer of optics and reprography products. Olympus was established on 12 October 1919, initially specializing in microscope and thermometer businesses. Its global headquarters are in Shinjuku, Tokyo, Japan, while its USA operations are based in Center Valley, Pennsylvania,...
Microscopy Resource Center (website), 2002. - "Immersion Oil Microscopy" by David B. Fankhauser, Biology at University of CincinnatiUniversity of CincinnatiThe University of Cincinnati is a comprehensive public research university in Cincinnati, Ohio, and a part of the University System of Ohio....
, Clermont College (website), December 30, 2004. - "History of Oil Immersion Lenses" by Jim Solliday, Southwest Museum of Engineering, Communications, and Computation (website), 2007.
- "Immersion Oil and the Microscope" by John J. Cargille, New York Microscopical Society Yearbook, 1964 (revised, 1985). (Archived at Cargille Labs (website).)