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Concentrated dyes as a source of two‐dimensional fluorescent field for characterization of a confocal microscope
Author(s) -
MODEL M. A.,
BLANK J. L.
Publication year - 2008
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.1365-2818.2007.01880.x
Subject(s) - microscope , confocal , fluorescence , opacity , 4pi microscope , optics , confocal microscopy , resolution (logic) , materials science , absorption (acoustics) , wavelength , fluorescence microscope , optical microscope , microscopy , fluorescein , excitation wavelength , characterization (materials science) , chemistry , optoelectronics , scanning electron microscope , nanotechnology , physics , multiphoton fluorescence microscope , artificial intelligence , computer science
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.