Pixel shift in unidirectional mode!

[Karuna Mittal]

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Dear Members,

I am a user of LSM 700 Ziess confocal microscope and I image the cell organelle centrosomes in human tissue samples on it. Stained with gamma tubulin (red channel) and nuclei with DAPI (blue channel). From past few days we are facing a problem as we can see there is vibration in our image and as we increase the scan speed it becomes worse. We have tried eliminating all the possible reasons like the vibration due to the continuous construction in building, building pressure sometimes causes it also, we worked with Zeiss tech to look at all other possible ways to correct it but still when it is unidirectional mode we see this pixel shift. We cannot move to bidirectional now as lot of our data is already acquired in unidirectional and all that is volume and not sure how much it will effect our data.

Please suggest how should we proceed. Your time and help is much appreciated.

Best Regards
Karuna Mittal

Sent from my iPhone

[PEARSON Matthew]

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Hi Karuna,

You didn't mention what kind of damping you have in place, assuming the microscope is on either a passive or active anti-vibration table and breadboard?  Again sorry to point out all the obvious things but are you sure the compressed air isolators are all pumped up.  Another thing to check is that there is nothing on the isolated breadboard that has contact with the table frame other than the isolators on which the breadboard sits.  The apparent worsening of the vibration with scan speed assuming it is not caused by aberrant movement somewhere on the microscope or within the scanhead could just be because you're starting to image at a sampling rate that is getting closer to the frequency of vibration.  We once had a vibration that came through a stage top piezo z insert, if this was removed it wasn't seen.  Turned out to be a dodgy fan in our camera causing it and the piezo happened to be more susceptible to it.

Thanks,

Matt

--
Matt Pearson
Microscopy Facility
MRC Human Genetics Unit
Institute of Genetics and Molecular Medicine (IGMM)
University of Edinburgh
Crewe Road
EH4 2XU




On 23 Nov 2017, at 14:45, [hidden email]<mailto:[hidden email]>
 wrote:

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Dear Members,

I am a user of LSM 700 Ziess confocal microscope and I image the cell organelle centrosomes in human tissue samples on it. Stained with gamma tubulin (red channel) and nuclei with DAPI (blue channel). From past few days we are facing a problem as we can see there is vibration in our image and as we increase the scan speed it becomes worse. We have tried eliminating all the possible reasons like the vibration due to the continuous construction in building, building pressure sometimes causes it also, we worked with Zeiss tech to look at all other possible ways to correct it but still when it is unidirectional mode we see this pixel shift. We cannot move to bidirectional now as lot of our data is already acquired in unidirectional and all that is volume and not sure how much it will effect our data.

Please suggest how should we proceed. Your time and help is much appreciated.

Best Regards
Karuna Mittal

Sent from my iPhone


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The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

[Jacqueline Ross]

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Hi Karuna,

Basic question I know but do you see zig-zags or pixel shift? Could you please post an example image? Also, which lens are you using and are you using a zoom?

Kind regards,

Jacqui

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Friday, 24 November 2017 3:46 a.m.
To: [hidden email]
Subject: Pixel shift in unidirectional mode!

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear Members,

I am a user of LSM 700 Ziess confocal microscope and I image the cell organelle centrosomes in human tissue samples on it. Stained with gamma tubulin (red channel) and nuclei with DAPI (blue channel). From past few days we are facing a problem as we can see there is vibration in our image and as we increase the scan speed it becomes worse. We have tried eliminating all the possible reasons like the vibration due to the continuous construction in building, building pressure sometimes causes it also, we worked with Zeiss tech to look at all other possible ways to correct it but still when it is unidirectional mode we see this pixel shift. We cannot move to bidirectional now as lot of our data is already acquired in unidirectional and all that is volume and not sure how much it will effect our data.

Please suggest how should we proceed. Your time and help is much appreciated.

