Spectral Detector Calibration

[Claire Brown]

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We just had our annual PM visit for our LSM710.
They recalibrated our 32-channel PMT array.

We have several people doing spectral imaging including some people doing 7 colour imaging.
They will need to repeat all of their reference spectra due to the recalibration.
Their new data will be unmixed with different reference spectra relative to data collected before the PM visit.

This has happened to us several times in the past. One time the users actually asked us to go back to the old calibration settings so they could finish out their study.

I was just wondering how other facilities handle this complex issue?
Do you inform your users that a PM was done and the instrument calibration has changed?
Have people thought about this in term of reproducibility? Every instrument is different.

Would love to hear people's thoughts.

Claire
Advanced BioImaging Facility, McGill University

[Jonkman, James]

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Hi, Claire.  That's so frustrating!  We have had the same experience many times with laser intensities: company XXX comes for a PM and as they leave they proudly announce that they have tweaked the alignment and XXX laser line is now, say, 25% or 50% higher intensity compared to yesterday.  We have learned that we have to measure laser powers before and after a PM, and then we post the old and new numbers right on the booking calendar so that users can scale their data.  Usually we remember to do this.  I don't know if I've ever mentioned this before (!), but I think it's pathetic that laser powers are completely uncalibrated on confocal microscopes.  Ok, maybe I mentioned it before.

In your case, if you have reference spectra from before and after the calibration you could create a transformation between the 2 cases.  Perhaps the calibration files that Zeiss produces can be used for this?  I don't know if they are readable with just a text editor or not?

Cheers,
James

-----------------------------------------------
   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 Claire Brown, Dr.
Sent: Thursday, December 10, 2020 2:52 PM
To: [hidden email]
Subject: [External] Spectral Detector Calibration

*****
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*****

We just had our annual PM visit for our LSM710.
They recalibrated our 32-channel PMT array.

We have several people doing spectral imaging including some people doing 7 colour imaging.
They will need to repeat all of their reference spectra due to the recalibration.
Their new data will be unmixed with different reference spectra relative to data collected before the PM visit.

This has happened to us several times in the past. One time the users actually asked us to go back to the old calibration settings so they could finish out their study.

I was just wondering how other facilities handle this complex issue?
Do you inform your users that a PM was done and the instrument calibration has changed?
Have people thought about this in term of reproducibility? Every instrument is different.

Would love to hear people's thoughts.

Claire
Advanced BioImaging Facility, McGill University

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[Cammer, Michael-2]

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One of the labs using a confocal here checks the power of lasers and AOTF output at sample at the beginning of every imaging session to make sure same power for all experiments.  This, however, does not guarantee same sensitivity of detectors over time and has been an issue when we have had detectors replaced.  I don't know whether they use dyes at known concentrations for calibration.  Somewhere I have uranyl glass for green fluorescence, but nobody has used this as a standard for over 7 years.  Fluorescent Plexiglas could probably be a similar standard (with a few caveats).
Cheers-
Michael Cammer

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jonkman, James
Sent: Thursday, December 10, 2020 3:10 PM
To: [hidden email]
Subject: Re: [External] Spectral Detector Calibration

[EXTERNAL]

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Hi, Claire.  That's so frustrating!  We have had the same experience many times with laser intensities: company XXX comes for a PM and as they leave they proudly announce that they have tweaked the alignment and XXX laser line is now, say, 25% or 50% higher intensity compared to yesterday.  We have learned that we have to measure laser powers before and after a PM, and then we post the old and new numbers right on the booking calendar so that users can scale their data.  Usually we remember to do this.  I don't know if I've ever mentioned this before (!), but I think it's pathetic that laser powers are completely uncalibrated on confocal microscopes.  Ok, maybe I mentioned it before.

In your case, if you have reference spectra from before and after the calibration you could create a transformation between the 2 cases.  Perhaps the calibration files that Zeiss produces can be used for this?  I don't know if they are readable with just a text editor or not?

