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Kohler illumination is a method used in transmission-or –reflection microscopy that optimizes the specimen illumination. It was first introduced by August Kohler in 1893 and it is also known as double diaphragm illumination. Prior to Kohler illumination technique, all the other techniques could not prevent the filament of the light bulb (light source) from being visible in the sample plane. Kohler’s technique gets rid of this problem by creating parallel light rays to pass through the specimen, so that they are not in focus when creating the image of the specimen. The advantages of Kohler Illumination are: an evenly illuminated field and a bright image without glare and minimum heating of the specimen.
The light is generated by the light source and it is first captured by the Collector Lens. The focal length from the collector to the light source to has to match the length of the filament. This way, it is ensured that the filament image is the right size when projected into the condenser aperture.
Then the light reaches the Field Lens, which brings the image of the filament into focus at the plane of the sub-stage condenser aperture diaphragm.
In some microscopes after the field lens there is a mirror that reflects the focused light that passed through the field lens and directs it to the Field Diaphragm.
The Field Diaphragm serves as filter and a virtual source of light since it regulates the amount of light that will reach the condenser. Therefore, it is one of the most important parts of the microscope. It also has the advantage of not affecting the optical resolution, numerical aperture, or the intensity of illumination. It can prevent glare as well.
After passing through the field diaphragm, the light reaches the Condenser. The function of a Condenser is to concentrate the light and generate what is known as a cone of light. On the condenser’s base there is another diaphragm called the Aperture Diaphragm. Its correct aperture is critical for proper Kohler illumination, since it determines the angle of the light cone reaching the specimen and consequently the Numerical Aperture.
Numerical Aperture is number that characterizes the range of angles over which the system can accept or emit light. It is given by the following equation:
Where, n is the index of refraction and θ is the half-angle of the maximum cone of light that can enter or exit the lens.
Something important to take into consideration is that the aperture or closure of the field diaphragm does not affect the angle and numerical aperture of the light cone. However, its closure slightly decreases the size of the lower portions of the light cones. Also, it is important to notice that the aperture or closure of the aperture diaphragm should not control the illumination intensity.
Set up for Kohler Illumination
“The first step for ray tracing is to test what objects are intersecting the ray. If the object has an intersection with the ray, the distance between the viewpoint and intersection has to be calculated. If the ray has more than one intersection, the smallest distance identifies the visible surface. Primary rays are rays from the view point to the nearest intersection point.” Primary Rays
Resolution is the smallest resolvable distance between two sample points. It is given by the following equation:
Where R is the resolution, λ is the wavelength andand are the numerical apertures of the objective and the condenser respectively. If this two are equal, the equation is reduced to: