training and bets practices for confocal

[Cammer, Michael]

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I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
http://nyulmc.org/micros  http://microscopynotes.com/

[Joshua Zachary Rappoport]

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Obviously there are a million other bits of info you could add, but this seems very reasonable as an essential primer
Under "If you see red pixels, you need to turn down the Gain or Laser.", you might want to add something like,
"Although increasing/decreasing gain and laser will make the image bright/dimmer, Gain refers to the detector, and increasing this can lead to more noise, while Laser refers to the illumination, and increasing this can lead to more photo-bleaching."
Cheers
Josh



--
Joshua Z. Rappoport PhD
Adjunct Faculty
Department of Cell and Molecular Biology
Northwestern University Feinberg School of Medicine

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]>
Sent: Thursday, October 10, 2019 11:15 AM
To: [hidden email]
Subject: training and bets practices for confocal

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*****

I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_imagej_saturation_index.html&d=DwIFAg&c=yHlS04HhBraes5BQ9ueu5zKhE7rtNXt_d012z2PA6ws&r=Tlex6a4rANueY3Z4ow0mcS-UVV1PpCZInmcJs49fQLOd1BLGJHg6nnZ85uZJf5RY&m=qJqPpt9SRyfT_ABEZPCiAINvhx1oMT4IceovC21eSyg&s=6NxuEXHOOJkpZI4fa-gQSEfMNQUdB3AsWiovV6sLhrc&e= >. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=yHlS04HhBraes5BQ9ueu5zKhE7rtNXt_d012z2PA6ws&r=Tlex6a4rANueY3Z4ow0mcS-UVV1PpCZInmcJs49fQLOd1BLGJHg6nnZ85uZJf5RY&m=qJqPpt9SRyfT_ABEZPCiAINvhx1oMT4IceovC21eSyg&s=oj8Bxluf59ESWtKCUlB4kjBb8qUrc7upnrTDePFORNM&e=   https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=yHlS04HhBraes5BQ9ueu5zKhE7rtNXt_d012z2PA6ws&r=Tlex6a4rANueY3Z4ow0mcS-UVV1PpCZInmcJs49fQLOd1BLGJHg6nnZ85uZJf5RY&m=qJqPpt9SRyfT_ABEZPCiAINvhx1oMT4IceovC21eSyg&s=Bz6CDGf6LkPlz68vJjWuOrT9sybpGhI0WPZJqqaCsz8&e=

[Armstrong, Brian]

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Hi, I completely agree.

*There are circumstances when the pinhole is not at 1AU such as when you are matching the optical sectioning thickness per wavelength in a z-stack. There are always going to be exceptions to a given set of rules.

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 Cammer, Michael
Sent: Thursday, October 10, 2019 9:16 AM
To: [hidden email]
Subject: training and bets practices for confocal

[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 http://www.imgur.com and include the link in your posting.
*****

I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
http://nyulmc.org/micros  http://microscopynotes.com/

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[mmodel]

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I think there are situations when opening the pinhole is acceptable (though perhaps not for first-time users). It's when one is more interested in quantification rather than in localizing the label - to capture thicker slices

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]>
Sent: Thursday, October 10, 2019 12:15 PM
To: [hidden email] <[hidden email]>
Subject: training and bets practices for confocal

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*****

I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2Fimagej%2Fsaturation%2Findex.html&amp;data=02%7C01%7Cmmodel%40KENT.EDU%7Cd0b72d63264e45d986ea08d74d9d3970%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637063209979705401&amp;sdata=a%2B1YcwLKvNh7uNFqddTo1K7wB1DlmFAC7XThWCLt09o%3D&amp;reserved=0>. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fnyulmc.org%2Fmicros&amp;data=02%7C01%7Cmmodel%40KENT.EDU%7Cd0b72d63264e45d986ea08d74d9d3970%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637063209979705401&amp;sdata=xKM%2FXPczy0nAK2JN6dtXrgDNajwrY5nqXwpet9tOO6I%3D&amp;reserved=0  https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2F&amp;data=02%7C01%7Cmmodel%40KENT.EDU%7Cd0b72d63264e45d986ea08d74d9d3970%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637063209979705401&amp;sdata=TLXHM80HQWUtSPjsKxBzUZuldFv4PWKgdIxYkcIy%2F5o%3D&amp;reserved=0

[Oshel, Philip Eugene]

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Generally I would agree, but:
Only Siths deal in absolutes. (Which is an absolute...) So, "Always use" and the like is not true. Sometimes one has to do something against the rules to get the best results. So you title "General Best Practices" should be understood as that, not hard rules.
* "No saturated pixels" should be "One or a few saturated and one or a few undersaturated pixels." If there isn't that "one or a few", then the user can't know if they are actually using the full dynamic range available, and therefore could be losing data.
But *only* one or a few.
* There is nothing wrong with saving files as TIFF (or other uncompressed file format), *but* also save the image/experiment in the confocal system's native file format (e.g. nd2 for Nikon).

I would add that initial training MUST include not just how to use the microscopy and software but WHY the various controls and software settings are being used when and as they are.
Meaning, training must include some physics and chemistry as well as electronics. What exactly are the Gain and Offset (Black level) adjusting and in what part of the system. What does varying laser power *really* adjust? The laser itself? a neutral density filter? an ATOF? Why and when would one turn up the Gain instead of the Laser. Etc. and so on.
Easy enough to cover in a class, but these can still be covered during a training session.

My explanation when training is the user is looking at the image, and wants to achieve a certain effect: clean up an image (and what "clean up' means - reduce noise or something else?), get a brighter signal, or whatever. The user should be able to look at the image and go to the correct control(s) and make the correct adjustment(s) without having to sit and think or page through notes or whatever and bleach or kill their sample. They can't do this if they don't understand the whys of the system, not only the hows.

Other than that, I just point out the things they can do that are Bad and Expensive and They Will Get To Pay For It. That works well.

Phil
-------------
Philip Oshel    
Imaging Facility Director
Biology Department
1304 Biosciences
1455 Calumet Ct.
Central Michigan University
Mt. Pleasant, MI 48859
989 774-3576 office

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> on behalf of "Cammer, Michael" <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Thursday,  10October, 2019 at 12:16
To: "[hidden email]" <[hidden email]>
Subject: training and bets practices for confocal

    *****
    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.
    *****
   
    I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
    Cheers-
    Michael
   
   
    General Confocal Best practices:
   
      *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
    If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
    Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
      *   Offset. Always use at 0 or 1.
    Other numbers are wrong.
      *   Digital gain. The preset is 1. Leave it there.
      *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
    Saving Files
    All files should be stored in Drive D:.
    Files left on the desktop, drive C, Pictures folder, etc will be deleted.
    .lsm or .czi always.
    CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
    If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
    Move data to your lab's shared server space.
   
   
   
   
    Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
    NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
    Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
    http://nyulmc.org/micros  http://microscopynotes.com/
   

[Ben Abrams-2]

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Hi,

Regarding the pinhole, I tell my users that 1 AU is almost always a good
place to start, but that there may be some cases where it is worth it to
increase the pinhole size to a degree.  It is still confocal, unless
open to the point where your orthogonal projections look like widefield.
You can get a significant bump in brightness this way ( I'd argue this
/is/ fair) and are still getting a defined optical section thickness, so
3D reconstruction is still possible, just at a lower axial resolution. 
It really depends on the goal of the experiment what the worthwhile
trade-offs are. Users just need to be sure to accurately explain what
they did in the methods section.

I also think offset setting have to be set carefully in a sample/scope
dependent way.  I tell my users to always use a range indicator LUT and
set the offset so they just see a few zero intensity pixels, and have
gain set just below saturation.  This way they are maximizing the
dynamic range of their images.  Samples that are to be compared
quantitatively should use the same settings.  I also talk to them about
the balance of gain and laser power, noise vs. bleaching etc. as Josh
mentioned.

