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 <[log in to unmask]> 
Reply-To: Confocal Microscopy List 
<[log in to unmask]> 
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