Well, the stretching should be 1.518 / 1.457 if simple RI difference is the cause - ie about 4%.  If you are seeing more than that you'd better look elsewhere - maybe a dodgy Z drive?  I suppose changes in SA with depth would add to the discrepancy, but that depends a lot on how great a depth you are measuring through.  Also, though it's true oil and coverslip should have the same RI, oil changes with temperature whereas glass doesn't (much) so if you  can use #1.5 and still have enough working distance it might be better.   

                                       Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Michal Gdula
Sent: Friday, 3 April 2009 9:21 PM
To: [log in to unmask]
Subject: Correction of Z-axis distortion- request for opinion

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

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