I think it is useful to have this discussion re best practices in
training, thanks.

-Ben

On 10/10/19 9:15 AM, 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.
> *****
>
> I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
> Cheers-
> Michael
>
>
> General Confocal Best practices:
>
>    *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
> If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
> Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
>    *   Offset. Always use at 0 or 1.
> Other numbers are wrong.
>    *   Digital gain. The preset is 1. Leave it there.
>    *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
> Saving Files
> All files should be stored in Drive D:.
> Files left on the desktop, drive C, Pictures folder, etc will be deleted.
> .lsm or .czi always.
> CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
> If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
> Move data to your lab's shared server space.
>
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
> Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/

--
Benjamin Abrams,  Ph.D.
Director
UCSC Life Sciences Microscopy Center

University of California, Santa Cruz
1156 High Street
150 Sinsheimer labs
Santa Cruz, CA 95064

Office/voicemail: [phone]
Mobile: [phone]

http://stemcell.soe.ucsc.edu/facilities/microscopy

Training Request Form:
http://goo.gl/forms/M9jd1ZHyohTnj0xk1

[mmodel]

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In cases when people are interested in relative brightness, I advise them first to image the "most positive" control and adjust the brightness such that there are no or few saturated pixels in the areas of interest. And then to keep the same settings for all other images that need to be compared, even if they look too dark

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Oshel, Philip Eugene <[hidden email]>
Sent: Thursday, October 10, 2019 1:00 PM
To: [hidden email] <[hidden email]>
Subject: Re: training and bets practices for confocal

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*****

Generally I would agree, but:
Only Siths deal in absolutes. (Which is an absolute...) So, "Always use" and the like is not true. Sometimes one has to do something against the rules to get the best results. So you title "General Best Practices" should be understood as that, not hard rules.
* "No saturated pixels" should be "One or a few saturated and one or a few undersaturated pixels." If there isn't that "one or a few", then the user can't know if they are actually using the full dynamic range available, and therefore could be losing data.
But *only* one or a few.
* There is nothing wrong with saving files as TIFF (or other uncompressed file format), *but* also save the image/experiment in the confocal system's native file format (e.g. nd2 for Nikon).

I would add that initial training MUST include not just how to use the microscopy and software but WHY the various controls and software settings are being used when and as they are.
Meaning, training must include some physics and chemistry as well as electronics. What exactly are the Gain and Offset (Black level) adjusting and in what part of the system. What does varying laser power *really* adjust? The laser itself? a neutral density filter? an ATOF? Why and when would one turn up the Gain instead of the Laser. Etc. and so on.
Easy enough to cover in a class, but these can still be covered during a training session.

My explanation when training is the user is looking at the image, and wants to achieve a certain effect: clean up an image (and what "clean up' means - reduce noise or something else?), get a brighter signal, or whatever. The user should be able to look at the image and go to the correct control(s) and make the correct adjustment(s) without having to sit and think or page through notes or whatever and bleach or kill their sample. They can't do this if they don't understand the whys of the system, not only the hows.

Other than that, I just point out the things they can do that are Bad and Expensive and They Will Get To Pay For It. That works well.

Phil
-------------
Philip Oshel
Imaging Facility Director
Biology Department
1304 Biosciences
1455 Calumet Ct.
Central Michigan University
Mt. Pleasant, MI 48859
989 774-3576 office

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> on behalf of "Cammer, Michael" <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Thursday,  10October, 2019 at 12:16
To: "[hidden email]" <[hidden email]>
Subject: training and bets practices for confocal

    *****
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    *****

    I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
    Cheers-
    Michael


    General Confocal Best practices:

      *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
    If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
    Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
      *   Offset. Always use at 0 or 1.
    Other numbers are wrong.
      *   Digital gain. The preset is 1. Leave it there.
      *   Use the Range Indicator button to make sure you have no saturated pixels<https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2Fimagej%2Fsaturation%2Findex.html&amp;data=02%7C01%7Cmmodel%40KENT.EDU%7C836b4364d165408312ed08d74da37a43%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637063236839198887&amp;sdata=Uq06XIU1GeU8V%2FmVDdIxXfJSBPfeJT9ehHAxV6AjWEk%3D&amp;reserved=0>. If you see red pixels, you need to turn down the Gain or Laser.
    Saving Files
    All files should be stored in Drive D:.
    Files left on the desktop, drive C, Pictures folder, etc will be deleted.
    .lsm or .czi always.
    CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
    If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
    Move data to your lab's shared server space.




    Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
    NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
    Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
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[Craig Brideau]

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*****

On Thu, Oct 10, 2019 at 10:16 AM Cammer, Michael <
[hidden email]> wrote:

> *****
>   *   The pinhole is what makes the confocal a confocal. Set at 1AU (which
> means 1 Airy unit) and click the 1AU button each time you change lenses.
> If you are opening it for imaging fixed samples, you should go use a
> widefield fluorescence scope instead.
> Except in special case of live cell imaging where you understand that
> images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES
> BRIGHTER. You won't hurt the instrument, but when you write your methods,
> you won't be accurately describing your microscopy as "confocal".
>

I would say it is not 'Airy matched'. It is still optically sectioning,
which is the point of a confocal, and there are foreseeable situations
where a user may be willing to compromise with a thicker optical slice for
additional signal. Live samples, where you are limited in the amount of
laser power you can use, does come to mind in this case.


>   *   Offset. Always use at 0 or 1.
> Other numbers are wrong.
>   *   Digital gain. The preset is 1. Leave it there.
>

I strongly agree with the above. Never do anything at acquisition time that
you couldn't do later in post processing if absolutely necessary. By using
offset you are throwing away data. Digital Gain simply multiplies the
captured numbers and is not valid as it does nothing for your
signal-to-noise. If you have to, once you have the complete data loaded in
the software of your choice you can play with offset, etc, but for image
ethics purposes any modifications should be clearly stated, and should only
be for the purposes of clarity.


>   *   Use the Range Indicator button to make sure you have no saturated
> pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you
> see red pixels, you need to turn down the Gain or Laser.
> Saving Files
>

I agree that saturation is bad, but there is a subtlety I always have to
emphasize with people who cling too tightly to this mantra: Do not saturate
your *areas of interest* in your sample. Example: your sample exhibits
overstained nuclei when all you care about is microtubules in the
cytoplasm; go ahead and saturate that nucleus if it means you are using
more dynamic range to image the tubulin that you are actually interested in.


> All files should be stored in Drive D:.
> Files left on the desktop, drive C, Pictures folder, etc will be deleted.
>

More generally I tell users to not expect data left on the microscope to
still be there later. I am somewhat flexible on this, but warn users that
if the drive is full beyond a certain threshold I will arbitrarily delete
any stray files to ensure operation of the equipment for the next user. I
also keep a stash of high-capacity thumb drives for users to sign out if
they are desperate for somewhere to temporarily store their data to
transfer it.

Craig

[Jason M. Kirk]

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Hi Michael,

A note on the offset item:

“ * Offset. Always use at 0 or 1.  Other numbers are wrong.”

This offset value is highly dependent on the calibration vendors do behind the scenes. So YMMV when using their defaults such as 0 or 1.

Ideally you just don’t want to clip the low end values in the final image.

What I typically recommend is when using your range indicator LUT, scan the sample with no laser active (AOTF off) and set the offset so all pixels are just above 0 (showing no blue - for Zeiss or green - for Leica).

I find that on a given system - the values wind up being fairly consistent - but there may be some variation between instruments.

Hope this helps!

