Köhler illumination
Encyclopedia
Köhler illumination
is a method of specimen
illumination
used for transmitted and reflected light (trans- and epi-illuminated) optical
microscopy
. Köhler illumination acts to generate an extremely even illumination of the sample and ensures that an image of the illumination source (for example a halogen lamp
filament) is not visible in the resulting image
. Köhler illumination is the predominant technique for sample illumination in modern scientific light microscopy although it requires additional optics which less expensive and simpler light microscopes may not have.
was the predominant technique for sample illumination. Critical illumination has the major limitation that the image of the light source (typically a light bulb) falls in the same plane as the image of the specimen, i.e. the bulb filament is visible in the final image. The image of the light source is often referred to as the filament image. Critical illumination therefore gives uneven illumination of the sample; bright regions in the filament image illuminate those region of the sample more strongly. Uneven illumination is undesirable as it can introduce artefacts such as glare
and shadowing in the image.
Various methods can be used to diffuse the filament image, including reducing power to the light source or using an opal glass bulb or an opal glass diffuser between the bulb and the sample. These methods are all, to some extent, functional at reducing the unevenness of illumination however they all reduce intensity of illumination and alter the range of wavelengths of light which reach the sample.
In order to address these limitations August Köhler
designed a new method of illumination which uses a perfectly defocused image of the light source to illuminate the sample. This work was published in 1893 in the Zeitschrift für wissenschaftliche Mikroskopie and was soon followed by publication of an English translation in the Journal of the Royal Microscopical Society .
Köhler illumination requires several optical components to function:
These components lie in this order between the light source and the specimen and control the illumination of the specimen. The collector/field lenses act to collect light from the light source and focus it at the plane of the condenser diaphragm. The condenser lens acts to project this light, without focusing it, through the sample. This illumination scheme creates two sets of conjugate image planes, one with the light source image and one with the specimen. These two sets of image planes are found at the following points:
By adjustment of the field diaphragm the amount of light entering the sample can be freely adjusted without altering the wavelengths of light present, in contrast to reducing power to the light source with critical illumination. Adjusting the condenser diaphragm alters sample contrast
. Furthermore altering the size of the condenser diaphragm allows adjustment of sample depth of field
by altering the effective numerical aperture
of the microscope. The role of the condenser diaphragm is analogous to the aperture
in photography
although the condenser diaphragm of a microscope functions by controlling illumination of the specimen, while the aperture of a camera functions by controlling illumination of the detector.
Alignment of optical components on the specimen image plane is typically performed by first loading a test specimen and bringing it into focus by moving the objective or the specimen. The field diaphragm is then partially closed; the edges of the diaphragm should be in the same conjugate image planes as the specimen, therefore should appear in focus. The focus can be adjusted by raising or lowering the condenser lenses and diaphragm. Finally, the field diaphragm is reopened to just beyond the field of view.
In order to test the alignment of components on the light source image plane, the eyepiece must be removed to allow observation of the intermediate image plane (the position of the eyepiece diaphragm) either directly or by using an phase telescope
/Bertrand lens. The light source (e.g. the bulb filament) and the edges of the condenser diaphragm should appear in focus. Any optical components at the back focal plane of the objective (e.g. the phase ring for phase contrast microscopy) and at the condenser diaphragm (e.g. the annulus for phase contrast microscopy) should also appear in focus.
Optical microscopy techniques which use Köhler illumination as a basis:
Köhler illumination
Köhler illumination is a method of specimen illumination used for transmitted and reflected light optical microscopy. Köhler illumination acts to generate an extremely even illumination of the sample and ensures that an image of the illumination source is not visible in the resulting image...
is a method of specimen
Specimen
A specimen is a portion/quantity of material for use in testing, examination, or study.BiologyA laboratory specimen is an individual animal, part of an animal, a plant, part of a plant, or a microorganism, used as a representative to study the properties of the whole population of that species or...
