http://confocal-microscopy-list.275.s1.nabble.com/Cy3-Cy5-registration-on-fluoview-1000-tp7579371p7579387.html
Well, that is going to depend on your Stokes shift. In widefield fluorescence it's only the emission that counts, but as you say in confocal both come into the picture. Many dyes (eg FITC) have a quite small Stokes shift so if you are exciting and detecting at optimal values the excursion between the two wavelengths should not be very great. However that is not the case for all dyes (wild type GFP for example) and also with fixed laser lines you may be exciting way off the optimum. Any focus shift between excitation & emission will spoil the confocality - the image of the spot will not be on the pinhole. Your 'best focus' (brightest image) will be somewhere between the two foci. It's the difference between the two 'best' positions that will make the optical sections appear out of step.
The only thing you can do is use the most highly corrected lens you can get and stay within the range of its correction. If you are using a 405 laser, for example, use a 'VC' (violet corrected) lens. With CY5 you are probably going outside the correction range at the red end. There may be lenses designed to cope with this. I recall Olympus announcing a 'super apochromat' a few years ago that had very small divergence over a very wide range. I've heard (but have not verified) that Nikon make lenses with four 'crossing points'. What this all emphasizes is that we need more information from the manufacturers. They seem to think this would harm them, but in fact it's probably the opposite. Researchers would realize that they need different lenses for different experiments and therefore buy more of these very expensive objectives!
>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
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
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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:
>>
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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