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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