widefield autofluorescence in unlabeled cells - a filter mystery?

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

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A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.

My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?

Doug

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Douglas W. Cromey, M.S. - Associate Scientific Investigator
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Life Sciences North, Room 463
1333 N. Martin Ave, Tucson, AZ  85721 USA

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[Peter Brunt AVR]

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Hi Doug,
Normally if the excitation filter is failing through burn-out, you'll see this as an increase in the overall noise reaching the camera. With that said, burn-out is usually very easy to spot visually when looking at the filter so if you haven't already, you may want to pull the filters out of the setup and check for obvious signs of degradation in the coating. They may be old filters (pre-hard-coated IBS days) which means it will be difficult to clean them, in which case they might need replacing.

As for seeing cells that aren't there, do the images you see coincide at all with the cells that you're trying to image? Or are they complete different?


Best Regards
 
Peter Brunt



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-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Cromey, Douglas W - (dcromey)
Sent: Monday, April 26, 2021 12:48 PM
To: [hidden email]
Subject: widefield autofluorescence in unlabeled cells - a filter mystery?

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A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.

My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?

Doug

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

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

UA Microscopy Alliance - http://microscopy.arizona.edu/ A collaborative effort to bring information about shared microscopy facilities to the University of Arizona and the community.

[Z.J. Zhang]

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Doug,

Your guess is likely correct.
I used to have a similar system. It is not hard to take the filter wheel off. You can then check the excitation filters and you will likely see physical damages ('turns') on the filter even  with naked eyes. If budget allows, it might be a good idea to change the light source as well.

Zhaojie Zhang
Director, Jenkins Microscopy Facility
University of Wyoming

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Subject: widefield autofluorescence in unlabeled cells - a filter mystery?

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A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.

My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?

Doug

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

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

UA Microscopy Alliance - http://microscopy.arizona.edu/ A collaborative effort to bring information about shared microscopy facilities to the University of Arizona and the community.

[Jonkman, James]

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Hi, Doug.  Are these your own samples or your colleague's samples?  I had someone with a similar problem on one of my microscopes a few months ago.  Their no-primary control cells showed pretty strong fluorescence, and so they brought some cells that had no labeling whatsoever and they still fluoresced.  Eventually we got talking about what kind of cells these were, and it turns out they were some kind of stem cells, which got me thinking that maybe they had previously done cell sorting.  Indeed, they had used fluorophores for cell sorting and for some reason they didn't realize that the fluorescence would persist after fixing for microscopy.  I had the advantage of knowing that that my microscope filters and everything was not the problem, but it still took several sessions before this truth came out.  Maybe your case is completely unrelated but I thought I would just throw this out there!  Good luck with your trouble-shooting,
James

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
Sent: Monday, April 26, 2021 12:48 PM
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Subject: [External] widefield autofluorescence in unlabeled cells - a filter mystery?

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A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.

My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?

Doug

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

office:  LSN 463        email: [hidden email]<mailto:[hidden email]>
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[Moulding, Dale]

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Hi Doug,

Does it have an LED for transmitted light? Is it the LED shining back at the sample? I had this problem on an old scope, when I put my hand between condenser and the 'fluorescence' went away. Fixed with a green interference filter in the transmitted light path.

Cheers

Dale

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Cromey, Douglas W - (dcromey)
Sent: 26 April 2021 17:48
To: [hidden email]
Subject: widefield autofluorescence in unlabeled cells - a filter mystery?

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A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.

My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?

Doug

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

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

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It looks like it is the auto fluorescence from the cell. Do you see Red (561nm excitation) or far red (647nm excitation) fluorescence? This could be due to the cell culture issue often encountered in a biology lab, do you feel difficulty in transfecting cells?  Best try new cell line, new media and make sure the incubator is running under normal condition, etc.




-----Original Message-----
From: Cromey, Douglas W - (dcromey) <[hidden email]>
Sent: Monday, April 26, 2021 12:48 PM
To: [hidden email]
Subject: widefield autofluorescence in unlabeled cells - a filter mystery?

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A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.

My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?

Doug

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

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

UA Microscopy Alliance - http://microscopy.arizona.edu/ A collaborative effort to bring information about shared microscopy facilities to the University of Arizona and the community.