Cheers,
James

-----------------------------------------------
   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]
   https://urldefense.proofpoint.com/v2/url?u=http-3A__www.aomf.ca&d=DwIFAw&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=6f9TNt3D2qeWCo1pedCt8G3ZQ9oI1tXjnwzkk_Z_1dI&s=lXqon7Bs5UK6uPyMSpBvXnolhtLwn_OeoAHn3Ixqoko&e=


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claire Brown, Dr.
Sent: Thursday, December 10, 2020 2:52 PM
To: [hidden email]
Subject: [External] Spectral Detector Calibration

*****
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*****

We just had our annual PM visit for our LSM710.
They recalibrated our 32-channel PMT array.

We have several people doing spectral imaging including some people doing 7 colour imaging.
They will need to repeat all of their reference spectra due to the recalibration.
Their new data will be unmixed with different reference spectra relative to data collected before the PM visit.

This has happened to us several times in the past. One time the users actually asked us to go back to the old calibration settings so they could finish out their study.

I was just wondering how other facilities handle this complex issue?
Do you inform your users that a PM was done and the instrument calibration has changed?
Have people thought about this in term of reproducibility? Every instrument is different.

Would love to hear people's thoughts.

Claire
Advanced BioImaging Facility, McGill University

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[leavesley]

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We have used the green fluorescent glass and fluorescent plexiglas as
well and they are nice because they are very stable. We have also tried
measuring the output of a NIST-traceable lamp by placing it at the
microscope stage - just be careful with the alignment if you try this.

Best regards,

Silas Leavesley
Professor
Department of Chemical and Biomolecular Engineering
Department of Pharmacology
Center for Lung Biology
University of South Alabama


On 12/10/2020 2:19 PM, Cammer, Michael 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.
> *****
>
> One of the labs using a confocal here checks the power of lasers and AOTF output at sample at the beginning of every imaging session to make sure same power for all experiments.  This, however, does not guarantee same sensitivity of detectors over time and has been an issue when we have had detectors replaced.  I don't know whether they use dyes at known concentrations for calibration.  Somewhere I have uranyl glass for green fluorescence, but nobody has used this as a standard for over 7 years.  Fluorescent Plexiglas could probably be a similar standard (with a few caveats).
> Cheers-
> Michael Cammer
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Jonkman, James
> Sent: Thursday, December 10, 2020 3:10 PM
> To: [hidden email]
> Subject: Re: [External] Spectral Detector Calibration
>
> [EXTERNAL]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAw&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=6f9TNt3D2qeWCo1pedCt8G3ZQ9oI1tXjnwzkk_Z_1dI&s=0KK4H-Dio0qyWMV1rf9-fy2JsPCQyieYuo_kl6j0ndQ&e=  and include the link in your posting.
> *****
>
> Hi, Claire.  That's so frustrating!  We have had the same experience many times with laser intensities: company XXX comes for a PM and as they leave they proudly announce that they have tweaked the alignment and XXX laser line is now, say, 25% or 50% higher intensity compared to yesterday.  We have learned that we have to measure laser powers before and after a PM, and then we post the old and new numbers right on the booking calendar so that users can scale their data.  Usually we remember to do this.  I don't know if I've ever mentioned this before (!), but I think it's pathetic that laser powers are completely uncalibrated on confocal microscopes.  Ok, maybe I mentioned it before.
>
> In your case, if you have reference spectra from before and after the calibration you could create a transformation between the 2 cases.  Perhaps the calibration files that Zeiss produces can be used for this?  I don't know if they are readable with just a text editor or not?
>
> Cheers,
> James
>
> -----------------------------------------------
>     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]
>     https://urldefense.proofpoint.com/v2/url?u=http-3A__www.aomf.ca&d=DwIFAw&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=6f9TNt3D2qeWCo1pedCt8G3ZQ9oI1tXjnwzkk_Z_1dI&s=lXqon7Bs5UK6uPyMSpBvXnolhtLwn_OeoAHn3Ixqoko&e=
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claire Brown, Dr.
> Sent: Thursday, December 10, 2020 2:52 PM
> To: [hidden email]
> Subject: [External] Spectral Detector Calibration
>
> *****
> 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!fdeBLqfMPIFa9KuVfgisdyjqDjhd30J-qjK6WCktoYjA49hoq68vI68hnuRtMo5C9D3d$ [lists[.]umn[.]edu] Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!fdeBLqfMPIFa9KuVfgisdyjqDjhd30J-qjK6WCktoYjA49hoq68vI68hnuRtMjdlS4Rh$ [imgur[.]com] and include the link in your posting.
> *****
>
> We just had our annual PM visit for our LSM710.
> They recalibrated our 32-channel PMT array.
>
> We have several people doing spectral imaging including some people doing 7 colour imaging.
> They will need to repeat all of their reference spectra due to the recalibration.
> Their new data will be unmixed with different reference spectra relative to data collected before the PM visit.
>
> This has happened to us several times in the past. One time the users actually asked us to go back to the old calibration settings so they could finish out their study.
>
> I was just wondering how other facilities handle this complex issue?
> Do you inform your users that a PM was done and the instrument calibration has changed?
> Have people thought about this in term of reproducibility? Every instrument is different.
>
> Would love to hear people's thoughts.
>
> Claire
> Advanced BioImaging Facility, McGill University
>
> 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.
>
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>
> ------------------------------------------------------------
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> =================================

