Acquisition depth (zPos)

This is a quantity expressed in micrometers (µm) that describes at what real depth inside the preparation is the focus of the microscope, measured from the coverslip (which is considered by the Huygens Software as Z=0).
The position of the first image plane can be set when imaging starts further from the coverslip (see Coverslip Position).

This figure is important in order to compute the amount of Spherical Aberration (SA), because it depends on the depth of the focus inside the sample. Spherical aberration happens due to a Refractive Index Mismatch, but it is negligible if the value of this parameter is near zero (i.e. when you are imaging very close to the coverslip).

If you acquire large volumes in the presence of SA, the deeper layers away from the coverslip will suffer from more aberration. The Point Spread Function (PSF) is therefore more distorted the deeper you focus into your sample. This distortion can be calculated and partially corrected but it is much better to avoid it during acquisition by using matching refractive indices.

To make the depth correction of the Theoretical Psf, the Huygens Software considers that the coverslip is placed at the bottom of the image, at lower Z coordinates (as in an inverted microscope). In the Nyquist Calculator you can obtain a PSF calculated for a given acquisition depth, and see how it is affected by a Refractive Index Mismatch.

Keywords: depth distance cover slip