-Jason

--------------------------------------------
Jason M. Kirk
Technical Director, Optical Imaging & Vital Microscopy Core (OiVM)
Baylor College of Medicine
Ph: 713.798.6486
Email: [hidden email]

http://www.bcm.edu/oivm
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]>
Sent: Thursday, October 10, 2019 11:15:46 AM
To: [hidden email] <[hidden email]>
Subject: training and bets practices for confocal

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*****

I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_imagej_saturation_index.html&d=DwIFAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=yXnfAE8uMdZaMvW02fOtqH6i45jUz8tcrQTMV7z8wSk&m=nsZwr_NZcagcR36b5iKA0OBES834aq7czMZyDVXHHEs&s=w53VhOV7aYHc2JbvneV55tVHLwioHN59G6XB36pQTxs&e= >. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
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[Arvydas Matiukas]

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*****

I support very much Phil's approach "that initial training MUST include
not just how to use the microscopy and software but WHY the various
controls and software settings are being used when and as they are".
However, on the other side I am dissapointed when PI/mentor expects
his/her student become almost a confocal expert after 3hr basic
training, and even suggests to ommit all that physics/optics/electronics
equating that to "rocket science". Quite often I am asked to provide
simple formal training just showing which knobs to turn and buttons to
push because more detail explanation  confuses the student with
microscopy advices given by other lab members and lab protocols. My
approach always is that to achieve the best results a researcher of any
level must understand the details of the techniques he/she is using.
Please share your arguments for in depth training.

My General Confocal Best practices (for optimal image):
*fine adjustment of confocal focus
*pinhole at 1AU (exception only when sample is dim)
*gain providing full detector range (few over/under saturated pixels in
ROI ; laser power, offset, digital gain at defaults)
*pixel size matches lens resolution (exception too long acquisition)
*max scan speed
*averaging to reduce noise
*check color
*z-stack interval >= 0.5 optical slice
*save file in lsm format
*quality samples provide quality images

  Finally, students often aim for a nice live/acquired image instead of
correct raw  image. It is important to obtain raw image with correct
intensities and it can be later processed in many  different ways  to
produce nice looking final image emphasizing details of interest.\

Best regards,
Arvydas

+++++++++++++++++++++++++++++
Arvydas Matiukas, Ph.D.
Manager of NRB Shared Equipment
Director of Confocal&Two-Photon Core
SUNY Upstate Medical University
Neuroscience & Physiology Dept



>>> "Oshel, Philip Eugene" <[hidden email]> 10/10/2019 1:00 PM >>>
*****
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Post images on
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 and include the link in your posting.

*****

Generally I would agree, but:
Only Siths deal in absolutes. (Which is an absolute...) So, "Always
use" and the like is not true. Sometimes one has to do something against
the rules to get the best results. So you title "General Best Practices"
should be understood as that, not hard rules.
* "No saturated pixels" should be "One or a few saturated and one or a
few undersaturated pixels." If there isn't that "one or a few", then the
user can't know if they are actually using the full dynamic range
available, and therefore could be losing data.
But *only* one or a few.
* There is nothing wrong with saving files as TIFF (or other
uncompressed file format), *but* also save the image/experiment in the
confocal system's native file format (e.g. nd2 for Nikon).

I would add that initial training MUST include not just how to use the
microscopy and software but WHY the various controls and software
settings are being used when and as they are.
Meaning, training must include some physics and chemistry as well as
electronics. What exactly are the Gain and Offset (Black level)
adjusting and in what part of the system. What does varying laser power
*really* adjust? The laser itself? a neutral density filter? an ATOF?
Why and when would one turn up the Gain instead of the Laser. Etc. and
so on.
Easy enough to cover in a class, but these can still be covered during
a training session.

My explanation when training is the user is looking at the image, and
wants to achieve a certain effect: clean up an image (and what "clean
up' means - reduce noise or something else?), get a brighter signal, or
whatever. The user should be able to look at the image and go to the
correct control(s) and make the correct adjustment(s) without having to
sit and think or page through notes or whatever and bleach or kill their
sample. They can't do this if they don't understand the whys of the
system, not only the hows.

Other than that, I just point out the things they can do that are Bad
and Expensive and They Will Get To Pay For It. That works well.

Phil
-------------
Philip Oshel
Imaging Facility Director
Biology Department
1304 Biosciences
1455 Calumet Ct.
Central Michigan University
Mt. Pleasant, MI 48859
989 774-3576 office

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> on
behalf of "Cammer, Michael" <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Thursday,  10October, 2019 at 12:16
To: "[hidden email]"
<[hidden email]>
Subject: training and bets practices for confocal

    *****
    To join, leave or search the confocal microscopy listserv, go to:
   
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    Post images on
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 and include the link in your posting.

    *****
   
    I have been providing the following guideline in introductory
confocal training which I thought were critical for data integrity.  But
other people have been disagreeing with all three points.  Interested
whether there is a consensus.  Does anyone disagree with the guidelines
below?  Any comments welcome.
    Cheers-
    Michael
   
   
    General Confocal Best practices:
   
          *   The pinhole is what makes the confocal a confocal. Set at
1AU (which means 1 Airy unit) and click the 1AU button each time you
change lenses.
    If you are opening it for imaging fixed samples, you should go use
a widefield fluorescence scope instead.
    Except in special case of live cell imaging where you understand
that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE
IMAGES BRIGHTER. You won't hurt the instrument, but when you write your
methods, you won't be accurately describing your microscopy as
"confocal".
          *   Offset. Always use at 0 or 1.
    Other numbers are wrong.
          *   Digital gain. The preset is 1. Leave it there.
          *   Use the Range Indicator button to make sure you have no
saturated
pixels<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_imagej_saturation_index.html&d=DwIGaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=pjCVoeD77yFjyEe_DZmcKZn4dP-ybv5rCgAwrdbCikc&s=1EDj55zzjOtnvlVuJH4BW5aZzIIuDWCliEAE3QctagM&e=
>. If you see red pixels, you need to turn down the Gain or Laser.

    Saving Files
    All files should be stored in Drive D:.
    Files left on the desktop, drive C, Pictures folder, etc will be
deleted.
    .lsm or .czi always.
    CZI is best. These files can be opened directly into image analysis
software. These files retain instrument settings, channel integrity, bit
depth, and spatial scale that may be necessary for image analysis.
    If you save files as TIFor other formats, the integrity of color
channels may be lost and you will have no metadata regarding instrument
settings and spatial scale.
    Move data to your lab's shared server space.
   
   
   
   
    Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
    NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New
York, NY  10016
    Office: 646-501-0567 Cell: 914-309-3270
[hidden email]<mailto:[hidden email]>
   
https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIGaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=pjCVoeD77yFjyEe_DZmcKZn4dP-ybv5rCgAwrdbCikc&s=muszPTN4AMHLYRyXQtHY1Xlc4MH7lhjtuwpbvXbsUt8&e=
 
https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIGaQ&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=pjCVoeD77yFjyEe_DZmcKZn4dP-ybv5rCgAwrdbCikc&s=w45b-QuTprUjF4xRrIaFuGZ6sVT47cl3NeK99_sjSP8&e=


   

[Arvydas Matiukas]

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Michael,

Thanks for bringing up for the discussion  general confocal  training principles. It was very useful.
 
I wonder how  .czi image format is better than  .lsm . I have been having problems importing .czi into ImageJ so I advise users on .lsm . Did I miss late development in this situation?


Thanks,
Arvydas
>>> "Cammer, Michael" <[hidden email]> 10/10/2019 12:15 PM >>>
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I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_imagej_saturation_index.html&d=DwIFAg&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=LFeOQi-hox2DT93qvqRrrcP4fZuHIMpsC1pqhPikh6U&s=IBOH5Kp8DzC98-n5vrmelr6womyIubyxQ4_5lZlO17A&e= >. If you see red pixels, you need to turn down the Gain or Laser.

Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=LFeOQi-hox2DT93qvqRrrcP4fZuHIMpsC1pqhPikh6U&s=0B88sZbLC6gptv0_YugIFqxTpSVwUOVHZfGLZkm8gcQ&e=   https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=LFeOQi-hox2DT93qvqRrrcP4fZuHIMpsC1pqhPikh6U&s=PJT9zYLyX3Wnq2p-xpE5g9mkT_z1AJ-H1Njw4GkUrsM&e= 

[Reece, Jeff (NIH/NIDDK) [E]-2]

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For the pinhole, I tell people to start with 1AU, but to feel free to optimize for the question being answered: adjust smaller to get more resolution (when signal allows); or larger if SNR, speed, and photostability requirements can't be met with other adjustments...  

When there is plenty of signal (most fixed specimens), I recommend reducing pinhole for better resolution, with a practical limit of roughly 0.5AU.  The sweet spot is usually 0.6-0.7AU.

I stress to the users, not to report methods merely as "confocal", or mentioning the AU only, but rather report the theoretical optical section thickness achieved with the pinhole, which has more meaning for the users' experiments and the readers.  It bothers me when I read a paper and all they say is "confocal".

Zeiss formats: In years past, I found that Zeiss lsm imports better than czi into Fiji, and even better at reporting the settings in ZEN (Black), although both formats appear to 'ReUse' correctly.  Based on other more recent reports, my feeling is that these problems with czi are less true of newer versions of ZEN and Fiji, with Zeiss focusing now much more on czi.  We have mostly older Zeiss scopes, so lsm is still my default, but I recommend people try both formats and see what works better for them.

Other things you didn't ask about, but I'll quickly mention:
...Find practical upper limit of PMT Gain by scanning with laser off and inspecting detector noise in the background, with contrast increased if it's important to preserve quantitation in dim pixels.
... Decide on a scan zoom and stick with it, so you don't have to deal with changing zoom later for figures.
... For nominally correct sampling in xy, use the 'Optimize' button to choose the # of pixels.  Same for z-stack delta which is for sampling in z.  These values can be changed somewhat, always exceptions, various reasons.  Generally multiply the answer by 1.5-2x if performing decon.
...Use the fastest scan speed possible.
... Don't hit any of the fancier 'automatic' buttons like auto-exposure, settings wizard, or auto-focus, which are limited as to the conditions in which they work correctly.  But thank you for trying, software engineers.
...If you lose your focus, remember where your pinhole setting is, then open it all the way to see and focus your sample again (then return pinhole setting).

Cheers,
Jeff

-----Original Message-----
From: Cammer, Michael <[hidden email]>
Sent: Thursday, October 10, 2019 12:16 PM
To: [hidden email]
Subject: training and bets practices for confocal

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I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
http://nyulmc.org/micros  http://microscopynotes.com/

[Tim Feinstein]

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Offset is useful if you are acquiring in 8-bit mode, since your dynamic range is so low that you can't afford background signal, but (in my opinion) people shouldn't acquire in 8-bit mode.  Even when someone just needs a snapshot for lab meeting I generally encourage folks to take it in 12-bit and post-process in ImageJ.  This seems like a side point, but are there any circumstances where you encourage people to acquire in 8-bit mode?  I understand that it's rare for >256 pixels to strike a detector in the space of one pixel, but electron counts can get pretty high so 12-bit seems more appropriate when you're not using photon counting mode.  

Re pinholes, I encourage people to know the XY/Z resolution limit of each objective on that system so they can keep the pinhole set to 1 AU when they're near that limit.  If resolution (especially in Z) is well below an objective's performance limits then a pinhole of AU=2 shouldn't impact image quality.  Mostly I tell people not to touch it since it's the last thing you should try after you've tried everything else.  

Best,


T


On 10/10/19, 1:29 PM, "Confocal Microscopy List on behalf of Craig Brideau" <[hidden email] on behalf of [hidden email]> wrote:

    *****
    To join, leave or search the confocal microscopy listserv, go to:
    https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7C1d89833f5d144db880cb08d74da774de%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637063253921137431&amp;sdata=tY3L914lPPyNZCTsgzO7PzXRkbePgnvmv6MhOARpZ2w%3D&amp;reserved=0
    Post images on https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7C1d89833f5d144db880cb08d74da774de%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637063253921137431&amp;sdata=aKsAwKn4aUs19kzNRSuPoV%2FJ6NPA2KiWVd6EYLVWqaM%3D&amp;reserved=0 and include the link in your posting.
    *****
   
    On Thu, Oct 10, 2019 at 10:16 AM Cammer, Michael <
    [hidden email]> wrote:
   
    > *****
    >   *   The pinhole is what makes the confocal a confocal. Set at 1AU (which
    > means 1 Airy unit) and click the 1AU button each time you change lenses.
    > If you are opening it for imaging fixed samples, you should go use a
    > widefield fluorescence scope instead.
    > Except in special case of live cell imaging where you understand that
    > images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES
    > BRIGHTER. You won't hurt the instrument, but when you write your methods,
    > you won't be accurately describing your microscopy as "confocal".
    >
   
    I would say it is not 'Airy matched'. It is still optically sectioning,
    which is the point of a confocal, and there are foreseeable situations
    where a user may be willing to compromise with a thicker optical slice for
    additional signal. Live samples, where you are limited in the amount of
    laser power you can use, does come to mind in this case.
   
   
    >   *   Offset. Always use at 0 or 1.
    > Other numbers are wrong.
    >   *   Digital gain. The preset is 1. Leave it there.
    >
   
    I strongly agree with the above. Never do anything at acquisition time that
    you couldn't do later in post processing if absolutely necessary. By using
    offset you are throwing away data. Digital Gain simply multiplies the
    captured numbers and is not valid as it does nothing for your
    signal-to-noise. If you have to, once you have the complete data loaded in
    the software of your choice you can play with offset, etc, but for image
    ethics purposes any modifications should be clearly stated, and should only
    be for the purposes of clarity.
   
   
    >   *   Use the Range Indicator button to make sure you have no saturated
    > pixels<https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2Fimagej%2Fsaturation%2Findex.html&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7C1d89833f5d144db880cb08d74da774de%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637063253921137431&amp;sdata=QcYIH8RK%2BjYKf2QNLCHJ6coQpuXUIlJ3e6KNC9kEq64%3D&amp;reserved=0>. If you
    > see red pixels, you need to turn down the Gain or Laser.
    > Saving Files
    >
   
    I agree that saturation is bad, but there is a subtlety I always have to
    emphasize with people who cling too tightly to this mantra: Do not saturate
    your *areas of interest* in your sample. Example: your sample exhibits
    overstained nuclei when all you care about is microtubules in the
    cytoplasm; go ahead and saturate that nucleus if it means you are using
    more dynamic range to image the tubulin that you are actually interested in.
   
   
    > All files should be stored in Drive D:.
    > Files left on the desktop, drive C, Pictures folder, etc will be deleted.
    >
   
    More generally I tell users to not expect data left on the microscope to
    still be there later. I am somewhat flexible on this, but warn users that
    if the drive is full beyond a certain threshold I will arbitrarily delete
    any stray files to ensure operation of the equipment for the next user. I
    also keep a stash of high-capacity thumb drives for users to sign out if
    they are desperate for somewhere to temporarily store their data to
    transfer it.
   
    Craig
   

[Pat Robison]

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8-bit acquisition can be advisable for extended time series/large volumes,
when a larger file may impact the ability of the acquisition computer to
manage the file. This is especially true if the question of interest is one
of morphology or timing and intensity quantification is not desired.

