Re: Cy3/Cy5 registration on fluoview 1000

Posted by Martin Wessendorf-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cy3-Cy5-registration-on-fluoview-1000-tp7579371p7579372.html

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Dear Jean-Pierre--

On 11/28/2012 4:26 PM, Jean-Pierre CLAMME wrote:

> 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.

Not sure it can be fixed but you may be able to get a different, better,
lens.

It's not uncommon to have some misregistration in the z-axis between
colors and in my experience the amount of misregistration will be
different among different objectives--it's an example of axial chromatic
aberration.

However, before you do anything else, clean your lens.  Lenses that have
mounting medium, glycerol or other substances other than the proper
immersion oil on them, can have markedly worse chromatic aberration.

Assuming it's clean, I'd quantify your chromatic aberration.  You can
test for the differences across different wavelengths by performing a
line-scan of a mirror--I'd do a simultaneous-excitation line-scan down
the z-axis and detect the reflectance of the different laser lines.
(Make sure you set up the confocal's filters to detect the laser lines
you're using, and turn down the voltage on the PMTs to under 300 v.) The
maximums of reflectance should all occur at roughly the same place in
the z-axis--with the closer they are, the better.

I'd then talk to the Olympus vendor and have them bring out several
different objectives to test.  You may find that some are better than
the one you have.  Depending on your negotiation skills and how bad
yours looks compared to the others, you may want to see about getting a
replacement.

Good luck!

Martin

--
Martin Wessendorf, Ph.D.                   office: [phone]
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