Posted by
Jeremy Adler-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Correction-of-Z-axis-distortion-request-for-opinion-tp2580156p2580914.html
A minor note of caution about using 4um microspheres to measure Z axis
dimensions - the microspheres have a refractive index(1.473)which differs
from your medium.
Why not use the thickness of your section as a reference, over a few
sections it should average out to 20um. Begs the question as to how the
cryostat is calibrated.
Dr Jeremy Adler
F451a
Cell Biologi
Wenner-Gren Inst.
The Arhenius Lab
Stockholm University
S-106 91 Stockholm
Sweden
tel +46 (0)8 16 2759
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On
Behalf Of Michal Gdula
Sent: den 3 april 2009 12:21
To:
[hidden email]
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
[hidden email]
Bradford University