<http://www.usahealthsystem.com/clb>

[Benjamin Smith]

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*****

Just to play devil's advocate, data critical calibrations should be
verified at a set interval, independent of system recalibrations.  Some
systems parameters can vary with humidity, temperature, debris on the
objective lens, or simple use, or a previous user may have put the system
into an odd configuration.  Regularly re-testing your assumptions about the
state of the system helps to ensure you don't waste time chasing after
false results.  Even if it is something as simple as re-imaging the same
reference slide, or Tetraspeck beads, or any other reference of your
choice, this can do wonders for catching changes in your system before they
become major issues.

Quite often I'll even include a quick validation of the control calibration
data in the analysis code, as a computer tends to be much better at
catching slight changes over time than the human eye (especially
considering our built in biases).  As such, I can tell you from a recent
project that the lateral chromatic aberration and axial point spread
function did indeed slightly drift over time on a commercial core
microscope during the couple of months we collected our data for a
super-resolution imaging experiment.

That said, I can also sympathize with having a complex, finely tuned
experiment getting thrown into the wringer can be stressful. especially if
the experiment is producing exciting results.  That said, assuring the
users that most any re-calibration should be a simple linear transform of
the previous configuration should relieve some of their stress.

Cheers,
   Ben Smith



On Thu, Dec 10, 2020 at 12:11 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, Claire.  That's so frustrating!  We have had the same experience many
> times with laser intensities: company XXX comes for a PM and as they leave
> they proudly announce that they have tweaked the alignment and XXX laser
> line is now, say, 25% or 50% higher intensity compared to yesterday.  We
> have learned that we have to measure laser powers before and after a PM,
> and then we post the old and new numbers right on the booking calendar so
> that users can scale their data.  Usually we remember to do this.  I don't
> know if I've ever mentioned this before (!), but I think it's pathetic that
> laser powers are completely uncalibrated on confocal microscopes.  Ok,
> maybe I mentioned it before.
>
> In your case, if you have reference spectra from before and after the
> calibration you could create a transformation between the 2 cases.  Perhaps
> the calibration files that Zeiss produces can be used for this?  I don't
> know if they are readable with just a text editor or not?
>
> Cheers,
> James
>
> -----------------------------------------------
>    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 Claire Brown, Dr.
> Sent: Thursday, December 10, 2020 2:52 PM
> To: [hidden email]
> Subject: [External] Spectral Detector Calibration
>
> *****
> 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!fdeBLqfMPIFa9KuVfgisdyjqDjhd30J-qjK6WCktoYjA49hoq68vI68hnuRtMo5C9D3d$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!fdeBLqfMPIFa9KuVfgisdyjqDjhd30J-qjK6WCktoYjA49hoq68vI68hnuRtMjdlS4Rh$
> [imgur[.]com] and include the link in your posting.
> *****
>
> We just had our annual PM visit for our LSM710.
> They recalibrated our 32-channel PMT array.
>
> We have several people doing spectral imaging including some people doing
> 7 colour imaging.
> They will need to repeat all of their reference spectra due to the
> recalibration.
> Their new data will be unmixed with different reference spectra relative
> to data collected before the PM visit.
>
> This has happened to us several times in the past. One time the users
> actually asked us to go back to the old calibration settings so they could
> finish out their study.
>
> I was just wondering how other facilities handle this complex issue?
> Do you inform your users that a PM was done and the instrument calibration
> has changed?
> Have people thought about this in term of reproducibility? Every
> instrument is different.
>
> Would love to hear people's thoughts.
>
> Claire
> Advanced BioImaging Facility, McGill University
>
> 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.
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