Best Regards
Karuna Mittal

Sent from my iPhone

[Moulding, Dale]

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Hi Karuna,

I've had this problem on our 880. As Matt said, check for local vibrations. Ours turned out to be the fan in the HXP lamp unit. We moved this off the air table and no more wobbly images.
It took a couple of Zeiss visits before we uncovered the culprit.

Cheers

Dale



Dale Moulding PhD
ICH Light Microscopy Facility,
Room W2.06, BDRC office,
UCL Great Ormond Street Institute of Child Health,
30 Guilford St,
London WC1N 1EH
Mob [phone]
http://www.ucl.ac.uk/ich/core-scientific-facilities-centres/confocal-microscopy



-------- Original message --------
From: [hidden email]
Date: 23/11/2017 14:47 (GMT+00:00)
To: [hidden email]
Subject: Pixel shift in unidirectional mode!

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Members,

I am a user of LSM 700 Ziess confocal microscope and I image the cell organelle centrosomes in human tissue samples on it. Stained with gamma tubulin (red channel) and nuclei with DAPI (blue channel). From past few days we are facing a problem as we can see there is vibration in our image and as we increase the scan speed it becomes worse. We have tried eliminating all the possible reasons like the vibration due to the continuous construction in building, building pressure sometimes causes it also, we worked with Zeiss tech to look at all other possible ways to correct it but still when it is unidirectional mode we see this pixel shift. We cannot move to bidirectional now as lot of our data is already acquired in unidirectional and all that is volume and not sure how much it will effect our data.

Please suggest how should we proceed. Your time and help is much appreciated.

Best Regards
Karuna Mittal

Sent from my iPhone

[Joseph Mckenna]

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Hello Karunta,

We had a similar issue with our 880. It was caused by the water bottle for
the temperature chamber being left on the breadboard. Only noticed it when
we were using the 100X objective and digital zoom. Hope this helps!

Cheers,

Joe

On 23 November 2017 at 20:37, Moulding, Dale <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Karuna,
>
> I've had this problem on our 880. As Matt said, check for local
> vibrations. Ours turned out to be the fan in the HXP lamp unit. We moved
> this off the air table and no more wobbly images.
> It took a couple of Zeiss visits before we uncovered the culprit.
>
> Cheers
>
> Dale
>
>
>
> Dale Moulding PhD
> ICH Light Microscopy Facility,
> Room W2.06, BDRC office,
> UCL Great Ormond Street Institute of Child Health,
> 30 Guilford St,
> London WC1N 1EH
> Mob [phone]
> http://www.ucl.ac.uk/ich/core-scientific-facilities-centres/
> confocal-microscopy
>
>
>
> -------- Original message --------
> From: [hidden email]
> Date: 23/11/2017 14:47 (GMT+00:00)
> To: [hidden email]
> Subject: Pixel shift in unidirectional mode!
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Members,
>
> I am a user of LSM 700 Ziess confocal microscope and I image the cell
> organelle centrosomes in human tissue samples on it. Stained with gamma
> tubulin (red channel) and nuclei with DAPI (blue channel). From past few
> days we are facing a problem as we can see there is vibration in our image
> and as we increase the scan speed it becomes worse. We have tried
> eliminating all the possible reasons like the vibration due to the
> continuous construction in building, building pressure sometimes causes it
> also, we worked with Zeiss tech to look at all other possible ways to
> correct it but still when it is unidirectional mode we see this pixel
> shift. We cannot move to bidirectional now as lot of our data is already
> acquired in unidirectional and all that is volume and not sure how much it
> will effect our data.
>
> Please suggest how should we proceed. Your time and help is much
> appreciated.
>
> Best Regards
> Karuna Mittal
>
> Sent from my iPhone
>

--
*Dr. Joseph F McKenna*

*Postdoctoral researcher in plant cell biology*

Oxford Brookes University
Sinclair Annex 1-01,
Gypsy lane,
Oxford, OX3 0BP, UK

Tel: [phone]

