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3‐D image formation in high‐aperture fluorescence confocal microscopy: a numerical analysis
Author(s) -
Voort H. T. M.,
Brakenhoff G. J.
Publication year - 1990
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.1365-2818.1990.tb02975.x
Subject(s) - optics , confocal , pinhole (optics) , physics , polarization (electrochemistry) , microscope , point spread function , image formation , diffraction , microscopy , confocal microscopy , image plane , light sheet fluorescence microscopy , ray , computer science , chemistry , computer vision , scanning confocal electron microscopy , image (mathematics)
SUMMARY The imaging properties of a confocal fluorescence microscope are considered on the basis of a theoretical model. The model takes into account high‐aperture objectives, the polarization state of the excitation light and a finite detector pinhole. Electromagnetic diffraction theory of the field near focus as developed by Richards and Wolf is used to compute the optical properties of the model. These are shown to be dependent on the polarization of the light. With the resulting three‐dimensional point spread function we have studied the imaging of point, line and plane objects as a function of their orientation with respect to the confocal plane. In addition, the effect of the pinhole size on the image formation of these objects is discussed. The results indicate the necessity to take object orientation into account during image processing activities such as segmentation or analysis.