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Computer‐Controlled Double‐Beam Scanning Microspectrophotometry For Rapid Microscopic Image Reconstructions
Author(s) -
Kuc̆era P.,
Ribaupierre Y.,
Ribaupierre F.
Publication year - 1979
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1979.tb00200.x
Subject(s) - absorbance , optics , horseradish peroxidase , personal computer , fluorescence , microscope , materials science , image processing , microscopy , biological system , photographic emulsion , computer science , biomedical engineering , chemistry , artificial intelligence , emulsion , image (mathematics) , physics , biology , computer hardware , silver halide , biochemistry , enzyme , medicine
SUMMARY A method for automated collection of various specific data from an entire microscopical preparation and their quantitative evaluation is described. Its application to the study of neuronal connections is discussed in some detail. Brain sections are scanned using a computer‐controlled microscope for reflectance, fluorescences or absorbance signals. Two illuminating beams are used, one of them being amplitude modulated. By means of a synchronous detection the two signals are recorded simultaneously: for example, in an autoradiograph, the reflectance (measuring the density of the silver grains in the emulsion) and the absorbance (allowing to localize the underlying counterstained cells). The data are stored in a computer. Various off‐line processing schemes allow the reconstruction of the data with respect to the corresponding spatial coordinates. Thus pseudo‐three‐dimensional, analogue or digital, graphic displays may be obtained in which the patterns of neuronal connections can be recognized and interpreted. A method for the detection of weakly labelled nerve fibres based on digital filtering is presented. The whole processing for a frontal section of the mouse brain (7 × 10 mm area) takes less than 1 h. In addition to the evaluation of microscopically labelled material (grains of auto‐radiographs, horseradish peroxidase, nucleic acids) the technique described has been successfully used for the study of naturally fluorescent intracellular components in living tissue cultures.