  Accurate distance measurement in 3D confocal microscopy is important for
quantitative analysis, volume visualization and image restoration. However,
axial distances can be distorted by both the point spread function and by a
refractive-index mismatch between the sample and immersion liquid, which are
difficult to separate. Additionally, accurate calibration of the axial
distances in confocal microscopy remains cumbersome, although several high-end
methods exist. In this paper we present two methods to calibrate axial
distances in 3D confocal microscopy that are both accurate and easily
implemented. With these methods, we measured axial scaling factors as a
function of refractive-index mismatch for high-aperture confocal microscopy
imaging. We found that our scaling factors are almost completely linearly
dependent on refractive index and that they were in good agreement with
theoretical predictions that take the full vectorial properties of light into
account. There was however a strong deviation with the theoretical predictions
using (high-angle) geometrical optics, which predict much lower scaling
factors. As an illustration, we measured the point-spread-function of a
point-scanning confocal microscope and showed that an index-matched,
micron-sized spherical object is still significantly elongated due to this PSF,
which confirms that single micron-sized spheres are not well suited to
determine accurate axial calibration nor axial scaling.
