Dear Confocalists,

I need opinion of somebody experienced in correcting spherical aberrations to 
asses my approach.
I am using Carl Zeiss LSM 510M microscope (with 63x apochromat objective, 
NA=1.4) and I noticed significant stretching in Z-axis. 
I found out from the literature that most probably it happens because of 
refraction index mismatch between immersion oil (Zeiss Immersol 518F 
ne=1.518) and mounting medium (Vectashield, Vector ne=1.457) or between 
refraction index of the specimen (20um thin skin cryosection processed 
according to FISH procedure). I have also discovered lately that we are 
supplied with the cover slips no. 1 with the thickness 130-160 um, whereas 
the optimal is 170um, however it is written in the Zeiss manual that immerse 
oil objective should be not sensitive to the differences in cover slip thikness.
One of my aims is to measure  distances between FISH signals in 3D and I 
have to be  as accurate as possible. 
So far I  have done chromatic shift correction using measurements of 
differences between centroids of 0.5 um Tetraspeck beads in different 
channels.
I am planning to measure z-axis distortion scanning 4um TetraSpeck beads 
and then calculate the differences between dimensions in the x,y and z-axis. I 
will prepare 2 slides with beads on the microscope slide and the second with 
the bead on the cover slip to check the difference of the aberration in 
different depths. This data will serve me to correct z-coordinates of FISH 
signals – I will calculate average ratio x-axis/z-axis and multiply the z-
coordinates.
I know that some scientists use some more sophisticated ways for correction 
of z-axis distortion. I would be grateful for any opinions and remarks.

Best wishes,

Michal Gdula
Research PhD student
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Bradford University