Olympus FV1000 horizontal lines problem

[mcammer]

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Colleagues have a new Olympus FV1000 with autofocus and are having difficulties with it and asked our advice.  Based on images they sent us, one example posted at https://www.flickr.com/photos/mcammer/14273268488/ , I told them that I thought that the problem of horizontal lines was adjustments made by the autofocus during the scan.  (Another possibility we entertained was laser fluctuation and still another would be sudden temperature change by the incubator unit.)

Questions:

1.       Do you think we have diagnosed the problem correctly?

2.       Do other people have this problem?

3.       Is there a software setting in the FV1000 software to tell it to check and set the focus only at the beginning of a scan and at the end of a scan with autofocus polling and adjusting suspended during the scan?


Thank you!!
===========================================================================
Michael Cammer, Microscopy Core & Dustin Lab , Skirball Institute, NYU Langone Medical Center
Cell:  914-309-3270   Lab: 212-263-3208
http://ocs.med.nyu.edu/microscopy & http://www.med.nyu.edu/skirball-lab/dustinlab/

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[Martin Wessendorf-2]

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Dear Michael--

On 6/19/2014 12:12 PM, Cammer, Michael wrote:
> Colleagues have a new Olympus FV1000 with autofocus and are having difficulties with it and asked our advice.  Based on images they sent us, one example posted at https://www.flickr.com/photos/mcammer/14273268488/ , I told them that I thought that the problem of horizontal lines was adjustments made by the autofocus during the scan.  (Another possibility we entertained was laser fluctuation and still another would be sudden temperature change by the incubator unit.)
>
> Questions:
>
> 1.       Do you think we have diagnosed the problem correctly?
I've seen lines similar to this when focused close to the surface of the
specimen and when the air table isn't damping vibration properly.  Any
chance that that's it?

For us, the issue turned out to be a sticky valve on the air table.

Good luck--

Martin Wessendorf

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Sathya]

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Dear Michael,
As Martin has mentioned, the vibration might be one of the causes. We had similar problems with our Nikon A1R recently and one of the legs was jammed to the top and was sending vibrations to the images. We were lucky to have Craig Brideau and he figured out the problem to be with a jammed leg of the anti vibration table. We were also checking the air flow if it is pushing in a wind gust to the microscope and we added a temporary screen near the vent to rule out that possibility. Our Director came up with a suggestion of electrical noise due to faulty grounding which he had experienced in his previous lab. Another possibility is the X Y galvo itself as we had with our C1si and the X galvo got jammed or was not moving properly. Again Craig figured it out of what was happening with the XY galvo scan head and we fixed the problem. A simple test if it is associated with the table is by tapping the table top or the microscope stage and if the lines appears and disappears then it is the table issue. The galvo problem will also occur due to the power supply to the controller and make sure to rule out that possibility by adding a UPS with automatic voltage regulator and surge protector.
 
Good luck
 
Sathya Srinivasan
Manager
RUN Microscopy Facility
(www.ucalgary.ca/runcore)
University of Calgary
Room 126 HMRB
3330 Hospital Drive, NW
Calgary, AB, Canada
 

> Date: Thu, 19 Jun 2014 12:19:53 -0500
> From: [hidden email]
> Subject: Re: Olympus FV1000 horizontal lines problem
> To: [hidden email]
>
> *****
> 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 Michael--
>
> On 6/19/2014 12:12 PM, Cammer, Michael wrote:
> > Colleagues have a new Olympus FV1000 with autofocus and are having difficulties with it and asked our advice.  Based on images they sent us, one example posted at https://www.flickr.com/photos/mcammer/14273268488/ , I told them that I thought that the problem of horizontal lines was adjustments made by the autofocus during the scan.  (Another possibility we entertained was laser fluctuation and still another would be sudden temperature change by the incubator unit.)
> >
> > Questions:
> >
> > 1.       Do you think we have diagnosed the problem correctly?
> I've seen lines similar to this when focused close to the surface of the
> specimen and when the air table isn't damping vibration properly.  Any
> chance that that's it?
>
> For us, the issue turned out to be a sticky valve on the air table.
>
> Good luck--
>
> Martin Wessendorf
>
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]

     

[Rosemary.White]

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Agree, it could be vibration. We saw similar recently (though stripes were
more even), very puzzled until we realised the air table had been switched
off...

cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 20/06/14 7:51 AM, "Sathya Srinivasan" <[hidden email]> wrote:

>*****
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>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Dear Michael,
>As Martin has mentioned, the vibration might be one of the causes. We had
>similar problems with our Nikon A1R recently and one of the legs was
>jammed to the top and was sending vibrations to the images. We were lucky
>to have Craig Brideau and he figured out the problem to be with a jammed
>leg of the anti vibration table. We were also checking the air flow if it
>is pushing in a wind gust to the microscope and we added a temporary
>screen near the vent to rule out that possibility. Our Director came up
>with a suggestion of electrical noise due to faulty grounding which he
>had experienced in his previous lab. Another possibility is the X Y galvo
>itself as we had with our C1si and the X galvo got jammed or was not
>moving properly. Again Craig figured it out of what was happening with
>the XY galvo scan head and we fixed the problem. A simple test if it is
>associated with the table is by tapping the table top or the microscope
>stage and if the lines appears and disappears then it is the table issue.
>The galvo problem will also occur due to the power supply to the
>controller and make sure to rule out that possibility by adding a UPS
>with automatic voltage regulator and surge protector.
>
>Good luck
>
>Sathya Srinivasan
>Manager
>RUN Microscopy Facility
>(www.ucalgary.ca/runcore)
>University of Calgary
>Room 126 HMRB
>3330 Hospital Drive, NW
>Calgary, AB, Canada
>
>> Date: Thu, 19 Jun 2014 12:19:53 -0500
>> From: [hidden email]
>> Subject: Re: Olympus FV1000 horizontal lines problem
>> To: [hidden email]
>>
>> *****
>> 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 Michael--
>>
>> On 6/19/2014 12:12 PM, Cammer, Michael wrote:
>> > Colleagues have a new Olympus FV1000 with autofocus and are having
>>difficulties with it and asked our advice.  Based on images they sent
>>us, one example posted at
>>https://www.flickr.com/photos/mcammer/14273268488/ , I told them that I
>>thought that the problem of horizontal lines was adjustments made by the
>>autofocus during the scan.  (Another possibility we entertained was
>>laser fluctuation and still another would be sudden temperature change
>>by the incubator unit.)
>> >
>> > Questions:
>> >
>> > 1.       Do you think we have diagnosed the problem correctly?
>> I've seen lines similar to this when focused close to the surface of
>>the
>> specimen and when the air table isn't damping vibration properly.  Any
>> chance that that's it?
>>
>> For us, the issue turned out to be a sticky valve on the air table.
>>
>> Good luck--
>>
>> Martin Wessendorf
>>
>> --
>> Martin Wessendorf, Ph.D.                   office: [phone]
>> Assoc Prof, Dept Neuroscience                 lab: [phone]
>> University of Minnesota             Preferred FAX: [phone]
>> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
>> Minneapolis, MN  55455                    e-mail: [hidden email]
>    

[Craig Brideau]

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*****

Yeah, in the case Sathya mentioned, the valve on one of the legs was set
too high and the table was at the extent of its travel. When the leg is
jammed at the top of its extent of motion it conducts vibration in from the
frame and floor. I adjusted the valve and leaned on the table to free the
stuck leg and the vibration instantly disappeared. It took us a while to
figure out what was going on because of course the jammed leg was at the
back of the table and not easily accessed. Make sure your height control
valves are set for the middle of the range and that no leg is either
bottomed out or overextended.