[Cammer, Michael-2]

*****
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*****

Of course, PMs and service calls are extremely important for all the good stuff.  We rely on them for operation and learning more about the instruments.
However, sometime we have problems.  Increased laser power is not a problem; it's always been a good thing for us, especially for people doing FRAP, but as already noted, we need to know what the laser powers are.  
The biggest problem we have after PMs or other service calls is that settings have been changed during the service.  For instance, a lens position has been reset to a calibration lens and not changed back.  Or the properties that are applied to the Reuse command are changed.  Or camera colors are swapped.  Essentially, there isn’t a checklist of resetting states.
Software updates with changed functionality, which sometimes includes elimination of functionality or buttons being moved, is another issue.
Cheers-
Michael Cammer

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Benjamin Smith
Sent: Thursday, December 10, 2020 3:27 PM
To: [hidden email]
Subject: Re: [External] Spectral Detector Calibration

[EXTERNAL]

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*****

Just to play devil's advocate, data critical calibrations should be verified at a set interval, independent of system recalibrations.  Some systems parameters can vary with humidity, temperature, debris on the objective lens, or simple use, or a previous user may have put the system into an odd configuration.  Regularly re-testing your assumptions about the state of the system helps to ensure you don't waste time chasing after false results.  Even if it is something as simple as re-imaging the same reference slide, or Tetraspeck beads, or any other reference of your choice, this can do wonders for catching changes in your system before they become major issues.

Quite often I'll even include a quick validation of the control calibration data in the analysis code, as a computer tends to be much better at catching slight changes over time than the human eye (especially considering our built in biases).  As such, I can tell you from a recent project that the lateral chromatic aberration and axial point spread function did indeed slightly drift over time on a commercial core microscope during the couple of months we collected our data for a super-resolution imaging experiment.

That said, I can also sympathize with having a complex, finely tuned experiment getting thrown into the wringer can be stressful. especially if the experiment is producing exciting results.  That said, assuring the users that most any re-calibration should be a simple linear transform of the previous configuration should relieve some of their stress.

Cheers,
   Ben Smith



On Thu, Dec 10, 2020 at 12:11 PM Jonkman, James < [hidden email]> wrote:

