http://confocal-microscopy-list.275.s1.nabble.com/Cy3-Cy5-registration-on-fluoview-1000-tp7579371p7579380.html
>Actually, Olympus is one of the very few
>companies who have released the curves of
>chromatic aberration of at least some of their
>objectives. Since this is, these days,
>absolutely critical information we should be
>putting pressure on all manufacturers to provide
>this as a datasheet with their objectives. They
>all have the data! To claim a lens as an
>achromat it needs to have two crossing points
>(where foci are the same) which are usually in
>the blue (~480nm) and the red (~650nm). The
>deviation between and beyond these points is not
>specified. It could easily be in the 430nm
>region at the midpoint between these
>wavelengths. However, I doubt if JP is using
>quite such a basic lens. A fluorite lens has
>the same two crossing points but a much lower
>deviation and should not give the depth
>difference JP sees. However, without a curve
>one cannot say much. An apochromat lens has
>three crossing points, and while these are
>traditionally red, green and blue the basic
>design principles allow any 3 points, and many
>makers now offer VC objectives corrected into
>the violet. There will still be deviations
>between the crossing points but they absolutely
>should not allow the focus shift JP is seeing.
>
>When we buy a car it could cost as little as
>just one objective, and you would get quite a
>supercar for the price of a complete microscope.
>The car would come with comprehensive
>specification sheet. Why doesn't the microscope?
>
> Guy
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:
[hidden email]] On
>Behalf Of Steffen Dietzel
>Sent: Thursday, 29 November 2012 9:37 PM
>To:
[hidden email]
>Subject: Re: Cy3/Cy5 registration on fluoview 1000
>
>*****
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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:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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>>
>> 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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