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Cy3/Cy5 registration on fluoview 1000

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Jean-Pierre CLAMME-2

Cy3/Cy5 registration on fluoview 1000

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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

Martin Wessendorf-2

Re: Cy3/Cy5 registration on fluoview 1000

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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: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [hidden email]

Steffen Dietzel

Re: Cy3/Cy5 registration on fluoview 1000

In reply to this post by Jean-Pierre CLAMME-2

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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

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27, München-Großhadern

Guy Cox-2

Re: Cy3/Cy5 registration on fluoview 1000

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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:

Oshel, Philip Eugene

Re: Cy3/Cy5 registration on fluoview 1000

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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.
Phil

>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:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>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
>
>Mail room:
>Marchioninistr. 15, D-81377 München
>
>Building location:
>Marchioninistr. 27, München-Großhadern

--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
(989) 774-3576

Claire Brown

Re: Cy3/Cy5 registration on fluoview 1000

In reply to this post by Jean-Pierre CLAMME-2

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The ABRF light microscopy research group (www.abrf.org/lmrg) did a study on
channel registration and the details are published in Microscopy and
Microanalysis including how to accurately measure this shift:

http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=8332637

I am not surprised it is high when using cy5 many lenses are not well
corrected out in the far red. If you follow the protocol and take high
resolution z-stacks of tetraspek beads you should be able to precisely
calculate the shift between Cy3 and Cy5 for your lens.

Good luck!

Claire

mcammer

Re: Cy3/Cy5 registration on fluoview 1000

In reply to this post by Oshel, Philip Eugene

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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.)

Regards,

Michael

________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208 Cell: (914) 309-3270

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Philip Oshel
Sent: Thursday, November 29, 2012 9:54 AM
To: [hidden email]
Subject: Re: Cy3/Cy5 registration on fluoview 1000

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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.
Phil

>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:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>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
>
>Mail room:
>Marchioninistr. 15, D-81377 München
>
>Building location:
>Marchioninistr. 27, München-Großhadern

--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
(989) 774-3576

Claire Brown

Re: Cy3/Cy5 registration on fluoview 1000

In reply to this post by Jean-Pierre CLAMME-2

*****
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*****

I guess my link is not working for some people.

You can find a link to the paper at our lmrg website

http://www.abrf.org/lmrg

of the paper is:

Quality Assurance Testing for Modern Optical Imaging Systems
Robert F. Stacka1, Carol J. Baylesa2, Anne-Marie Girarda3, Karen Martina4,
Cynthia Opanskya5, Katherine Schulza5 and Richard W. Colea1 c1

Claire

Jean-Pierre CLAMME-2

Re: Cy3/Cy5 registration on fluoview 1000

In reply to this post by Jean-Pierre CLAMME-2

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Thank you all for your comments.

The objective I used is the 60x water UPLASAPO 1.2 NA which is what I
would consider to be one of their high end objectives for the confocal.
That's why I was a little surprised by the extend of the shift.

Also what software are you using to do a good Z registration ? We have
imaris and I found imaris unable to do subpixel registration (At my
previous place I was using Slidebook to do subpixel registration).

Thank you

JP

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Jean-Pierre CLAMME, PhD
Chief Scientist
Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: 1-760-696-9428

Guy Cox-2

Re: Cy3/Cy5 registration on fluoview 1000

In reply to this post by Oshel, Philip Eugene

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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!

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Philip Oshel
Sent: Friday, 30 November 2012 1:54 AM
To: [hidden email]
Subject: Re: Cy3/Cy5 registration on fluoview 1000

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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.
Phil

--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
(989) 774-3576

Steffen Dietzel

Re: Cy3/Cy5 registration on fluoview 1000

In reply to this post by Jean-Pierre CLAMME-2

*****
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On 29.11.2012 18:08, Jean-Pierre CLAMME wrote:
> Also what software are you using to do a good Z registration ? We have
> imaris and I found imaris unable to do subpixel registration (At my
> previous place I was using Slidebook to do subpixel registration).

The Sync Measure tool in ImageJ or Fiji was designed with that
application in mind. You need stacks of mentioned multicolorbeads (or
other signals that show up in the channels in questions) and the tool
will evaluate the gravity centers in the various color channels in
parallel.
For a short description see
http://rsbweb.nih.gov/ij/plugins/sync-windows.html

Steffen

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27, München-Großhadern