> *****
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> *****
>
> Hi, Claire.  That's so frustrating!  We have had the same experience
> many times with laser intensities: company XXX comes for a PM and as
> they leave they proudly announce that they have tweaked the alignment
> and XXX laser line is now, say, 25% or 50% higher intensity compared
> to yesterday.  We have learned that we have to measure laser powers
> before and after a PM, and then we post the old and new numbers right
> on the booking calendar so that users can scale their data.  Usually
> we remember to do this.  I don't know if I've ever mentioned this
> before (!), but I think it's pathetic that laser powers are completely
> uncalibrated on confocal microscopes.  Ok, maybe I mentioned it before.
>
> In your case, if you have reference spectra from before and after the
> calibration you could create a transformation between the 2 cases.  
> Perhaps the calibration files that Zeiss produces can be used for
> this?  I don't know if they are readable with just a text editor or not?
>
> Cheers,
> James
>
> -----------------------------------------------
>    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]
>    
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.aomf.ca&d=DwIB
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>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of Claire Brown, Dr.
> Sent: Thursday, December 10, 2020 2:52 PM
> To: [hidden email]
> Subject: [External] Spectral Detector Calibration
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confoca
> lmicroscopy__;!!CjcC7IQ!fdeBLqfMPIFa9KuVfgisdyjqDjhd30J-qjK6WCktoYjA49
> hoq68vI68hnuRtMo5C9D3d$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!fdeBLqfMP
> IFa9KuVfgisdyjqDjhd30J-qjK6WCktoYjA49hoq68vI68hnuRtMjdlS4Rh$
> [imgur[.]com] and include the link in your posting.
> *****
>
> We just had our annual PM visit for our LSM710.
> They recalibrated our 32-channel PMT array.
>
> We have several people doing spectral imaging including some people
> doing
> 7 colour imaging.
> They will need to repeat all of their reference spectra due to the
> recalibration.
> Their new data will be unmixed with different reference spectra
> relative to data collected before the PM visit.
>
> This has happened to us several times in the past. One time the users
> actually asked us to go back to the old calibration settings so they
> could finish out their study.
>
> I was just wondering how other facilities handle this complex issue?
> Do you inform your users that a PM was done and the instrument
> calibration has changed?
> Have people thought about this in term of reproducibility? Every
> instrument is different.
>
> Would love to hear people's thoughts.
>
> Claire
> Advanced BioImaging Facility, McGill University
>
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--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
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[Armstrong, Brian]

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*****

Hi Claire, for the settings there are values in the database. We reset these values back to the uncalibrated settings for a particular user so that they could continue with their existing experiment. We reset the values back to the calibrated values for new users. Your service engineer should be able to show you where these values are in the database.

Cheers,

Brian Armstrong PhD
Associate Research Professor
Developmental and Stem Cell Biology
Diabetes and Metabolic Diseases
Director, Light Microscopy Core
Beckman Research Institute, City of Hope



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claire Brown, Dr.
Sent: Thursday, December 10, 2020 11:52 AM
To: [hidden email]
Subject: Spectral Detector Calibration

[Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.]

----------------------------------------------------------------------
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Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!Fou38LsQmgU!9Agl2E0yeQulx6Zubij1VPkDE9Tcx28EEGvncT_ZVNE4yLXS1jLJJcRXFKXXaCE$  and include the link in your posting.
*****

We just had our annual PM visit for our LSM710.
They recalibrated our 32-channel PMT array.

We have several people doing spectral imaging including some people doing 7 colour imaging.
They will need to repeat all of their reference spectra due to the recalibration.
Their new data will be unmixed with different reference spectra relative to data collected before the PM visit.

This has happened to us several times in the past. One time the users actually asked us to go back to the old calibration settings so they could finish out their study.

I was just wondering how other facilities handle this complex issue?
Do you inform your users that a PM was done and the instrument calibration has changed?
Have people thought about this in term of reproducibility? Every instrument is different.

Would love to hear people's thoughts.

Claire
Advanced BioImaging Facility, McGill University

----------------------------------------------------------------------
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-SECURITY/CONFIDENTIALITY WARNING-  

This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (LCP301)
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[George McNamara]

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*****

That the 32-channel detector is may be changed could have implications
for AiryScan and AiryScan2 performance over time, since same 32-channel
detector type is used for these (I suppose the field service engineer
could be adjusting exactly where the spectrum is being dispersed across
the detector). Hopefully the customer has access to the Airy's raw data.

On the Zeiss 510 META 32-channel detector (generation before 710 version
of Quasar), the Zeiss field service engineer could -- and occasionally
did -- adjust the offset (and maybe the gain) of EACH element
independently. I do not know whether Quasar has the same feature.

