Hi

Yes, using NA objectives with the wrong mounting medium leads to big 
problems for quantification...
Why not use a glycerol objective? Zeiss now has a high NA (1.3 I think) 
glycerol objective available. If you go tis route you will need to pay 
attention to coverslip correction collar setting so adding a few 
~0.5-1.0 um beads to your mountant might be useful for visual check on 
spherical aberration correction.

Regards Mark Cannell





 think)Michal Gdula wrote:
> 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
> [log in to unmask]
> Bradford University
>