LSM 510 troubleshooting - horizontal stripes

[Andrew-2]

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Hello all,

I have an old LSM 510 that recently started having a major issue where it
shows a horizontal banding pattern with the laser either on or off and was
just wondering if anyone has any idea what may be occuring. It's also much
less sensitive than it used to be (normal fluorescence samples don't work,
only reflected light shows up) and the PMT gain does not seem to affect
anything. Here are links to some images showing the issue:

Scan with laser off <https://imgur.com/lgdNXTY>

Scan with laser on <https://imgur.com/wOlTTIg>

I originally thought this was a laser overheating problem, until I
discovered it occurred even with the lasers off. I've already tried all the
basic troubleshooting, and checked all the voltages from the CANcheck
utility, checked the connections for all the cables, but can't find
anything indicating any problem. Sadly, not worth the money to have a
technician come to look at it, so trying to see if it's something I can fix
myself. Any tips/guidance would be appreciated.

Thanks,


Andrew
--

Andrew McCall, PhD

Director, Optical Imaging and Analysis Facility

406 Biomedical Research Building

University at Buffalo

[Cromey, Douglas W - (dcromey)-2]

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Andrew,

Any chance that your table is no longer floating? That's a simple check, just in case.

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
Life Sciences North, Room 463
1333 N. Martin Ave, Tucson, AZ  85721 USA

office:  LSN 463        email: [hidden email]
voice:  520-626-2824       fax:  [phone]

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A collaborative effort to bring information about shared microscopy
facilities to the University of Arizona and the community.

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Andrew
Sent: Monday, September 14, 2020 10:24 AM
To: [hidden email]
Subject: [EXT]LSM 510 troubleshooting - horizontal stripes

External Email

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Hello all,

I have an old LSM 510 that recently started having a major issue where it shows a horizontal banding pattern with the laser either on or off and was just wondering if anyone has any idea what may be occuring. It's also much less sensitive than it used to be (normal fluorescence samples don't work, only reflected light shows up) and the PMT gain does not seem to affect anything. Here are links to some images showing the issue:

Scan with laser off <https://imgur.com/lgdNXTY>

Scan with laser on <https://imgur.com/wOlTTIg>

I originally thought this was a laser overheating problem, until I discovered it occurred even with the lasers off. I've already tried all the basic troubleshooting, and checked all the voltages from the CANcheck utility, checked the connections for all the cables, but can't find anything indicating any problem. Sadly, not worth the money to have a technician come to look at it, so trying to see if it's something I can fix myself. Any tips/guidance would be appreciated.

Thanks,


Andrew
--

Andrew McCall, PhD

Director, Optical Imaging and Analysis Facility

406 Biomedical Research Building

University at Buffalo

[Jonkman, James]

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Hi, Andrew.  I wonder if the AOTF or the AOTF driver is shot?  I had similar horizontal stripes on a couple of instruments over the years.  In my cases, though, the laser still came through and you could get a fluorescence image, but with these stripes superimposed.  If you have more than 1 AOTF (i.e., perhaps a separate fiber for the DAPI laser?)  then you could test this.  Alternatively the PMT is dead and just giving you noise essentially, unless hit by a large signal from reflected light - again you could check another PMT.  There are probably plenty of parts around for old LSM510s (I have some myself here in Toronto and would be happy to donate them), but I'm not sure that you'll find anyone who will do the servicing and realignment for you.  Good luck and let us know if you ever solve it!

Cheers,
James
PS: you can check out Figure 9h and i in our Nat Prot paper, "Tutorial: Guidance for Quantitative Confocal Microscopy" and associated text on pg 19.  Happy to send you a copy offline if you email me.

-----------------------------------------------
   James Jonkman, Staff Scientist
   Advanced Optical Microscopy Facility (AOMF)
   and Wright Cell Imaging Facility (WCIF)
   University Health Network
   MaRS, PMCRT tower, 101 College St., Room 15-305
   Toronto, ON, CANADA    M5G 1L7
  [hidden email]  Tel: [phone]
   www.aomf.ca


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew
Sent: Monday, September 14, 2020 1:24 PM
To: [hidden email]
Subject: [External] LSM 510 troubleshooting - horizontal stripes

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

Hello all,

I have an old LSM 510 that recently started having a major issue where it shows a horizontal banding pattern with the laser either on or off and was just wondering if anyone has any idea what may be occuring. It's also much less sensitive than it used to be (normal fluorescence samples don't work, only reflected light shows up) and the PMT gain does not seem to affect anything. Here are links to some images showing the issue:

Scan with laser off <https://urldefense.com/v3/__https://imgur.com/lgdNXTY__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgM5zAUutf$ [imgur[.]com]>

Scan with laser on <https://urldefense.com/v3/__https://imgur.com/wOlTTIg__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgM7PqDu8v$ [imgur[.]com]>

I originally thought this was a laser overheating problem, until I discovered it occurred even with the lasers off. I've already tried all the basic troubleshooting, and checked all the voltages from the CANcheck utility, checked the connections for all the cables, but can't find anything indicating any problem. Sadly, not worth the money to have a technician come to look at it, so trying to see if it's something I can fix myself. Any tips/guidance would be appreciated.

