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Microphotography, Image Processing with Video Cameras, AVEC-DIC and AVEC-POL

Background subtraction

The development of microscopy created a need for the documentation of the images. The classical approach that is practized by every biology student is drawing. A help was the construction of a drawing device invented by E. ABBÉ, that is placed upon the eyepiece and projects the image via prism and mirror onto a drawing pad. An alternative came into being with microphotography, that has the disadvantage of a very small depth of field and captures consequently only single planes of the object. This is the reason why it is until today viewed rather critically by some biologists. But it has nevertheless been established as an independent method. Numerous images on our web pages show that an exact image of what can be seen is given by a photo. If it is necessary, several images of different planes of the object can be taken. The important advantages compared to drawings are the speed of documentation and the exclusion of subjective perceptions (freedom of the artist), whereby a lot of damage can be caused. One and the same object can be studied in parallel with with different microscopic methods, corresponding pictures can be compared in order to determine their orientation. The film material of today brings astonishing results even at the very poor light conditions of fluorescence microscopy.

The possibility of cinematography has already been mentioned shortly. Many documentaries of cellular motion, of cells, their coupling etc. exist. Most of the documentaries produced in Germany were done in co-operation with the Institut für den Wissenschaftlichen Film in Göttingen (Institute for Scientific Film at Göttingen). It is possible for other institutes and other scientific institutions to borrow them.

Video cameras, image intensification and computer-controlled image-storing and -interpretation do also belong to modern microscopy. They have first been used at the beginning of the fifties, but the important developments started during the last years of the seventies. Two processes especially that have been developed by R. D. ALLEN, N. STRÖMGREN-ALLEN and J. F. TRAVIS (Dartmouth College, Hanover, New Hampshire, USA) have to be mentioned:

AVEC-DIC (Allen video-enhanced contrast-differential interference contrast) and

AVEC-POL (Allen video-enhanced contrast-polarization microscopy)

Both methods are based on the fact that certain video cameras (the right choice of the model is decisive) can process differences in brightness on a completely different scale than the human eye or a photographic film. Depending on the study or the object can different adjustments of the camera be chosen. E. ABBÉ defined the resolution limit of the light microscope as that distance between two adjoining points, at which they can only just be perceived as different unities. The limiting factor is the wavelength of the used light. This resolution limit does not mean that smaller structures stay invisible. Fluorochromization, for example, allows single molecular complexes to be seen, if the distance between molecules of the same kind is above the resolution limit of the microscope.

Instead of fluorochromization contrast-enhancement by AVEC-DIC can be used and although the contrast produced by microtubuli is not big enough to be perceived by the human eye it can be recorded and identified by a camera.

Image-storing and -interpretation allow the emphasizing of movable particles. The appropriate computer program subtracts the content of one image from that recorded seconds or minutes afterwards of the same spot. All immovable structures vanish and only the moveable ones are depicted.

Cameras with image intensification are used in fluorescence microscopy to document fluorochromes with a fluorescence that is far below that perceivable by the human eye. Again it is possible to use the already mentioned method of vital staining, because far more specific sondes are known today. Since their concentration is smaller than that used in the forties or fifties the cells are damaged less.

While in the nowadays common fluorescence microscopy photons (hny) are emitted and recorded after the illumination of the object, a photoelectron microscope allows the tracing of secondary electrons, that are set free after exposure to UV radiation. This microscope has electronic optics (see next paragraph) and the resolution is higher than that of fluorescence microscopes. The technique has only recently been developed, but first results with biological objects exist already. They suggest that the method will be widespread within short time (O. GRIFFITH, G. B. BIRRELL, 1985).

© Peter v. Sengbusch - Impressum