Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses

The effect of the specimen refractive index on the resolution, image brightness and axial scaling in confocal fluorescence microscopy is theoretically investigated for ten of the most popular fluorophores. The calculations are based on a vectorial theory based on Fermat's principle. We show that the axial scaling of the image does not depend on the fluorophore used. Axial scaling factors are derived for objects embedded in glycerol and water. For each fluorophore, the maximum intensity and the full-width-half-maxima of the confocal point spread function are given as a function of the focusing depth. The full-width-half-maxima allow for an assessment of the axial and lateral resolution when deeper regions of the specimen are to be investigated.