Thanks,


Andrew
--

Andrew McCall, PhD

Director, Optical Imaging and Analysis Facility

406 Biomedical Research Building

University at Buffalo

This e-mail may contain confidential and/or privileged information for the sole use of the intended recipient.
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[Andrew-2]

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Thank you all for the responses. We had some issues with the AOTF a while
ago that presented quite differently and I managed to find a workaround
for it, though I will check it's basic functionality again as it's not too
difficult. I suspect Michael is right though and it is the PMT power
supply, as it happened quite suddenly and to all the PMT channels at once
(something I forgot to mention, apologies).


Andrew

On Mon, Sep 14, 2020 at 2:02 PM Jonkman, James <[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, Andrew.  I wonder if the AOTF or the AOTF driver is shot?  I had
> similar horizontal stripes on a couple of instruments over the years.  In
> my cases, though, the laser still came through and you could get a
> fluorescence image, but with these stripes superimposed.  If you have more
> than 1 AOTF (i.e., perhaps a separate fiber for the DAPI laser?)  then you
> could test this.  Alternatively the PMT is dead and just giving you noise
> essentially, unless hit by a large signal from reflected light - again you
> could check another PMT.  There are probably plenty of parts around for old
> LSM510s (I have some myself here in Toronto and would be happy to donate
> them), but I'm not sure that you'll find anyone who will do the servicing
> and realignment for you.  Good luck and let us know if you ever solve it!
>
> Cheers,
> James
> PS: you can check out Figure 9h and i in our Nat Prot paper, "Tutorial:
> Guidance for Quantitative Confocal Microscopy" and associated text on pg
> 19.  Happy to send you a copy offline if you email me.
>
> -----------------------------------------------
>    James Jonkman, Staff Scientist
>    Advanced Optical Microscopy Facility (AOMF)
>    and Wright Cell Imaging Facility (WCIF)
>    University Health Network
>    MaRS, PMCRT tower, 101 College St., Room 15-305
>    Toronto, ON, CANADA    M5G 1L7
>   [hidden email]  Tel: [phone]
>    www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Andrew
> Sent: Monday, September 14, 2020 1:24 PM
> To: [hidden email]
> Subject: [External] LSM 510 troubleshooting - horizontal stripes
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgMwy3KQ8F$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgMzcQ-gp2$
> [imgur[.]com] and include the link in your posting.
> *****
>
> Hello all,
>
> I have an old LSM 510 that recently started having a major issue where it
> shows a horizontal banding pattern with the laser either on or off and was
> just wondering if anyone has any idea what may be occuring. It's also much
> less sensitive than it used to be (normal fluorescence samples don't work,
> only reflected light shows up) and the PMT gain does not seem to affect
> anything. Here are links to some images showing the issue:
>
> Scan with laser off <
> https://urldefense.com/v3/__https://imgur.com/lgdNXTY__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgM5zAUutf$
> [imgur[.]com]>
>
> Scan with laser on <
> https://urldefense.com/v3/__https://imgur.com/wOlTTIg__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgM7PqDu8v$
> [imgur[.]com]>
>
> I originally thought this was a laser overheating problem, until I
> discovered it occurred even with the lasers off. I've already tried all the
> basic troubleshooting, and checked all the voltages from the CANcheck
> utility, checked the connections for all the cables, but can't find
> anything indicating any problem. Sadly, not worth the money to have a
> technician come to look at it, so trying to see if it's something I can fix
> myself. Any tips/guidance would be appreciated.
>
> Thanks,
>
>
> Andrew
> --
>
> Andrew McCall, PhD
>
> Director, Optical Imaging and Analysis Facility
>
> 406 Biomedical Research Building
>
> University at Buffalo
>
> This e-mail may contain confidential and/or privileged information for the
> sole use of the intended recipient.
> Any review or distribution by anyone other than the person for whom it was
> originally intended is strictly prohibited.
> If you have received this e-mail in error, please contact the sender and
> delete all copies.
> Opinions, conclusions or other information contained in this e-mail may
> not be that of the organization.
>
> If you feel you have received an email from UHN of a commercial nature and
> would like to be removed from the sender's mailing list please do one of
> the following:
> (1) Follow any unsubscribe process the sender has included in their email
> (2) Where no unsubscribe process has been included, reply to the sender
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> Please note that we are unable to automatically unsubscribe individuals
> from all UHN mailing lists.
>

[Craig Brideau]

*****
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*****

Easy way to confirm if it is the lasers or the detectors is to
back-illuminate a piece of paper with a dim light and scan the signal
coming from the paper. Assuming your test light doesn't flicker, you should
record a very even illumination across the FOV. If you still see the lines
then you know it is a gain/detector issue. Otherwise, as others have said,
it is most likely your laser power control (AOTF etc).

