Köhler illumination

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. It requires additional optical elements which are more expensive and may not be present in more basic light microscopes.

Prior to Köhler illumination critical 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 regions 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

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