-Pat Robison

On Thu, Oct 10, 2019, 2:54 PM Reece, Jeff (NIH/NIDDK) [E] <
[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.
> *****
>
> For the pinhole, I tell people to start with 1AU, but to feel free to
> optimize for the question being answered: adjust smaller to get more
> resolution (when signal allows); or larger if SNR, speed, and
> photostability requirements can't be met with other adjustments...
>
> When there is plenty of signal (most fixed specimens), I recommend
> reducing pinhole for better resolution, with a practical limit of roughly
> 0.5AU.  The sweet spot is usually 0.6-0.7AU.
>
> I stress to the users, not to report methods merely as "confocal", or
> mentioning the AU only, but rather report the theoretical optical section
> thickness achieved with the pinhole, which has more meaning for the users'
> experiments and the readers.  It bothers me when I read a paper and all
> they say is "confocal".
>
> Zeiss formats: In years past, I found that Zeiss lsm imports better than
> czi into Fiji, and even better at reporting the settings in ZEN (Black),
> although both formats appear to 'ReUse' correctly.  Based on other more
> recent reports, my feeling is that these problems with czi are less true of
> newer versions of ZEN and Fiji, with Zeiss focusing now much more on czi.
> We have mostly older Zeiss scopes, so lsm is still my default, but I
> recommend people try both formats and see what works better for them.
>
> Other things you didn't ask about, but I'll quickly mention:
> ...Find practical upper limit of PMT Gain by scanning with laser off and
> inspecting detector noise in the background, with contrast increased if
> it's important to preserve quantitation in dim pixels.
> ... Decide on a scan zoom and stick with it, so you don't have to deal
> with changing zoom later for figures.
> ... For nominally correct sampling in xy, use the 'Optimize' button to
> choose the # of pixels.  Same for z-stack delta which is for sampling in
> z.  These values can be changed somewhat, always exceptions, various
> reasons.  Generally multiply the answer by 1.5-2x if performing decon.
> ...Use the fastest scan speed possible.
> ... Don't hit any of the fancier 'automatic' buttons like auto-exposure,
> settings wizard, or auto-focus, which are limited as to the conditions in
> which they work correctly.  But thank you for trying, software engineers.
> ...If you lose your focus, remember where your pinhole setting is, then
> open it all the way to see and focus your sample again (then return pinhole
> setting).
>
> Cheers,
> Jeff
>
> -----Original Message-----
> From: Cammer, Michael <[hidden email]>
> Sent: Thursday, October 10, 2019 12:16 PM
> To: [hidden email]
> Subject: training and bets practices for confocal
>
> *****
> 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.
> *****
>
> I have been providing the following guideline in introductory confocal
> training which I thought were critical for data integrity.  But other
> people have been disagreeing with all three points.  Interested whether
> there is a consensus.  Does anyone disagree with the guidelines below?  Any
> comments welcome.
> Cheers-
> Michael
>
>
> General Confocal Best practices:
>
>   *   The pinhole is what makes the confocal a confocal. Set at 1AU (which
> means 1 Airy unit) and click the 1AU button each time you change lenses.
> If you are opening it for imaging fixed samples, you should go use a
> widefield fluorescence scope instead.
> Except in special case of live cell imaging where you understand that
> images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES
> BRIGHTER. You won't hurt the instrument, but when you write your methods,
> you won't be accurately describing your microscopy as "confocal".
>   *   Offset. Always use at 0 or 1.
> Other numbers are wrong.
>   *   Digital gain. The preset is 1. Leave it there.
>   *   Use the Range Indicator button to make sure you have no saturated
> pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you
> see red pixels, you need to turn down the Gain or Laser.
> Saving Files
> All files should be stored in Drive D:.
> Files left on the desktop, drive C, Pictures folder, etc will be deleted.
> .lsm or .czi always.
> CZI is best. These files can be opened directly into image analysis
> software. These files retain instrument settings, channel integrity, bit
> depth, and spatial scale that may be necessary for image analysis.
> If you save files as TIFor other formats, the integrity of color channels
> may be lost and you will have no metadata regarding instrument settings and
> spatial scale.
> Move data to your lab's shared server space.
>
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
> Office: 646-501-0567 Cell: 914-309-3270  [hidden email]
> <mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/
>

[0000001ed7f52e4a-dmarc-request]

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Hi Arvydas, Michael and others,

We switched to czi when using the Elyra super-resolution microscope, as the lsm format did not handle the small pixel sizes properly. As I understand czi has other advantages such as handling very large images, I mean not only large data sets but single images with e.g. 60,000 x 60,000 pixels which exceeds the 2^31 limit. Maybe not so common in confocal microscopy, but happens when you image a whole coverslip with 300 nm resolution.

A Zeiss application specialist once told me that for the newer Zeiss microscopes (710 onwards?) the digital gain does more than just multiplying by a factor, not sure what exactly, but it seems to me important where the multiplication happens, if it is before the A/D conversion, it might actually help to reduce digitalisation noise. I found it helpful in multi-photon microscopy when the signal is low. 

For the pinhole setting, on the Leica microscopes there is a wavelength setting to which the 1 AU refers (usually hidden), the preset seems to be 580 nm, so you might want to change it for green or blue dyes. They also automatically resets to 1 AU when you change the objective which helps the inexperienced user, but can be very annoying when working with other pinhole sizes. When the signal is very bright we sometimes close the pinhole to increase resolution, easier than working with silly low laser powers like 0.03%

best wishes

Andreas


-----Original Message-----
From: Arvydas Matiukas <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, 10 Oct 2019 19:44
Subject: Re: training and bets practices for confocal

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Michael,

Thanks for bringing up for the discussion  general confocal  training principles. It was very useful.
 
I wonder how  .czi image format is better than  .lsm . I have been having problems importing .czi into ImageJ so I advise users on .lsm . Did I miss late development in this situation?


Thanks,
Arvydas
>>> "Cammer, Michael" <[hidden email]> 10/10/2019 12:15 PM >>>
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*****

I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *  The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *  Offset. Always use at 0 or 1.
Other numbers are wrong.
  *  Digital gain. The preset is 1. Leave it there.
  *  Use the Range Indicator button to make sure you have no saturated pixels<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_imagej_saturation_index.html&d=DwIFAg&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=LFeOQi-hox2DT93qvqRrrcP4fZuHIMpsC1pqhPikh6U&s=IBOH5Kp8DzC98-n5vrmelr6womyIubyxQ4_5lZlO17A&e= >. If you see red pixels, you need to turn down the Gain or Laser.

Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
https://urldefense.proofpoint.com/v2/url?u=http-3A__nyulmc.org_micros&d=DwIFAg&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=LFeOQi-hox2DT93qvqRrrcP4fZuHIMpsC1pqhPikh6U&s=0B88sZbLC6gptv0_YugIFqxTpSVwUOVHZfGLZkm8gcQ&e=   https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_&d=DwIFAg&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=LFeOQi-hox2DT93qvqRrrcP4fZuHIMpsC1pqhPikh6U&s=PJT9zYLyX3Wnq2p-xpE5g9mkT_z1AJ-H1Njw4GkUrsM&e= 

[Mark Scott-3]

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Hi Michael,

I have always tended to teach the offset to be one of either two things, low enough so you just get a few "zero" pixels, or the next step up that results in no zero pixels - my thought is that ensuring the offset is high enough reduces potential for lost information, however part of me always worries about "zero" values when doing any quantification.

Most settings like pinhole size and how much you go into it (along with the reasoning) tends to stem from the user you are training as well.  Often for novice users it is just easier to get them started by claiming 1AU is the optimal confocal setting, especially those who are just interested in getting their images and getting out of there.  I do normally run through what the pinhole actually does to cut out out-of-focus light and the bigger the pinhole the thicker the slice and the more out of focus light they will see, then if they are trying out things like Airyscan, Hyvolution then I will explain more.

I'm with you on not touching Digital Gain, no matter how much vendors try and convince me that it is fine to use these days - in my view, if you need to raise this to see your fluorescence then you probably should be going back to re-optimise your antibody labelling.

Cheers
Mark

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael
Sent: Friday, 11 October 2019 2:16 AM
To: [hidden email]
Subject: training and bets practices for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
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I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
http://nyulmc.org/micros  http://microscopynotes.com/

[George McNamara]

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*****

Hi Jeff,

Confocal Sweet Spot! I love your phrase!!!

