Posted by
Michal Gdula-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Correction-of-Z-axis-distortion-request-for-opinion-tp2580156p2593142.html
Thank you for your response!
I am almost sure that our cryostate is not extremly precise, especially with
thicker cryosections. I can see this because I need to make from 90 up to 140
2d Scans to get whole cryosection (0.2 um of step between the optical
sections).
What should I expect after difference of Refractive index (RI) between
tetraspeck beads and a medium? I know the ideas behind refraction, and
breaking of the light beam when light passes through substances with
different RI. But since I want to scan surface of the bead?
Notabene when I scan beads covered with oil (I prepared different slides
mounted with vectashield, oil and water) They appeared to be 1um smaller in
xy plane... Do you think that different RI can have anything in common with
this? Is it possible that they are soluble in oil? - some of them looked damaged
Kind regards,
Michal Gdula
Subject: Re: Correction of Z-axis distortion- request for opinion
From: Jeremy Adler <
[hidden email]>
Reply-To: Confocal Microscopy List
<
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Date: Fri, 3 Apr 2009 16:08:25 +0200
Content-Type: text/plain
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