Craig

On Mon, Sep 14, 2020 at 12:02 PM Jonkman, James <
[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, Andrew.  I wonder if the AOTF or the AOTF driver is shot?  I had
> similar horizontal stripes on a couple of instruments over the years.  In
> my cases, though, the laser still came through and you could get a
> fluorescence image, but with these stripes superimposed.  If you have more
> than 1 AOTF (i.e., perhaps a separate fiber for the DAPI laser?)  then you
> could test this.  Alternatively the PMT is dead and just giving you noise
> essentially, unless hit by a large signal from reflected light - again you
> could check another PMT.  There are probably plenty of parts around for old
> LSM510s (I have some myself here in Toronto and would be happy to donate
> them), but I'm not sure that you'll find anyone who will do the servicing
> and realignment for you.  Good luck and let us know if you ever solve it!
>
> Cheers,
> James
> PS: you can check out Figure 9h and i in our Nat Prot paper, "Tutorial:
> Guidance for Quantitative Confocal Microscopy" and associated text on pg
> 19.  Happy to send you a copy offline if you email me.
>
> -----------------------------------------------
>    James Jonkman, Staff Scientist
>    Advanced Optical Microscopy Facility (AOMF)
>    and Wright Cell Imaging Facility (WCIF)
>    University Health Network
>    MaRS, PMCRT tower, 101 College St., Room 15-305
>    Toronto, ON, CANADA    M5G 1L7
>   [hidden email]  Tel: [phone]
>    www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Andrew
> Sent: Monday, September 14, 2020 1:24 PM
> To: [hidden email]
> Subject: [External] LSM 510 troubleshooting - horizontal stripes
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgMwy3KQ8F$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgMzcQ-gp2$
> [imgur[.]com] and include the link in your posting.
> *****
>
> Hello all,
>
> I have an old LSM 510 that recently started having a major issue where it
> shows a horizontal banding pattern with the laser either on or off and was
> just wondering if anyone has any idea what may be occuring. It's also much
> less sensitive than it used to be (normal fluorescence samples don't work,
> only reflected light shows up) and the PMT gain does not seem to affect
> anything. Here are links to some images showing the issue:
>
> Scan with laser off <
> https://urldefense.com/v3/__https://imgur.com/lgdNXTY__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgM5zAUutf$
> [imgur[.]com]>
>
> Scan with laser on <
> https://urldefense.com/v3/__https://imgur.com/wOlTTIg__;!!CjcC7IQ!ai3nI3uqqqBbjIbfBFj80ObyfddfrJ8k8olaFwfpMtCocgUNq21UoMD40JOPlhWgM7PqDu8v$
> [imgur[.]com]>
>
> I originally thought this was a laser overheating problem, until I
> discovered it occurred even with the lasers off. I've already tried all the
> basic troubleshooting, and checked all the voltages from the CANcheck
> utility, checked the connections for all the cables, but can't find
> anything indicating any problem. Sadly, not worth the money to have a
> technician come to look at it, so trying to see if it's something I can fix
> myself. Any tips/guidance would be appreciated.
>
> Thanks,
>
>
> Andrew
> --
>
> Andrew McCall, PhD
>
> Director, Optical Imaging and Analysis Facility
>
> 406 Biomedical Research Building
>
> University at Buffalo
>
> This e-mail may contain confidential and/or privileged information for the
> sole use of the intended recipient.
> Any review or distribution by anyone other than the person for whom it was
> originally intended is strictly prohibited.
> If you have received this e-mail in error, please contact the sender and
> delete all copies.
> Opinions, conclusions or other information contained in this e-mail may
> not be that of the organization.
>
> If you feel you have received an email from UHN of a commercial nature and
> would like to be removed from the sender's mailing list please do one of
> the following:
> (1) Follow any unsubscribe process the sender has included in their email
> (2) Where no unsubscribe process has been included, reply to the sender
> and type "unsubscribe" in the subject line. If you require additional
> information please go to our UHN Newsletters and Mailing Lists page.
> Please note that we are unable to automatically unsubscribe individuals
> from all UHN mailing lists.
>