illumination
Illumination
Illumination, an observable property and effect of light, may also refer to:* Illumination , the use of light sources* Illumination , the use of light and shadow in art...
used for transmitted and reflected light (trans- and epi-illuminated) optical
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...
microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
. Köhler illumination acts to generate an extremely even illumination of the sample and ensures that an image of the illumination source (for example a halogen lamp
Halogen lamp
A halogen lamp, also known as a tungsten halogen lamp, is an incandescent lamp with a tungsten filament contained within an inert gas and a small amount of a halogen such as iodine or bromine. The chemical halogen cycle redeposits evaporated tungsten back on to the filament, extending the life of...
filament) is not visible in the resulting image
Image
An image is an artifact, for example a two-dimensional picture, that has a similar appearance to some subject—usually a physical object or a person.-Characteristics:...
. Köhler illumination is the predominant technique for sample illumination in modern scientific light microscopy although it requires additional optics which less expensive and simpler light microscopes may not have.
History and motivation
Prior to Köhler illumination critical illuminationCritical illumination
Critical illumination or Nelsonian illumination is a method of specimen illumination used for transmitted and reflected light optical microscopy. Critical illumination focuses an image of a light source on to the specimen for bright illumination...
was the predominant technique for sample illumination. Critical illumination has the major limitation that the image of the light source (typically a light bulb) falls in the same plane as the image of the specimen, i.e. the bulb filament is visible in the final image. The image of the light source is often referred to as the filament image. Critical illumination therefore gives uneven illumination of the sample; bright regions in the filament image illuminate those region of the sample more strongly. Uneven illumination is undesirable as it can introduce artefacts such as glare
Glare
Glare may refer to:* Glare is difficulty seeing in the presence of very bright light* A glare is a facial expression of squinted eyes and look of contempt* A call collision in telecommunications* GLARE, an advanced aerospace material...
and shadowing in the image.
Various methods can be used to diffuse the filament image, including reducing power to the light source or using an opal glass bulb or an opal glass diffuser between the bulb and the sample. These methods are all, to some extent, functional at reducing the unevenness of illumination however they all reduce intensity of illumination and alter the range of wavelengths of light which reach the sample.
In order to address these limitations August Köhler
August Köhler
August Karl Johann Valentin Köhler was a German professor and early staff member of Carl Zeiss AG in Jena, Germany. He is best known for his development of the microscopy technique of Köhler illumination, an important principle in optimizing microscopic resolution power by evenly illuminating the...
designed a new method of illumination which uses a perfectly defocused image of the light source to illuminate the sample. This work was published in 1893 in the Zeitschrift für wissenschaftliche Mikroskopie and was soon followed by publication of an English translation in the Journal of the Royal Microscopical Society .
Optical principles
The primary limitation of critical illumination is the formation of an image of the light source in the specimen image plane. Köhler illumination addresses this by ensuring the image of the light source is perfectly defocused in the sample plane and its conjugate image planes. In a ray diagram of the illumination light path this can be seen as the image-forming rays passing parallel through the sample.Köhler illumination requires several optical components to function:
- Collector lens and/or field lens
- Field diaphragm
- Condenser diaphragm
- Condenser lens
These components lie in this order between the light source and the specimen and control the illumination of the specimen. The collector/field lenses act to collect light from the light source and focus it at the plane of the condenser diaphragm. The condenser lens acts to project this light, without focusing it, through the sample. This illumination scheme creates two sets of conjugate image planes, one with the light source image and one with the specimen. These two sets of image planes are found at the following points:
- Light source image planes:
- Lamp filament
- Condenser diaphragm
- Back focal plane of the objective
- The eyepoint
- Specimen image planes:
- Field diaphragm
- Specimen
- Intermediate image plane (the eyepiece diaphragm)
- The eye retina or camera sensor
Advantages
The primary advantage of Köhler illumination is the extremely even illumination of the sample. This reduces image artifacts and provides high sample contrast. Even illumination of the sample is also critical for advanced illumination techniques such as phase contrast and differential interference contrast microscopy.By adjustment of the field diaphragm the amount of light entering the sample can be freely adjusted without altering the wavelengths of light present, in contrast to reducing power to the light source with critical illumination. Adjusting the condenser diaphragm alters sample contrast
Contrast
Contrast may refer to:* Contrast , the difference in color and light between parts of an image* Contrast , expressing distinctions between words...
