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High‐precision distance measurements and volume‐conserving segmentation of objects near and below the resolution limit in three‐dimensional confocal fluorescence microscopy
Author(s) -
Bornfleth,
Sätzler,
Eils,
Crémer
Publication year - 1998
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 - 10.1046/j.1365-2818.1998.00276.x
Subject(s) - confocal , optics , segmentation , microscope , confocal microscopy , volume (thermodynamics) , point spread function , resolution (logic) , calibration , microscopy , point (geometry) , physics , materials science , biological system , computer science , artificial intelligence , mathematics , geometry , quantum mechanics , biology
This study presents a method for high‐precision distance measurements and for the volume‐conserving segmentation of fluorescent objects with a size of the order of the microscopic observation volume. The segmentation was performed via a model‐based approach, using an algorithm that was calibrated by the microscopic point spread function. Its performance was evaluated for three different fluorochromes using model images and fluorescent microspheres as test targets. The fundamental limits which the microscopic imaging process imposes on the accuracy of volume and distance measurements were evaluated in detail. A method for the calibration of the axial stepwidth of a confocal microscope is presented. The results suggest that in biological applications, 3D distances and radii of objects in cell nuclei can be determined with an accuracy of ≤ 60 nm. Using objects of different spectral signature, 3D distance measurements substantially below the lateral half width of the confocal point spread function are feasible. This is shown both theoretically and experimentally.