Furthermore, altering the size of the condenser diaphragm allows adjustment of sample depth of field by altering the effective numerical aperture of the microscope. Uniform illumination of the sample is also critical for advanced illumination techniques such as phase contrast and differential interference contrast microscopy.Īdjusting the condenser diaphragm alters sample contrast.
This reduces image artifacts and provides high sample contrast. The primary advantage of Köhler illumination is the uniform illumination of the sample. Intermediate image plane (the eyepiece diaphragm) (C).Light source image planes (labeled with light green bar in image): These two sets of image planes are found at the following points (see image for numbers and letters): This illumination scheme creates two sets of conjugate image planes, one with the light source image and one with the specimen. The condenser lens acts to project this light, without focusing it, through the sample. The collector/field lenses act to collect light from the light source and focus it at the plane of the condenser diaphragm. These components lie in this order between the light source and the specimen and control the illumination of the specimen. Conjugated planes marked with light blue bar See text for further details. Conjugated planes marked with light green bar. Köhler illumination requires several optical components to function: 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 addresses this by ensuring the image of the light source is perfectly defocused in the sample plane and its conjugate image planes.
The primary limitation of critical illumination is the formation of an image of the light source in the specimen image plane. Köhler illumination has also been developed in the context of nonimaging optics. This work was published in 1893 in the Zeitschrift für wissenschaftliche Mikroskopie Īnd was soon followed by publication of an English translation in the Journal of the Royal Microscopical Society. To address these limitations August Köhler designed a method of illumination which uses a perfectly defocused image of the light source to illuminate 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. 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. Uneven illumination is undesirable as it can introduce artifacts such as glare and shadowing in the image. Critical illumination therefore gives uneven illumination of the sample bright regions in the filament image illuminate those regions of the sample more strongly. The image of the light source is often referred to as the filament image. the bulb filament is visible in the final image. 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. Prior to Köhler illumination critical illumination was the predominant technique for sample illumination.
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