Craig


On Thu, Jun 19, 2014 at 4:14 PM, <[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.
> *****
>
> Agree, it could be vibration. We saw similar recently (though stripes were
> more even), very puzzled until we realised the air table had been switched
> off...
>
> cheers,
> Rosemary
>
> Dr Rosemary White
> CSIRO Plant Industry
> GPO Box 1600
> Canberra, ACT 2601
> Australia
>
> T 61 2 6246 5475
> F 61 2 6246 5334
> E [hidden email]
>
>
> On 20/06/14 7:51 AM, "Sathya Srinivasan" <[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 Michael,
> >As Martin has mentioned, the vibration might be one of the causes. We had
> >similar problems with our Nikon A1R recently and one of the legs was
> >jammed to the top and was sending vibrations to the images. We were lucky
> >to have Craig Brideau and he figured out the problem to be with a jammed
> >leg of the anti vibration table. We were also checking the air flow if it
> >is pushing in a wind gust to the microscope and we added a temporary
> >screen near the vent to rule out that possibility. Our Director came up
> >with a suggestion of electrical noise due to faulty grounding which he
> >had experienced in his previous lab. Another possibility is the X Y galvo
> >itself as we had with our C1si and the X galvo got jammed or was not
> >moving properly. Again Craig figured it out of what was happening with
> >the XY galvo scan head and we fixed the problem. A simple test if it is
> >associated with the table is by tapping the table top or the microscope
> >stage and if the lines appears and disappears then it is the table issue.
> >The galvo problem will also occur due to the power supply to the
> >controller and make sure to rule out that possibility by adding a UPS
> >with automatic voltage regulator and surge protector.
> >
> >Good luck
> >
> >Sathya Srinivasan
> >Manager
> >RUN Microscopy Facility
> >(www.ucalgary.ca/runcore)
> >University of Calgary
> >Room 126 HMRB
> >3330 Hospital Drive, NW
> >Calgary, AB, Canada
> >
> >> Date: Thu, 19 Jun 2014 12:19:53 -0500
> >> From: [hidden email]
> >> Subject: Re: Olympus FV1000 horizontal lines problem
> >> To: [hidden email]
> >>
> >> *****
> >> 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 Michael--
> >>
> >> On 6/19/2014 12:12 PM, Cammer, Michael wrote:
> >> > Colleagues have a new Olympus FV1000 with autofocus and are having
> >>difficulties with it and asked our advice.  Based on images they sent
> >>us, one example posted at
> >>https://www.flickr.com/photos/mcammer/14273268488/ , I told them that I
> >>thought that the problem of horizontal lines was adjustments made by the
> >>autofocus during the scan.  (Another possibility we entertained was
> >>laser fluctuation and still another would be sudden temperature change
> >>by the incubator unit.)
> >> >
> >> > Questions:
> >> >
> >> > 1.       Do you think we have diagnosed the problem correctly?
> >> I've seen lines similar to this when focused close to the surface of
> >>the
> >> specimen and when the air table isn't damping vibration properly.  Any
> >> chance that that's it?
> >>
> >> For us, the issue turned out to be a sticky valve on the air table.
> >>
> >> Good luck--
> >>
> >> Martin Wessendorf
> >>
> >> --
> >> Martin Wessendorf, Ph.D.                   office: [phone]
> >> Assoc Prof, Dept Neuroscience                 lab: [phone]
> >> University of Minnesota             Preferred FAX: [phone]
> >> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> >> Minneapolis, MN  55455                    e-mail: [hidden email]
> >
>

[Stanislav Vitha-2]

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*****

If the stripes show with multiple lasers, then it is likely the vibrations as
others mentioned.
A long time ago we had a problem with stripes with our FV1000, but the
pattern looked different, it was a regular pattern of diagonal stripes that
changed direction depending on the scanning speed. This turned out to be
an issue with the optical fiber mount from the laser combiner;

I have also had laser stability issues (a dying HeNe laser) but that was
again a different pattern, some lines would be completely black.

Do the stripes show if you turn off the ZDC?


Good luck!

Stan Vitha
Microscopy and Imaging Center
Texas A&M University

[Paul Rigby-2]

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Hi Michael,
Your problem is creating a very unusual reflection image. A couple of questions/suggestions:
1. Given that the image is affected in similar x-axis positions over multiple scan lines, I am suspicious of the x galvo scanning. Is it possible that the laser alignment onto the x galvo mirror is not hitting the centre of the mirror? Perhaps try changing the number of scan lines and see if the pattern changes in the reflection image. Or change the dwell time?
2. Try opening the pinhole and see if the "banding" changes - this might help decide if it is a vibration problem.
3. The transmitted light image looks good - is this image generated from laser transmission? If so, perhaps you have a problem with the reflected light PMT (and the fluorescence PMT as well, although to a lesser extent). Maybe an issue with the  power supply to the PMT's?. Do the intensity fluctuations from the reflected and fluorescence images coincide?
4. Can you image using a different dichroic? Do you still get the patterns in the fluorescence image if you don't image in reflection mode?

