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. 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.