[Zdenek Svindrych-2]

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Hi Doug,
picture = 1000 words. Post a (link to a) picture.
It could be autofluorescence. Do the unlabeled cells look green on other
widefield fluorescence microscopes?
It could be transmission. Block all light between the sample and the
condenser. Does that help? Even a room light on the ceiling could work as
darkfield illumination. Btw, what's the objective lens?
It does not look like filter bleed through. That usually looks like (quite
non-uniform) signal independent of the cells.
Are the cells live or fixed? In PBS or in index-matching mounting medium?
Best, zdenek

On Mon, Apr 26, 2021 at 12:50 PM Cromey, Douglas W - (dcromey) <
[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.
> *****
>
> A colleague asked me to take a look at their widefield microscope. It is
> an inverted microscope with a 100W Hg source, excitation filter wheel, a
> couple of choices for dichroics in the microscope filter changer and a
> filter wheel in front of the camera. They are seeing unlabeled cells
> fluoresce green (FITC/GFP set) with an otherwise black background where
> there are no cells. The microscope is approximately 15 years old.
>
> My guess is that the excitation filters have failed (or are failing) after
> being on the receiving end of a 100W Hg lamp for all this time. Any other
> thoughts?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
>
> office:  LSN 463        email: [hidden email]<mailto:
> [hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> UA Microscopy Alliance - http://microscopy.arizona.edu/
> A collaborative effort to bring information about shared microscopy
> facilities to the University of Arizona and the community.
>

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

[Carol Heckman]

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Unlabeled cells do fluoresce in the yellow-green range.   I have always thought it is because they have soluble molecules such as flavenoids in the cytoplasm.  If the cells are permeabilized, for example, to use them in immunofluorescence procedures, there is much less of this background.
Carol Heckman
Bowling Green State University

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From: Confocal Microscopy List <[hidden email]> on behalf of Cromey, Douglas W - (dcromey) <[hidden email]>
Sent: Monday, April 26, 2021 12:47 PM
To: [hidden email] <[hidden email]>
Subject: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?

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A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.

My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?

Doug

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

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

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When I first learned immunofluorescence, we added a small amount of glut to the PFA, and this caused autofluorescence.  Therefore, we bleached the cells (I think after Saponin extraction) with NaBH4 (which is not compatible with Bodipy).  After 4 washes, we would check the cells on a fluorescence microscope to assure that we had bleached them sufficiently.  I believe other labs similarly use NaIO4  &/or glycine.


This discussion is particularly apropos to us now because a lab asked for my help with what I first thought was a simple autofluorescence problem, but appears to be something more difficult.  It also harkens to a discussion I saw elsewhere about imaging dapi after imaging green because the dapi may photoconvert.


I was going to post a more detailed description of the problem, but the timing is right to post about it now.


Rather than tell the story how we got to this point and discovered the problem, I will jump directly to the problem.


Cell cultures that are fixed with fresh PFA in PBS and Triton extracted have a very faint background when excited with 488 nm light and similarly faint background with secondary ab or isotype labeling.  The background is weak enough to not need further bleaching before the specific labeling.  (So far, no problem.)


The problem is that exposure to UV light (365 ex dapi filter with Hg lamp, 370 nm dapi filter with X-Cite LED, or 395nm or 405 nm LED or laser) causes the cells to emit brightly when subsequently excited at 488 nm.  Practically this means that any imaging of dapi or Hoechst prevents subsequent imaging of green.  This is dose dependent, so more exposure to UV means brighter green (no, we have not plotted a proper curve or looked for saturation, although we have a few data points).  This is not due to dapi photoconverting; we ran controls of completely unlabeled cells mounted both in Prolong Diamond and glycerol (where the response appears to be stronger).   They have seen this problem with a few cells types, so it's not something like a line mistakenly expressing a photoconvertible protein.


Of course there are ways around this.  Stop using dapi as a convenient way to find cells.  Always take picture of green first with the last exposure being dapi.  Switch nucleic acid labels to another color.  But while these work when limiting the staining to three colors, this problem eliminates blue as a possible color.  By widefield fluorescence, tiling is ruled out because the exposed circle is larger than the rectangular camera FOV.


Have other people seen this problem?  Any ideas?


Thank you!!


Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

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________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Carol Heckman <[hidden email]>
Sent: Monday, April 26, 2021 9:02 PM
To: [hidden email]
Subject: Re: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?

[EXTERNAL]

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Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!MXfaZl3l!JWKj7hLYJdK7cci5dKsFff3ry6VmzUy9DjlmxOHN7QCvwbXqdkWHqobavWDtXk9yU6nQ-HA$  and include the link in your posting.
*****

Unlabeled cells do fluoresce in the yellow-green range.   I have always thought it is because they have soluble molecules such as flavenoids in the cytoplasm.  If the cells are permeabilized, for example, to use them in immunofluorescence procedures, there is much less of this background.
Carol Heckman
Bowling Green State University

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cromey, Douglas W - (dcromey) <[hidden email]>
Sent: Monday, April 26, 2021 12:47 PM
To: [hidden email] <[hidden email]>
Subject: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&amp;data=04%7C01%7Checkman%40BGSU.EDU%7Cf170296dad8f4744d55f08d908d3772c%7Ccdcb729d51064d7cb75ba30c455d5b0a%7C1%7C0%7C637550526594680955%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=2losqYJ9Z9qpo5yOu5KBsmNg4AM6XWpo5p1oBLSDCTg%3D&amp;reserved=0 and include the link in your posting.
*****

A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.

