http://confocal-microscopy-list.275.s1.nabble.com/Cy3-Cy5-registration-on-fluoview-1000-tp7579371p7579382.html
This is normal and is different for each objective. Any time we get a new objective for we check for registration in the Z axis. This especially is an issue if you want to calculate colocalization of small puncta. The Z axis needs to be offset to correct for this aberration.
We have also found this to be an issue with TIRF and widefield. I have not noticed issues in the green-red range, but adding a channel below 500 nm or above 640 nm requires focal offsets above 200 nm depending on the objective and color (we have found this with Olympus 60X, Olympus 150X and Nikon 100X TIRF objectives). When taking sequential images a focus offset may be programmed or with the DualView we sandwiched a lens in with one of the emission filters to compensate. (I guess this could also be done with a confocal.)
How much of the focus shift is likely due to chromatic aberrations of the excitation light, and how much to the emitted light?
The colors may be chosen so that the design foci are the same for 2 excitation colors, but then they'd be wrong for the emitted light.
>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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