On 12/10/2020 2:51 PM, Claire Brown, Dr. 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.
> *****
>
> We just had our annual PM visit for our LSM710.
> They recalibrated our 32-channel PMT array.
>
> We have several people doing spectral imaging including some people doing 7 colour imaging.
> They will need to repeat all of their reference spectra due to the recalibration.
> Their new data will be unmixed with different reference spectra relative to data collected before the PM visit.
>
> This has happened to us several times in the past. One time the users actually asked us to go back to the old calibration settings so they could finish out their study.
>
> I was just wondering how other facilities handle this complex issue?
> Do you inform your users that a PM was done and the instrument calibration has changed?
> Have people thought about this in term of reproducibility? Every instrument is different.
>
> Would love to hear people's thoughts.
>
> Claire
> Advanced BioImaging Facility, McGill University

[Zdenek Svindrych-2]

*****
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*****

Hi George,

and it's going to be even worse with the Airyscan, as there is one
particular channel that always gets the most light (yes, the one in the
middle).

Now the question is whether in Claire's case they're correcting for the
wavelength drift (unrelated to the detector), or the detector response
itself...

Either way, the GaAsP will age unevenly, which won't really matter in the
case of the spectral detector (just don't use the calibration spectra you
acquired four years ago), but it will throw off the math behind airyscan.

It should be possible to devise an experiment to test this issue, maybe
something like "Pinhole wide open, uniformly fluorescent sample, perhaps
way out of focus...", and compare your very new machine with the old one
:-). I'm sure Zeiss folks know better. Are they willing to share?

Best, zdenek

On Thu, Dec 10, 2020 at 6:39 PM George McNamara <[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.
> *****
>
> That the 32-channel detector is may be changed could have implications
> for AiryScan and AiryScan2 performance over time, since same 32-channel
> detector type is used for these (I suppose the field service engineer
> could be adjusting exactly where the spectrum is being dispersed across
> the detector). Hopefully the customer has access to the Airy's raw data.
>
> On the Zeiss 510 META 32-channel detector (generation before 710 version
> of Quasar), the Zeiss field service engineer could -- and occasionally
> did -- adjust the offset (and maybe the gain) of EACH element
> independently. I do not know whether Quasar has the same feature.
>
> On 12/10/2020 2:51 PM, Claire Brown, Dr. 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.
> > *****
> >
> > We just had our annual PM visit for our LSM710.
> > They recalibrated our 32-channel PMT array.
> >
> > We have several people doing spectral imaging including some people
> doing 7 colour imaging.
> > They will need to repeat all of their reference spectra due to the
> recalibration.
> > Their new data will be unmixed with different reference spectra relative
> to data collected before the PM visit.
> >
> > This has happened to us several times in the past. One time the users
> actually asked us to go back to the old calibration settings so they could
> finish out their study.
> >
> > I was just wondering how other facilities handle this complex issue?
> > Do you inform your users that a PM was done and the instrument
> calibration has changed?
> > Have people thought about this in term of reproducibility? Every
> instrument is different.
> >
> > Would love to hear people's thoughts.
> >
> > Claire
> > Advanced BioImaging Facility, McGill University
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Craig Brideau]

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*****

We shoot a dim, fiber-coupled tungsten or tungsten-deuterium lamp into the
objective at low gain settings and record the spectrum. The lamp is
stabilized and gives a constant spectral power density, so you can use it
to monitor the stability of your detector over time. Additionally, if your
system is "recalibrated" then you can compare the previous lamp spectrum to
the post-recalibration spectrum to get a transform to safely compare data
acquired pre and post. Our lab has used spectral imaging extensively for
over a decade and it seems like only now other labs are realizing the full
advantages and disadvantages of the technique. I also feel that spectral
detection was unleashed on the user base by the various vendors without any
real consideration for long-term stability or calibration. I have found
that most systems drift considerably over time and that the users need to
monitor this for anything quantitative.

