Posted by
Michal Gdula-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Correction-of-Z-axis-distortion-request-for-opinion-tp2580156p2593509.html
Thank you for your answer!
I am aware that people estimate resolution of confocal microscope to 200-
300nm in xy and 600-700nm in z-axis. I have lately measured 4um Tetraspeck
beads and they appeared to be more than 1um longer in z-axis when mounted
with Vectashield, even more streched with water, and less when mounted
with immersion oil, which would tell that it is not only about resolution.
As far as I understand stretching caused by lower resolution in z-axis and by
some other phenomena can be removed by deconvolution after measuring the
point spread function (PSF).
The points that are below resolution of the confocal microscope will have the
PSF-like appearence. I think that this may be the reason of stretching caused
by lower resolution, in this case we should expect stretching of particular
points 300-400 nm (300-400nm from the top and bottom part of z-ahis would
give 600-800nm extension). I have stretching of more than 500 um (more than
1um i diameter of the spheres, which also would tell that there shoould be
some other factor besides resolution.
Do you think tha deconvolution is able to remove effects of spherical
abberration? Soonner or later I will deconvolute my scans, I bought beads so
far.
So as to software which one do you think is the best? My co-workers say that
Amira is not user friendly, but it gives good results for surface rendering. Do
you think that deconvolution with plugins of imagej or with freeware
Imagesurfer is not reliable?
I am relatively new to all this topics and I would be gratefull for any remarks.
Best wishes,
Michal Gdula
Subject: Re: Correction of Z-axis distortion- request for opinion
From: Armstrong, Brian
Reply-To: Confocal Microscopy List
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Date: Fri, 3 Apr 2009 09:30:20 -0700
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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-Imaging/Pages/default.aspx