. Furthermore altering the size of the condenser diaphragm allows adjustment of sample depth of field
Depth of field
In optics, particularly as it relates to film and photography, depth of field is the distance between the nearest and farthest objects in a scene that appear acceptably sharp in an image...
by altering the effective 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 microscope. The role of the condenser diaphragm is analogous to the aperture
Aperture
In optics, an aperture is a hole or an opening through which light travels. More specifically, the aperture of an optical system is the opening that determines the cone angle of a bundle of rays that come to a focus in the image plane. The aperture determines how collimated the admitted rays are,...
in photography
Photography
Photography is the art, science and practice of creating durable images by recording light or other electromagnetic radiation, either electronically by means of an image sensor or chemically by means of a light-sensitive material such as photographic film...
although the condenser diaphragm of a microscope functions by controlling illumination of the specimen, while the aperture of a camera functions by controlling illumination of the detector.
Testing for and setting up Köhler illumination
Microscopes using Köhler illumination must be routinely checked for correct alignment. The realignment procedure tests whether the correct optical components are in focus at the two sets of conjugate image planes; the light source image planes and the specimen image planes.Alignment of optical components on the specimen image plane is typically performed by first loading a test specimen and bringing it into focus by moving the objective or the specimen. The field diaphragm is then partially closed; the edges of the diaphragm should be in the same conjugate image planes as the specimen, therefore should appear in focus. The focus can be adjusted by raising or lowering the condenser lenses and diaphragm. Finally, the field diaphragm is reopened to just beyond the field of view.
In order to test the alignment of components on the light source image plane, the eyepiece must be removed to allow observation of the intermediate image plane (the position of the eyepiece diaphragm) either directly or by using an phase telescope
Phase telescope
A phase telescope or Bertrand lens is an optical device used in aligning the various optical components of a light microscope. In particular it allows observation of the back focal plane of the objective lens and its conjugate focal planes...
/Bertrand lens. The light source (e.g. the bulb filament) and the edges of the condenser diaphragm should appear in focus. Any optical components at the back focal plane of the objective (e.g. the phase ring for phase contrast microscopy) and at the condenser diaphragm (e.g. the annulus for phase contrast microscopy) should also appear in focus.
See also
- Optical microscopy
- August KöhlerAugust KöhlerAugust Karl Johann Valentin Köhler was a German professor and early staff member of Carl Zeiss AG in Jena, Germany. He is best known for his development of the microscopy technique of Köhler illumination, an important principle in optimizing microscopic resolution power by evenly illuminating the...
- Nonimaging optics#Kohler integration
Optical microscopy techniques which use Köhler illumination as a basis:
- Bright field microscopyBright field microscopyBright field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted white light and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample...
- Phase contrast microscopyPhase contrast microscopyPhase contrast microscopy is an optical microscopy illumination technique of great importance to biologists in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image.A phase contrast microscope does not require...
- Dark field microscopyDark field microscopyDark field microscopy describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen Dark field microscopy (dark ground microscopy) describes microscopy methods, in both light and electron...
- Differential interference contrast microscopyDifferential interference contrast microscopyDifferential interference contrast microscopy , also known as Nomarski Interference Contrast or Nomarski microscopy, is an optical microscopy illumination technique used to enhance the contrast in unstained, transparent samples...
- Polarized light microscopyPolarized light microscopyPolarized light microscopy can mean any of a number of optical microscopy techniques involving polarized light. Simple techniques include illumination of the sample with polarized light. Directly transmitted light can, optionally, be blocked with a polariser orientated at 90 degrees to the...
- Hoffman modulation contrast microscopy