Craig

On Thu, Dec 10, 2020 at 2:27 PM Brian Armstrong <[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 Claire, for the settings there are values in the database. We reset
> these values back to the uncalibrated settings for a particular user so
> that they could continue with their existing experiment. We reset the
> values back to the calibrated values for new users. Your service engineer
> should be able to show you where these values are in the database.
>
> Cheers,
>
> Brian Armstrong PhD
> Associate Research Professor
> Developmental and Stem Cell Biology
> Diabetes and Metabolic Diseases
> Director, Light Microscopy Core
> Beckman Research Institute, City of Hope
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Claire Brown, Dr.
> Sent: Thursday, December 10, 2020 11:52 AM
> To: [hidden email]
> Subject: Spectral Detector Calibration
>
> [Attention: This email came from an external source. Do not open
> attachments or click on links from unknown senders or unexpected emails.]
>
> ----------------------------------------------------------------------
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!Fou38LsQmgU!9Agl2E0yeQulx6Zubij1VPkDE9Tcx28EEGvncT_ZVNE4yLXS1jLJJcRXx72m5TQ$
> Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!Fou38LsQmgU!9Agl2E0yeQulx6Zubij1VPkDE9Tcx28EEGvncT_ZVNE4yLXS1jLJJcRXFKXXaCE$
> and include the link in your posting.
> *****
>
> We just had our annual PM visit for our LSM710.
> They recalibrated our 32-channel PMT array.
>
> We have several people doing spectral imaging including some people doing
> 7 colour imaging.
> They will need to repeat all of their reference spectra due to the
> recalibration.
> Their new data will be unmixed with different reference spectra relative
> to data collected before the PM visit.
>
> This has happened to us several times in the past. One time the users
> actually asked us to go back to the old calibration settings so they could
> finish out their study.
>
> I was just wondering how other facilities handle this complex issue?
> Do you inform your users that a PM was done and the instrument calibration
> has changed?
> Have people thought about this in term of reproducibility? Every
> instrument is different.
>
> Would love to hear people's thoughts.
>
> Claire
> Advanced BioImaging Facility, McGill University
>
> ----------------------------------------------------------------------
> ------------------------------------------------------------
> -SECURITY/CONFIDENTIALITY WARNING-
>
> This message and any attachments are intended solely for the individual or
> entity to which they are addressed. This communication may contain
> information that is privileged, confidential, or exempt from disclosure
> under applicable law (e.g., personal health information, research data,
> financial information). Because this e-mail has been sent without
> encryption, individuals other than the intended recipient may be able to
> view the information, forward it to others or tamper with the information
> without the knowledge or consent of the sender. If you are not the intended
> recipient, or the employee or person responsible for delivering the message
> to the intended recipient, any dissemination, distribution or copying of
> the communication is strictly prohibited. If you received the communication
> in error, please notify the sender immediately by replying to this message
> and deleting the message and any accompanying files from your system. If,
> due to the security risks, you do not wish to receive further
> communications via e-mail, please reply to this message and inform the
> sender that you do not wish to receive further e-mail from the sender.
> (LCP301)
> ------------------------------------------------------------
>

[Craig Brideau]

*****
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*****

I hadn't previously considered the stability of the multi-anode array in
the Airyscan before; thank you for bringing this to my attention. Our
multi-anode systems for spectral detection do clearly drift over time, with
the gain of the individual elements drifting differently over time. We
calibrate this periodically with a stabilized tungsten lamp (see other
thread), but in the case of the Airyscan the information is spatial and not
spectral. I suppose one could periodically measure a sub-resolution bead,
but it would have to be a very consistent sample.
I find the various companies that have implemented these multi-anode array
PMTs have not given much thought to the stability over time. The new GaAsP
systems will also age more rapidly than "classic" PMTs so I believe the
newer, more sensitive arrays will require more monitoring over time. I
would be interested in how the vendors actually field recalibrate these
spatial systems.

