I would also consider switching to a mounting medium with correct refractive 
index (=same RI as immersion oil)  2,2-thiodiethanol comes to mind.  See 
Staudt, 2007.  A 500 ml bottle can be had for ~$30.

1.	Staudt, T., M.C. Lang, R. Medda, J. Engelhardt, and S.W. Hell, 2,2'-
thiodiethanol: a new water soluble mounting medium for high resolution optical 
microscopy. Microsc Res Tech, 2007. 70(1): p. 1-9.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University
 
On Fri, 3 Apr 2009 09:30:20 -0700, Armstrong, Brian <[log in to unmask]> 
wrote:

>Michael, to be clear, your Z resolution will be approximately 3 times
>worse than your X,Y. Therefore, your Z will appear stretched. To address
>this you will need to perform deconvolution on your Z-Stacks. I would
>not worry too much the about RI of your oil and glycerin based mountant,
>as this is a very common procedure to use an oil imm objective on a
>glycerin based mounted sample and a glycerin imm objective will run
>around $10K US. I would however insist on a 1.5 coverslip that is 170um
>because your lens is designed for the angle created by that thickness
>and it is easy and cheap to use the right coverglass. After
>deconvolution I would recommend you use image software designed for
>accurate 3D visualization such as Imaris or Amira or Volocity.
>Cheers, 
>
>Brian D Armstrong PhD
>Light Microscopy Core Manager
>Beckman Research Institute
>City of Hope
>Dept of Neuroscience
>1450 E Duarte Rd
>Duarte, CA 91010
>626-256-4673 x62872
>http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
>ing/Pages/default.aspx
>
>-----Original Message-----
>From: Confocal Microscopy List 
[mailto:[log in to unmask]]
>On Behalf Of Michal Gdula
>Sent: Friday, April 03, 2009 4:21 AM
>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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