Twitter: @Joey_McK

http://oxfordbrookesbioimaging.weebly.com/

[Karuna Mittal]

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*****

Dear All,

Thanks a lot for the response. I am trying to sum up the possible answers I
have from all the responses:

1. Our damping system is the electronic self leveling isolation system.
2. We checked for the possible leakage in the pumps and cylinder -
everything is fine.
3. The board is not in touch with anything except the microscope -(no
wires, no lamp).
4. Our stage is inbuild and have not changed it since past 4 years.
5. I am using 63 X and will attach a sample image today.
6. The Zeiss tech has checked the calibrations properly a day before this
problem started.

I will attach the sample image in my next email. Thanks again for all the
responses.

Best Regards
Karuna Mittal

On Fri, Nov 24, 2017 at 11:08 AM, Joseph Mckenna <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello Karunta,
>
> We had a similar issue with our 880. It was caused by the water bottle for
> the temperature chamber being left on the breadboard. Only noticed it when
> we were using the 100X objective and digital zoom. Hope this helps!
>
> Cheers,
>
> Joe
>
> On 23 November 2017 at 20:37, Moulding, Dale <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi Karuna,
> >
> > I've had this problem on our 880. As Matt said, check for local
> > vibrations. Ours turned out to be the fan in the HXP lamp unit. We moved
> > this off the air table and no more wobbly images.
> > It took a couple of Zeiss visits before we uncovered the culprit.
> >
> > Cheers
> >
> > Dale
> >
> >
> >
> > Dale Moulding PhD
> > ICH Light Microscopy Facility,
> > Room W2.06, BDRC office,
> > UCL Great Ormond Street Institute of Child Health,
> > 30 Guilford St,
> > London WC1N 1EH
> > Mob [phone]
> > http://www.ucl.ac.uk/ich/core-scientific-facilities-centres/
> > confocal-microscopy
> >
> >
> >
> > -------- Original message --------
> > From: [hidden email]
> > Date: 23/11/2017 14:47 (GMT+00:00)
> > To: [hidden email]
> > Subject: Pixel shift in unidirectional mode!
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear Members,
> >
> > I am a user of LSM 700 Ziess confocal microscope and I image the cell
> > organelle centrosomes in human tissue samples on it. Stained with gamma
> > tubulin (red channel) and nuclei with DAPI (blue channel). From past few
> > days we are facing a problem as we can see there is vibration in our
> image
> > and as we increase the scan speed it becomes worse. We have tried
> > eliminating all the possible reasons like the vibration due to the
> > continuous construction in building, building pressure sometimes causes
> it
> > also, we worked with Zeiss tech to look at all other possible ways to
> > correct it but still when it is unidirectional mode we see this pixel
> > shift. We cannot move to bidirectional now as lot of our data is already
> > acquired in unidirectional and all that is volume and not sure how much
> it
> > will effect our data.
> >
> > Please suggest how should we proceed. Your time and help is much
> > appreciated.
> >
> > Best Regards
> > Karuna Mittal
> >
> > Sent from my iPhone
> >
>
>
>
> --
> *Dr. Joseph F McKenna*
>
> *Postdoctoral researcher in plant cell biology*
>
> Oxford Brookes University
> Sinclair Annex 1-01,
> Gypsy lane,
> Oxford, OX3 0BP, UK
>
> Tel: [phone]
>
> Twitter: @Joey_McK
>
> http://oxfordbrookesbioimaging.weebly.com/
>

--
Best Regards
Karuna Mittal

[Guy Hagen]

*****
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*****

Dear Colleagues:

Don't forget the possibility that there could actually be something wrong
with the microscope. The scanner could be broken, no matter how many times
the vendor tells you this is impossible (it happened to me on a Leica SP5).
When this happened the scan was stable at low speeds, but the image started
to get badly messed up at highest speeds. Vibrations, if present, will
probably be detectable if you put your hand on the microscope stage. Also a
vibration should probably look like a constant effect throughout the image,
not something that comes and goes.