Craig

On Thu, Dec 10, 2020 at 5:15 PM Zdenek Svindrych <[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 George,
>
> and it's going to be even worse with the Airyscan, as there is one
> particular channel that always gets the most light (yes, the one in the
> middle).
>
> Now the question is whether in Claire's case they're correcting for the
> wavelength drift (unrelated to the detector), or the detector response
> itself...
>
> Either way, the GaAsP will age unevenly, which won't really matter in the
> case of the spectral detector (just don't use the calibration spectra you
> acquired four years ago), but it will throw off the math behind airyscan.
>
> It should be possible to devise an experiment to test this issue, maybe
> something like "Pinhole wide open, uniformly fluorescent sample, perhaps
> way out of focus...", and compare your very new machine with the old one
> :-). I'm sure Zeiss folks know better. Are they willing to share?
>
> Best, zdenek
>
> On Thu, Dec 10, 2020 at 6:39 PM George McNamara <[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.
> > *****
> >
> > That the 32-channel detector is may be changed could have implications
> > for AiryScan and AiryScan2 performance over time, since same 32-channel
> > detector type is used for these (I suppose the field service engineer
> > could be adjusting exactly where the spectrum is being dispersed across
> > the detector). Hopefully the customer has access to the Airy's raw data.
> >
> > On the Zeiss 510 META 32-channel detector (generation before 710 version
> > of Quasar), the Zeiss field service engineer could -- and occasionally
> > did -- adjust the offset (and maybe the gain) of EACH element
> > independently. I do not know whether Quasar has the same feature.
> >
> > On 12/10/2020 2:51 PM, Claire Brown, Dr. 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.
> > > *****
> > >
> > > We just had our annual PM visit for our LSM710.
> > > They recalibrated our 32-channel PMT array.
> > >
> > > We have several people doing spectral imaging including some people
> > doing 7 colour imaging.
> > > They will need to repeat all of their reference spectra due to the
> > recalibration.
> > > Their new data will be unmixed with different reference spectra
> relative
> > to data collected before the PM visit.
> > >
> > > This has happened to us several times in the past. One time the users
> > actually asked us to go back to the old calibration settings so they
> could
> > finish out their study.
> > >
> > > I was just wondering how other facilities handle this complex issue?
> > > Do you inform your users that a PM was done and the instrument
> > calibration has changed?
> > > Have people thought about this in term of reproducibility? Every
> > instrument is different.
> > >
> > > Would love to hear people's thoughts.
> > >
> > > Claire
> > > Advanced BioImaging Facility, McGill University
> >
>
>
> --
> --
> Zdenek Svindrych, Ph.D.
> Research Scientist - Microscopy Imaging Specialist
> Department of Biochemistry and Cell Biology
> Geisel School of Medicine at Dartmouth
>

[Sylvie Le Guyader]

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Hi Claire

It would be interesting to know if the 7 colour samples gives significantly different results when unmixed with the 2 different calibrations and ref spectra. Have they saved the original, not unmixed data ?

This raises 2 questions :
- it points to the importance of defining 'original' data in microscopy (that we are supposed to store for 10 years after publication). Is it enough to save the unmixed data? It is enough to save the data before unmixing without saving the instrument calibration data?
- if the same sample, unmixed with the 2 calibration/ref spectra, shows different results and that influences the interpretation so that the conclusion changes, one can argue that the experimental design might have been flawed from the start, i.e. the instrument 'noise' in a wide sense, was not considered in the interpretation of the result.

Re-acquiring the ref spectra sounds wise.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Claire Brown, Dr. <[hidden email]>
Sent: Thursday, December 10, 2020 8:51:31 PM
To: [hidden email] <[hidden email]>
Subject: Spectral Detector Calibration

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We just had our annual PM visit for our LSM710.
They recalibrated our 32-channel PMT array.

We have several people doing spectral imaging including some people doing 7 colour imaging.
They will need to repeat all of their reference spectra due to the recalibration.
Their new data will be unmixed with different reference spectra relative to data collected before the PM visit.

This has happened to us several times in the past. One time the users actually asked us to go back to the old calibration settings so they could finish out their study.

I was just wondering how other facilities handle this complex issue?
Do you inform your users that a PM was done and the instrument calibration has changed?
Have people thought about this in term of reproducibility? Every instrument is different.

Would love to hear people's thoughts.

Claire
Advanced BioImaging Facility, McGill University



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