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Absorption and scattering correction in fluorescence confocal microscopy
Author(s) -
Visser T. D.,
Groen F. C. A.,
Brakenhoff G. J.
Publication year - 1991
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.1365-2818.1991.tb03171.x
Subject(s) - extinction (optical mineralogy) , attenuation coefficient , scattering , absorption (acoustics) , optics , attenuation , molar absorptivity , fluorescence , confocal , microscope , light scattering , excitation , microscopy , materials science , chemistry , molecular physics , physics , quantum mechanics
SUMMARY In three‐dimensional (3‐D) fluorescence images produced by a confocal scanning laser microscope (CSLM), the contribution of the deeper layers is attenuated due to absorption and scattering of both the excitation and the fluorescence light. Because of these effects a quantitative analysis of the images is not always possible without restoration. Both scattering and absorption are governed by an exponential decay law. Using only one (space‐dependent) extinction coefficient, the total attenuation process can be described. Given the extinction coefficient we calculate within a non‐uniform object the relative intensity of the excitation light at its deeper layers. We also give a method to estimate the extinction coefficients which are required to restore 3‐D images. An implementation of such a restoration filter is discussed and an example of a successful restoration is given.