Guy Hagen

On Mon, Nov 27, 2017 at 4:01 AM, Karuna Mittal <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear All,
>
> Thanks a lot for the response. I am trying to sum up the possible answers I
> have from all the responses:
>
> 1. Our damping system is the electronic self leveling isolation system.
> 2. We checked for the possible leakage in the pumps and cylinder -
> everything is fine.
> 3. The board is not in touch with anything except the microscope -(no
> wires, no lamp).
> 4. Our stage is inbuild and have not changed it since past 4 years.
> 5. I am using 63 X and will attach a sample image today.
> 6. The Zeiss tech has checked the calibrations properly a day before this
> problem started.
>
> I will attach the sample image in my next email. Thanks again for all the
> responses.
>
> Best Regards
> Karuna Mittal
>
> On Fri, Nov 24, 2017 at 11:08 AM, Joseph Mckenna <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hello Karunta,
> >
> > We had a similar issue with our 880. It was caused by the water bottle
> for
> > the temperature chamber being left on the breadboard. Only noticed it
> when
> > we were using the 100X objective and digital zoom. Hope this helps!
> >
> > Cheers,
> >
> > Joe
> >
> > On 23 November 2017 at 20:37, Moulding, Dale <[hidden email]>
> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > Hi Karuna,
> > >
> > > I've had this problem on our 880. As Matt said, check for local
> > > vibrations. Ours turned out to be the fan in the HXP lamp unit. We
> moved
> > > this off the air table and no more wobbly images.
> > > It took a couple of Zeiss visits before we uncovered the culprit.
> > >
> > > Cheers
> > >
> > > Dale
> > >
> > >
> > >
> > > Dale Moulding PhD
> > > ICH Light Microscopy Facility,
> > > Room W2.06, BDRC office,
> > > UCL Great Ormond Street Institute of Child Health,
> > > 30 Guilford St,
> > > London WC1N 1EH
> > > Mob [phone]
> > > http://www.ucl.ac.uk/ich/core-scientific-facilities-centres/
> > > confocal-microscopy
> > >
> > >
> > >
> > > -------- Original message --------
> > > From: [hidden email]
> > > Date: 23/11/2017 14:47 (GMT+00:00)
> > > To: [hidden email]
> > > Subject: Pixel shift in unidirectional mode!
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > Dear Members,
> > >
> > > I am a user of LSM 700 Ziess confocal microscope and I image the cell
> > > organelle centrosomes in human tissue samples on it. Stained with gamma
> > > tubulin (red channel) and nuclei with DAPI (blue channel). From past
> few
> > > days we are facing a problem as we can see there is vibration in our
> > image
> > > and as we increase the scan speed it becomes worse. We have tried
> > > eliminating all the possible reasons like the vibration due to the
> > > continuous construction in building, building pressure sometimes causes
> > it
> > > also, we worked with Zeiss tech to look at all other possible ways to
> > > correct it but still when it is unidirectional mode we see this pixel
> > > shift. We cannot move to bidirectional now as lot of our data is
> already
> > > acquired in unidirectional and all that is volume and not sure how much
> > it
> > > will effect our data.
> > >
> > > Please suggest how should we proceed. Your time and help is much
> > > appreciated.
> > >
> > > Best Regards
> > > Karuna Mittal
> > >
> > > Sent from my iPhone
> > >
> >
> >
> >
> > --
> > *Dr. Joseph F McKenna*
> >
> > *Postdoctoral researcher in plant cell biology*
> >
> > Oxford Brookes University
> > Sinclair Annex 1-01,
> > Gypsy lane,
> > Oxford, OX3 0BP, UK
> >
> > Tel: [phone]
> >
> > Twitter: @Joey_McK
> >
> > http://oxfordbrookesbioimaging.weebly.com/
> >
>
>
>
> --
> Best Regards
> Karuna Mittal
>