I posted a visual at linkedin,
https://www.linkedin.com/posts/georgemcnamara_confocal-sweet-spot-jeff-reese-nih-activity-6588214058011410432-IBV7/ 


I suggest a slight simplification to one, easy to remember value: 0.666
Airy units.

GPU deconvolve all the data, please!!!

Best confocal file formats: OIR and LIF (Olympus FV3000RS and Leica SP8)
... of course I've retired two Zeiss LSM510's. Good to know that CZI
supports 60,000x60,000 pixels (another reply in thread).

PMT gain (HV = High Voltage): my Olympus rep, Jason Brenner, simplified
this for our FV3000RS: the optimal HV setting for our four GaAsP and two
GaAs PMTs is 500 (that is, 500 mV). Yes there are times I suggest
UNoptimal values (usually in steps of 50, but maybe I'll try 666 mV next
time I am at 0.666 Airy Units), but if these PMTs have an optimum,
likely everyone's has an optimum. Since the user has control over the
HV, and the HV controls the amplification of electrons in the PMT (and
ignoring a lot of PMT details), I'll just claim that very few readers
(or my) PMT based confocal microscope are really reporting a 2x change
in fluorophore for intensity level 1000 vs 2000 (after subtracting the
PMT offset floor from the raw data output). More on quantitation later.

PMT offset: depends on the detector, mode (FV3000RS galvo mode is 12-bit
range, resonant scan mode is 10-bit range), I usually teach with Hi-Lo
LUT enabled, "no blue, no red", while live, no averaging.

PMT multiplier: 1 ... and a request to confocal companies with this:
please kill this GUI element!

Better than PMT: Leica HyD in 'counting mode' counts photons. Yes, there
is some counting saturation limit (we have 2nd gen, ask your Leica rep
how much faster counting SMD HyDs are), and other companies have similar
Hybrid detectors, with their own tweaks (and benefits and price points),
I believe first with Hybrid was Becker&Hickl (FLIM, now fast FLIM), now
also ISS, PicoQuant, probably others.

Disclosure: I am registered for the free (fast) TCSPC workshop hosted by
Becker&Hickl and Boston Electronics, Oct 31-Nov 1, 2019, Bethesda MD.
I've also hosted a 2018 Leica FALCON demo, and various microscope demo's
and talks.

https://www.becker-hickl.com/events/13th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-science/

I agree with Jeff: fastest scan speed possible. In particular, Leica SP8
starts up with default of 400 Hz, I tell all users to use 600 Hz (or
faster if their objective lens choice and zoom <--> field of view is
appropriate).

Best way to make live cells brighter: drop EGFP ---- soooooo 1996 ---
Nathan Shaner's AausFP1 is 5x brighter and ultra narrow (for FPs)
emission peak (10x with tandem dimer)
https://www.biorxiv.org/content/10.1101/677344v2.full Plasmids not in
addgene yet (its a preprint), sequence in paper and supplemental info
(and user's might want to use their own codon and/or other expression
optimizations).

***
Confocal quantitation - some thoughts:

-- the lasers on confocal microscopes are not perfectly stable, and
often very unstable (could be the AOTF intensity controller more than
the laser modules - my recollection is Bob Zucker, US EPA, explained
this to me ... and no reason to think any of the confocal companies have
made any serious effort to prevent these fluctiations). So no reason to
think AM vs PM, or even a few minutes apart, stable (to say nothing of
corner to corner at low zoom).

-- I pointed out above there is no reason to think that anyone's PMT
based confocal microscope value 1000 vs 2000 is really a 2x change in
fluorophore concentration (per voxel etc). Or that similar values would
appear if the specimen was scanned AM vs PM (see previous paragraph).

So: most -- that is PMT point scanner - confocal data is UNquantitative
with respect to whatever intensity measurement the user thinks they are
measuring of (a major use of confocal) single cells.

Photon counting is better (some PMTs can be operated in photon counting,
APDs are, but often slow, various Hybrid detectors nice behavior,
trusting Hamamatsu and whoever does the electronics to 'get it right',
or at least not mess it up too badly), but still subject to laser/AOTF
intensity fluctuation issues.


enjoy,
George
p.s. one of the other posts in this thread mentioned Leica SP8 pinhole
value control is hard to find and by default is set to 580 nm: In Leica
LAS X the drop down is easy to find, the "Airy 1.0" is easy to find, the
button uses the midpoint of the emission range when a single detector is
enabled (ex. 510-550 nm range --> 530 nm mid-point). If two detectors
(we have two HyDs) are on, it uses the midpoitn of those. Easy to turn
off one detector, click "1" using the other, then turn on both (user
choice).

On 10/10/2019 2:46 PM, Reece, Jeff (NIH/NIDDK) [E] 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.
> *****
>
> For the pinhole, I tell people to start with 1AU, but to feel free to optimize for the question being answered: adjust smaller to get more resolution (when signal allows); or larger if SNR, speed, and photostability requirements can't be met with other adjustments...
>
> When there is plenty of signal (most fixed specimens), I recommend reducing pinhole for better resolution, with a practical limit of roughly 0.5AU.  The sweet spot is usually 0.6-0.7AU.
>
> I stress to the users, not to report methods merely as "confocal", or mentioning the AU only, but rather report the theoretical optical section thickness achieved with the pinhole, which has more meaning for the users' experiments and the readers.  It bothers me when I read a paper and all they say is "confocal".
>
> Zeiss formats: In years past, I found that Zeiss lsm imports better than czi into Fiji, and even better at reporting the settings in ZEN (Black), although both formats appear to 'ReUse' correctly.  Based on other more recent reports, my feeling is that these problems with czi are less true of newer versions of ZEN and Fiji, with Zeiss focusing now much more on czi.  We have mostly older Zeiss scopes, so lsm is still my default, but I recommend people try both formats and see what works better for them.
>
> Other things you didn't ask about, but I'll quickly mention:
> ...Find practical upper limit of PMT Gain by scanning with laser off and inspecting detector noise in the background, with contrast increased if it's important to preserve quantitation in dim pixels.
> ... Decide on a scan zoom and stick with it, so you don't have to deal with changing zoom later for figures.
> ... For nominally correct sampling in xy, use the 'Optimize' button to choose the # of pixels.  Same for z-stack delta which is for sampling in z.  These values can be changed somewhat, always exceptions, various reasons.  Generally multiply the answer by 1.5-2x if performing decon.
> ...Use the fastest scan speed possible.
> ... Don't hit any of the fancier 'automatic' buttons like auto-exposure, settings wizard, or auto-focus, which are limited as to the conditions in which they work correctly.  But thank you for trying, software engineers.
> ...If you lose your focus, remember where your pinhole setting is, then open it all the way to see and focus your sample again (then return pinhole setting).
>
> Cheers,
> Jeff
>
> -----Original Message-----
> From: Cammer, Michael <[hidden email]>
> Sent: Thursday, October 10, 2019 12:16 PM
> To: [hidden email]
> Subject: training and bets practices for confocal
>
> *****
> 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.
> *****
>
> I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
> Cheers-
> Michael
>
>
> General Confocal Best practices:
>
>    *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
> If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
> Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
>    *   Offset. Always use at 0 or 1.
> Other numbers are wrong.
>    *   Digital gain. The preset is 1. Leave it there.
>    *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
> Saving Files
> All files should be stored in Drive D:.
> Files left on the desktop, drive C, Pictures folder, etc will be deleted.
> .lsm or .czi always.
> CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
> If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
> Move data to your lab's shared server space.
>
>
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
> Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
> http://nyulmc.org/micros  http://microscopynotes.com/

[Jacqueline Ross]

*****
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 everyone,

I'm late to this discussion but have the following comments. We have both Olympus and Zeiss confocals and previously had Leica as well.

