Re: Correction of Z-axis distortion- request for opinion

Posted by Mark Cannell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Correction-of-Z-axis-distortion-request-for-opinion-tp2580156p2580607.html

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
> [hidden email]
> Bradford University
>