Posted by
Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cy3-Cy5-registration-on-fluoview-1000-tp7579371p7579374.html
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Hi Jean Pierre,
although .43 microns is a bit much, (if you were using oil immersion)
the phenomenon in general is normal. As Martin wrote, the chromatic
aberration is mainly caused by the objective. For example, we would find
that green (520 nm) and deep red (670 nm) were aligned pretty well, but
the orange channel might be off about 200 - 300 nm in z (and up to 70 nm
in xy). Color correction by the microscope optics works only so well,
and for a certain range. It's one of those things you would like to know
about your objective but the companies won't tell you. It also might
change over time. When using objectives in the infrared, I measured up
to 10 microns aberration in z.
For testing, we used fluorescent multicolor beads and compared intensity
centers in ImageJ. The beads don't need to be subresolution for this
purpose, 0.5 µm TetraSpeck (Molecular Probes) dried on a glass surface
do the trick, there are probably others. Be sure to embed them in the
same mounting medium you use for your samples. And you might want to
attach them to the coverslip *and* the slide and compare both, in case
it gets worse with depth.
Regards
Steffen
On 28.11.2012 23:26, Jean-Pierre CLAMME wrote:
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> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hi,
>
> I noticed that on our fluoview 1000, the Cy3 and Cy5 channels are off by
> one slice when doing (0.43 um/slice Z stacks). I can correct it by
> software but I was wondering if anyone else see that on their instrument
> and If I should /can get it fixed.
>
> Thank you
>
> JP
>
--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy
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