Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope

The axial spread function is a useful tool for evaluation of a confocal microscope. It can be obtained experimentally by scanning a uniform fluorescent layer whose thickness is significantly below the resolution limit. Previous researchers have created thin fluorescent films by chemical synthesis. W...

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Bibliographic Details
Published in:Journal of microscopy (Oxford) 2008, Vol.229 (1), p.12-16
Main Authors: MODEL, M.A, BLANK, J.L
Format: Article
Language:English
Subjects:
Absorption cofficients
axial resolution
concentrated dyes
confocal microscopy
full width at half maximum
sectioning imaging property
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Summary:The axial spread function is a useful tool for evaluation of a confocal microscope. It can be obtained experimentally by scanning a uniform fluorescent layer whose thickness is significantly below the resolution limit. Previous researchers have created thin fluorescent films by chemical synthesis. We show here that concentrated fluorescent dyes with a strong absorption at the excitation wavelength can serve as a good approximation of thin fluorescent films. The vertical intensity profiles of such dyes are symmetrical and represent the true axial resolution of a microscope. Solutions of dyes sufficiently opaque to test confocal microscopes with high-NA objectives can be prepared from sodium fluorescein, acid fuchsin and acid blue 9 for excitation at 488 nm, 543 nm and 633 nm, respectively.
ISSN:0022-2720
1365-2818
DOI:10.1111/j.1365-2818.2007.01880.x