Print

Focal shift correction


In the presence of a Refractive Index Mismatch the axial movement of the microscope stage is not followed by the focus. This causes a geometrical distortion of the acquired dataset. See the Fishtank Effect.

The Huygens Software considers the refractive index mismatch to
adapt the calculated theoretical Point Spread Function (PSF) when Doing Deconvolution, but does not correct this geometrical distortion afterwards. This can be done simply by manually changing the Sampling Distance later. Do not
do it before deconvolution when using a Theoretical Psf, because its
calculation will take the mismatch into account and distort it accordingly. If the image is corrected but the PSF is not, the deconvolution will
return bad results.

A more difficult case happens when you use an air lens corrected to work as a, let's say, glycerol lens. See Air Lens Correction for details.

When using an Experimental Psf there is no need to modify it, because
if it was measured using the same conditions the distortion is also
present there. What matters at the end is that both the PSF and the
image have the same distortion. Correcting the image geometry
afterwards can be seen as simply a matter of data representation. In
this case, though, the correction can be applied before deconvolution
to both the image and the PSF, by simply setting the Z step to the
real value.

Other problems


Remind that on top of this geometrical distortion there is also
the effect of the Spherical Aberration. Its effect is to elongate the
PSF intrinsically, and therefore you have two "overlapped"
elongations. The intrinsic elongation will also affect the image in a
way that deconvolution will manage to reduce, but it is always a
better idea to minimize the mismatch during acquisition, e.g. using a
water lens instead of an oil one. More details in Mismatch Distorts Psf.