Sorry, no solutions but maybe a couple of things to try. Good luck.
Cheers
Paul

Assoc. Prof. Paul Rigby
Centre for Microscopy, Characterisation & Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6007
Australia



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael
Sent: Friday, 20 June 2014 1:13 AM
To: [hidden email]
Subject: Olympus FV1000 horizontal lines problem

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Colleagues have a new Olympus FV1000 with autofocus and are having difficulties with it and asked our advice.  Based on images they sent us, one example posted at https://www.flickr.com/photos/mcammer/14273268488/ , I told them that I thought that the problem of horizontal lines was adjustments made by the autofocus during the scan.  (Another possibility we entertained was laser fluctuation and still another would be sudden temperature change by the incubator unit.)

Questions:

1.       Do you think we have diagnosed the problem correctly?

2.       Do other people have this problem?

3.       Is there a software setting in the FV1000 software to tell it to check and set the focus only at the beginning of a scan and at the end of a scan with autofocus polling and adjusting suspended during the scan?


Thank you!!
===========================================================================
Michael Cammer, Microscopy Core & Dustin Lab , Skirball Institute, NYU Langone Medical Center
Cell:  914-309-3270   Lab: 212-263-3208
http://ocs.med.nyu.edu/microscopy & http://www.med.nyu.edu/skirball-lab/dustinlab/

------------------------------------------------------------
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
=================================

[MORONE Diego RIC]

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Hi Michael, it could also be a fiber problem. I had a damaged fiber connector on a Nikon A1R with descanned pmts that caused random stripes and a loss of signal similar to yours. At first we thought it was a vibration or a loss of focus from the piezo-Z drive

Best,
Diego


------------------------------------------
DAI IL TUO 5x1000 ALLA RICERCA HUMANITAS.

Codice fiscale 10125410158 (Ricerca sanitaria)

http://www.humanitas.it/5x1000
------------------------------------------
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Rigby
Sent: lunedì 23 giugno 2014 07:06
To: [hidden email]
Subject: Re: Olympus FV1000 horizontal lines problem

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

Hi Michael,
Your problem is creating a very unusual reflection image. A couple of questions/suggestions:
1. Given that the image is affected in similar x-axis positions over multiple scan lines, I am suspicious of the x galvo scanning. Is it possible that the laser alignment onto the x galvo mirror is not hitting the centre of the mirror? Perhaps try changing the number of scan lines and see if the pattern changes in the reflection image. Or change the dwell time?
2. Try opening the pinhole and see if the "banding" changes - this might help decide if it is a vibration problem.
3. The transmitted light image looks good - is this image generated from laser transmission? If so, perhaps you have a problem with the reflected light PMT (and the fluorescence PMT as well, although to a lesser extent). Maybe an issue with the  power supply to the PMT's?. Do the intensity fluctuations from the reflected and fluorescence images coincide?
4. Can you image using a different dichroic? Do you still get the patterns in the fluorescence image if you don't image in reflection mode?

Sorry, no solutions but maybe a couple of things to try. Good luck.
Cheers
Paul

Assoc. Prof. Paul Rigby
Centre for Microscopy, Characterisation & Analysis (M510) The University of Western Australia
35 Stirling Highway
Crawley  WA  6007
Australia



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael
Sent: Friday, 20 June 2014 1:13 AM
To: [hidden email]
Subject: Olympus FV1000 horizontal lines problem

*****
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.
*****

Colleagues have a new Olympus FV1000 with autofocus and are having difficulties with it and asked our advice.  Based on images they sent us, one example posted at https://www.flickr.com/photos/mcammer/14273268488/ , I told them that I thought that the problem of horizontal lines was adjustments made by the autofocus during the scan.  (Another possibility we entertained was laser fluctuation and still another would be sudden temperature change by the incubator unit.)

Questions:

1.       Do you think we have diagnosed the problem correctly?

2.       Do other people have this problem?

3.       Is there a software setting in the FV1000 software to tell it to check and set the focus only at the beginning of a scan and at the end of a scan with autofocus polling and adjusting suspended during the scan?


Thank you!!
===========================================================================
Michael Cammer, Microscopy Core & Dustin Lab , Skirball Institute, NYU Langone Medical Center
Cell:  914-309-3270   Lab: 212-263-3208
http://ocs.med.nyu.edu/microscopy & http://www.med.nyu.edu/skirball-lab/dustinlab/

------------------------------------------------------------
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
=================================