Pinhole setting
I explain the function of the pinhole and recommend that they use Auto/1 AU but I also explain that there are times when you might open or close it. For example, we have people doing live imaging of zebrafish embryos who need to do z stacks at each time point. In this case, we often open up the pinhole a bit to get a thicker optical section so that we don't need to take as small a step size to cover the entire depth. It also means we can get away with less laser power as well as lower voltage, resulting in less noisy images. We always discuss the impact on the resolution, i.e. it's not really confocal but it's still much better than widefield as there's still much less out of focus blur. In fact, this morning I opened the pinhole up to double and we managed to get a good enough image to demonstrate that the staining was probably working but needed to be stronger. Sometimes I close the pinhole down, e.g. for fine structures (if there's plenty of signal) or for reflection imaging (always heaps of signal).

Offset
I do teach people to adjust the offset to make sure that they don't have zero pixel values in their image. One or two zero values (blue) are not a big deal but if they have a lot, then it's not good. If they want to do image analysis (intensity-based), deconvolution, then they should try to ensure no black pixels (or white).

Digital gain
I usually don't use it unless the voltage is very high (resulting in high noise) and I'm unable to increase laser power or slow speed down or do averaging because of the temporal resolution I want or because of photobleaching/phototoxicity.

Range indicator/HiLo/QLUT
I tell users always to use the Look Up Tables used to indicate over and under saturation. This is extremely important and I'm always disappointed if I come into a room and see people setting up their imaging without using these LUTs. The same applies (in my opinion) to widefield systems in regard to saturation. People should use LUTs, pixel saturation indicators or check out the histogram.

Files
We're quite nice and a little disorganised. Ideally all files should be gone from the hard drive after a  month at the latest but we don't tidy up files this often. We do have a shared network drive that people can use to transfer files. I do not like any files left on the Desktop though and I will move these into the correct location if I find them there.

File format
For Olympus, I stress that they must always save as .oif or .oib before exporting to TIFF. The same applies for the Zeiss but we also use .lsm for our LSM 710 because I've had issues in the past with the colours coming up incorrectly when using other software with CZI. We now have an LSM 800 and that doesn't allow .lsm so I'm having to change my policy for that system!

Cheers,

Jacqui



Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI:  [phone]

Website:    http://www.auckland.ac.nz/biru

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael
Sent: Friday, 11 October 2019 5:16 a.m.
To: [hidden email]
Subject: training and bets practices for confocal

*****
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.
*****

I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
http://nyulmc.org/micros  http://microscopynotes.com/

[Jacqueline Ross]

*****
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 everyone,

One more thing in regard to setting offset. In my opinion, this very much depends on the sample. We have a lot of people working with very thick tissue sections with a large amount of background staining (DAPI/Hoechst leaching out) and/or autofluorescence (often fixative-induced). For this reason, we do adjust the offset but not to get rid of this completely, just to get to the point where we are still within the range (no zeros).

When I have people with cells on coverslips, they often have to go the other way as the image is too black when offset is set to zero.

Cheers,

Jacqui

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jacqueline Ross
Sent: Friday, 11 October 2019 6:12 p.m.
To: [hidden email]
Subject: Re: training and bets practices for confocal

*****
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 everyone,

I'm late to this discussion but have the following comments. We have both Olympus and Zeiss confocals and previously had Leica as well.

Pinhole setting
I explain the function of the pinhole and recommend that they use Auto/1 AU but I also explain that there are times when you might open or close it. For example, we have people doing live imaging of zebrafish embryos who need to do z stacks at each time point. In this case, we often open up the pinhole a bit to get a thicker optical section so that we don't need to take as small a step size to cover the entire depth. It also means we can get away with less laser power as well as lower voltage, resulting in less noisy images. We always discuss the impact on the resolution, i.e. it's not really confocal but it's still much better than widefield as there's still much less out of focus blur. In fact, this morning I opened the pinhole up to double and we managed to get a good enough image to demonstrate that the staining was probably working but needed to be stronger. Sometimes I close the pinhole down, e.g. for fine structures (if there's plenty of signal) or for reflection imaging (always heaps of signal).

Offset
I do teach people to adjust the offset to make sure that they don't have zero pixel values in their image. One or two zero values (blue) are not a big deal but if they have a lot, then it's not good. If they want to do image analysis (intensity-based), deconvolution, then they should try to ensure no black pixels (or white).

Digital gain
I usually don't use it unless the voltage is very high (resulting in high noise) and I'm unable to increase laser power or slow speed down or do averaging because of the temporal resolution I want or because of photobleaching/phototoxicity.

Range indicator/HiLo/QLUT
I tell users always to use the Look Up Tables used to indicate over and under saturation. This is extremely important and I'm always disappointed if I come into a room and see people setting up their imaging without using these LUTs. The same applies (in my opinion) to widefield systems in regard to saturation. People should use LUTs, pixel saturation indicators or check out the histogram.

Files
We're quite nice and a little disorganised. Ideally all files should be gone from the hard drive after a  month at the latest but we don't tidy up files this often. We do have a shared network drive that people can use to transfer files. I do not like any files left on the Desktop though and I will move these into the correct location if I find them there.

File format
For Olympus, I stress that they must always save as .oif or .oib before exporting to TIFF. The same applies for the Zeiss but we also use .lsm for our LSM 710 because I've had issues in the past with the colours coming up incorrectly when using other software with CZI. We now have an LSM 800 and that doesn't allow .lsm so I'm having to change my policy for that system!

Cheers,

Jacqui



Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit (BIRU)
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Telephone: Ext 87438; DDI:  [phone]

Website:    http://www.auckland.ac.nz/biru

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael
Sent: Friday, 11 October 2019 5:16 a.m.
To: [hidden email]
Subject: training and bets practices for confocal

*****
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.
*****

I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
Cheers-
Michael


General Confocal Best practices:

  *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  *   Offset. Always use at 0 or 1.
Other numbers are wrong.
  *   Digital gain. The preset is 1. Leave it there.
  *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
Saving Files
All files should be stored in Drive D:.
Files left on the desktop, drive C, Pictures folder, etc will be deleted.
.lsm or .czi always.
CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
Move data to your lab's shared server space.




Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
http://nyulmc.org/micros  http://microscopynotes.com/

[Mark Cannell-2]

*****
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*****

Hi George

I'm puzzled as to why you suggest PMTs detectors are non-linear. With proper dynode chain design linearity exists over ~5 orders of magnitude brightness. If the filtering into the A/D converter is too fast for the sampling you could get aliasing effects but that's another story. Photon counting mode is more likely to be non-linear due to pulse pile up at high rates. The intrinsic linearity of PMTs was carefully explored a while ago as they formed the core of all photometry.

Regards Mark

On 10/11/19, 2:17 AM, "Confocal Microscopy List on behalf of George McNamara" <[hidden email] on behalf of [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 Jeff,
   
    Confocal Sweet Spot! I love your phrase!!!
   
    I posted a visual at linkedin,
    https://www.linkedin.com/posts/georgemcnamara_confocal-sweet-spot-jeff-reese-nih-activity-6588214058011410432-IBV7/ 
   
   
    I suggest a slight simplification to one, easy to remember value: 0.666
    Airy units.
   
    GPU deconvolve all the data, please!!!
   
    Best confocal file formats: OIR and LIF (Olympus FV3000RS and Leica SP8)
    ... of course I've retired two Zeiss LSM510's. Good to know that CZI
    supports 60,000x60,000 pixels (another reply in thread).
   
    PMT gain (HV = High Voltage): my Olympus rep, Jason Brenner, simplified
    this for our FV3000RS: the optimal HV setting for our four GaAsP and two
    GaAs PMTs is 500 (that is, 500 mV). Yes there are times I suggest
    UNoptimal values (usually in steps of 50, but maybe I'll try 666 mV next
    time I am at 0.666 Airy Units), but if these PMTs have an optimum,
    likely everyone's has an optimum. Since the user has control over the
    HV, and the HV controls the amplification of electrons in the PMT (and
    ignoring a lot of PMT details), I'll just claim that very few readers
    (or my) PMT based confocal microscope are really reporting a 2x change
    in fluorophore for intensity level 1000 vs 2000 (after subtracting the
    PMT offset floor from the raw data output). More on quantitation later.
   
    PMT offset: depends on the detector, mode (FV3000RS galvo mode is 12-bit
    range, resonant scan mode is 10-bit range), I usually teach with Hi-Lo
    LUT enabled, "no blue, no red", while live, no averaging.
   
    PMT multiplier: 1 ... and a request to confocal companies with this:
    please kill this GUI element!
   
    Better than PMT: Leica HyD in 'counting mode' counts photons. Yes, there
    is some counting saturation limit (we have 2nd gen, ask your Leica rep
    how much faster counting SMD HyDs are), and other companies have similar
    Hybrid detectors, with their own tweaks (and benefits and price points),
    I believe first with Hybrid was Becker&Hickl (FLIM, now fast FLIM), now
    also ISS, PicoQuant, probably others.
   
    Disclosure: I am registered for the free (fast) TCSPC workshop hosted by
    Becker&Hickl and Boston Electronics, Oct 31-Nov 1, 2019, Bethesda MD.
    I've also hosted a 2018 Leica FALCON demo, and various microscope demo's
    and talks.
   
    https://www.becker-hickl.com/events/13th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-science/
   
    I agree with Jeff: fastest scan speed possible. In particular, Leica SP8
    starts up with default of 400 Hz, I tell all users to use 600 Hz (or
    faster if their objective lens choice and zoom <--> field of view is
    appropriate).
   
    Best way to make live cells brighter: drop EGFP ---- soooooo 1996 ---
    Nathan Shaner's AausFP1 is 5x brighter and ultra narrow (for FPs)
    emission peak (10x with tandem dimer)
    https://www.biorxiv.org/content/10.1101/677344v2.full Plasmids not in
    addgene yet (its a preprint), sequence in paper and supplemental info
    (and user's might want to use their own codon and/or other expression
    optimizations).
   
    ***
    Confocal quantitation - some thoughts:
   
    -- the lasers on confocal microscopes are not perfectly stable, and
    often very unstable (could be the AOTF intensity controller more than
    the laser modules - my recollection is Bob Zucker, US EPA, explained
    this to me ... and no reason to think any of the confocal companies have
    made any serious effort to prevent these fluctiations). So no reason to
    think AM vs PM, or even a few minutes apart, stable (to say nothing of
    corner to corner at low zoom).
   
    -- I pointed out above there is no reason to think that anyone's PMT
    based confocal microscope value 1000 vs 2000 is really a 2x change in
    fluorophore concentration (per voxel etc). Or that similar values would
    appear if the specimen was scanned AM vs PM (see previous paragraph).
   
    So: most -- that is PMT point scanner - confocal data is UNquantitative
    with respect to whatever intensity measurement the user thinks they are
    measuring of (a major use of confocal) single cells.
   
    Photon counting is better (some PMTs can be operated in photon counting,
    APDs are, but often slow, various Hybrid detectors nice behavior,
    trusting Hamamatsu and whoever does the electronics to 'get it right',
    or at least not mess it up too badly), but still subject to laser/AOTF
    intensity fluctuation issues.
   
   
    enjoy,
    George
    p.s. one of the other posts in this thread mentioned Leica SP8 pinhole
    value control is hard to find and by default is set to 580 nm: In Leica
    LAS X the drop down is easy to find, the "Airy 1.0" is easy to find, the
    button uses the midpoint of the emission range when a single detector is
    enabled (ex. 510-550 nm range --> 530 nm mid-point). If two detectors
    (we have two HyDs) are on, it uses the midpoitn of those. Easy to turn
    off one detector, click "1" using the other, then turn on both (user
    choice).
   
    On 10/10/2019 2:46 PM, Reece, Jeff (NIH/NIDDK) [E] 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.
    > *****
    >
    > For the pinhole, I tell people to start with 1AU, but to feel free to optimize for the question being answered: adjust smaller to get more resolution (when signal allows); or larger if SNR, speed, and photostability requirements can't be met with other adjustments...
    >
    > When there is plenty of signal (most fixed specimens), I recommend reducing pinhole for better resolution, with a practical limit of roughly 0.5AU.  The sweet spot is usually 0.6-0.7AU.
    >
    > I stress to the users, not to report methods merely as "confocal", or mentioning the AU only, but rather report the theoretical optical section thickness achieved with the pinhole, which has more meaning for the users' experiments and the readers.  It bothers me when I read a paper and all they say is "confocal".
    >
    > Zeiss formats: In years past, I found that Zeiss lsm imports better than czi into Fiji, and even better at reporting the settings in ZEN (Black), although both formats appear to 'ReUse' correctly.  Based on other more recent reports, my feeling is that these problems with czi are less true of newer versions of ZEN and Fiji, with Zeiss focusing now much more on czi.  We have mostly older Zeiss scopes, so lsm is still my default, but I recommend people try both formats and see what works better for them.
    >
    > Other things you didn't ask about, but I'll quickly mention:
    > ...Find practical upper limit of PMT Gain by scanning with laser off and inspecting detector noise in the background, with contrast increased if it's important to preserve quantitation in dim pixels.
    > ... Decide on a scan zoom and stick with it, so you don't have to deal with changing zoom later for figures.
    > ... For nominally correct sampling in xy, use the 'Optimize' button to choose the # of pixels.  Same for z-stack delta which is for sampling in z.  These values can be changed somewhat, always exceptions, various reasons.  Generally multiply the answer by 1.5-2x if performing decon.
    > ...Use the fastest scan speed possible.
    > ... Don't hit any of the fancier 'automatic' buttons like auto-exposure, settings wizard, or auto-focus, which are limited as to the conditions in which they work correctly.  But thank you for trying, software engineers.
    > ...If you lose your focus, remember where your pinhole setting is, then open it all the way to see and focus your sample again (then return pinhole setting).
    >
    > Cheers,
    > Jeff
    >
    > -----Original Message-----
    > From: Cammer, Michael <[hidden email]>
    > Sent: Thursday, October 10, 2019 12:16 PM
    > To: [hidden email]
    > Subject: training and bets practices for confocal
    >
    > *****
    > 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.
    > *****
    >
    > I have been providing the following guideline in introductory confocal training which I thought were critical for data integrity.  But other people have been disagreeing with all three points.  Interested whether there is a consensus.  Does anyone disagree with the guidelines below?  Any comments welcome.
    > Cheers-
    > Michael
    >
    >
    > General Confocal Best practices:
    >
    >    *   The pinhole is what makes the confocal a confocal. Set at 1AU (which means 1 Airy unit) and click the 1AU button each time you change lenses.
    > If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
    > Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
    >    *   Offset. Always use at 0 or 1.
    > Other numbers are wrong.
    >    *   Digital gain. The preset is 1. Leave it there.
    >    *   Use the Range Indicator button to make sure you have no saturated pixels<http://microscopynotes.com/imagej/saturation/index.html>. If you see red pixels, you need to turn down the Gain or Laser.
    > Saving Files
    > All files should be stored in Drive D:.
    > Files left on the desktop, drive C, Pictures folder, etc will be deleted.
    > .lsm or .czi always.
    > CZI is best. These files can be opened directly into image analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.
    > If you save files as TIFor other formats, the integrity of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.
    > Move data to your lab's shared server space.
    >
    >
    >
    >
    > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
    > Office: 646-501-0567 Cell: 914-309-3270  [hidden email]<mailto:[hidden email]>
    > http://nyulmc.org/micros  http://microscopynotes.com/