My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?

Doug

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

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

UA Microscopy Alliance - https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopy.arizona.edu%2F&amp;data=04%7C01%7Checkman%40BGSU.EDU%7Cf170296dad8f4744d55f08d908d3772c%7Ccdcb729d51064d7cb75ba30c455d5b0a%7C1%7C0%7C637550526594680955%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=qV%2BaW43NPHbyhJnzTKGCzr2KRicX1uPf%2BxkC2%2Fnx3yg%3D&amp;reserved=0
A collaborative effort to bring information about shared microscopy
facilities to the University of Arizona and the community.

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

[George McNamara]

*****
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The solution to all of these problems may be to change the solution: use
PBS (pH 7.4) or 80% glycerol : 20% PBS (pH 7.4) instead of whatever is
being used now (and gently stir with pipet tip - not shaken, not
vortexed) the Glycerol : PBS). Old mounting media often oxidize the
various "antifade" abbreviations/acronyms (DABCO, PPD, etc). Sure, PBS
may permit more rapid photobleaching of some fluorophores; mitigated by
efficient operation of the microscope.

In 2021 there should be very few users who really need DAPI as their DNA
counterstain. Consider using Zombie NIR (BioLegend, and a much cooler
name than DAPI ... need appropriate emission path for 750nm). Perhaps
ThermoFisher can un-retire To-Pro-5. Eliminating DAPI also enables using
the wavelengths for Brilliant Violet 421 (BD Biosciences, also available
from BioLegend and in one flavor [streptavidin] Jackson ImmunoResearch),
or SuperBright 436 (ThermoFisher) or SuperNova v428 (Beckman Coulter).
And then multiplex with tandems. I also note that ThremoFisher recently
acquired Phitonex, so various NovaFluors now available from them (40plex
by flow using NovaFluors, Brilliants, more have been posted online by
Phitonex, most fluorescence microcsopy experiments are stuck at 3plex +
DAPI). See PubMed 33376221 for a recent 65plex microscopy paper with ~10
fluorophores per iteration (Radtke ... Germain 2020 PNAS ... not open
access - can always email Ron Germain for the PDF (emails on that page).

Happy 2021,

George

On 4/26/2021 9:31 PM, Cammer, Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> When I first learned immunofluorescence, we added a small amount of glut to the PFA, and this caused autofluorescence.  Therefore, we bleached the cells (I think after Saponin extraction) with NaBH4 (which is not compatible with Bodipy).  After 4 washes, we would check the cells on a fluorescence microscope to assure that we had bleached them sufficiently.  I believe other labs similarly use NaIO4  &/or glycine.
>
>
> This discussion is particularly apropos to us now because a lab asked for my help with what I first thought was a simple autofluorescence problem, but appears to be something more difficult.  It also harkens to a discussion I saw elsewhere about imaging dapi after imaging green because the dapi may photoconvert.
>
>
> I was going to post a more detailed description of the problem, but the timing is right to post about it now.
>
>
> Rather than tell the story how we got to this point and discovered the problem, I will jump directly to the problem.
>
>
> Cell cultures that are fixed with fresh PFA in PBS and Triton extracted have a very faint background when excited with 488 nm light and similarly faint background with secondary ab or isotype labeling.  The background is weak enough to not need further bleaching before the specific labeling.  (So far, no problem.)
>
>
> The problem is that exposure to UV light (365 ex dapi filter with Hg lamp, 370 nm dapi filter with X-Cite LED, or 395nm or 405 nm LED or laser) causes the cells to emit brightly when subsequently excited at 488 nm.  Practically this means that any imaging of dapi or Hoechst prevents subsequent imaging of green.  This is dose dependent, so more exposure to UV means brighter green (no, we have not plotted a proper curve or looked for saturation, although we have a few data points).  This is not due to dapi photoconverting; we ran controls of completely unlabeled cells mounted both in Prolong Diamond and glycerol (where the response appears to be stronger).   They have seen this problem with a few cells types, so it's not something like a line mistakenly expressing a photoconvertible protein.
>
>
> Of course there are ways around this.  Stop using dapi as a convenient way to find cells.  Always take picture of green first with the last exposure being dapi.  Switch nucleic acid labels to another color.  But while these work when limiting the staining to three colors, this problem eliminates blue as a possible color.  By widefield fluorescence, tiling is ruled out because the exposed circle is larger than the rectangular camera FOV.
>
>
> Have other people seen this problem?  Any ideas?
>
>
> Thank you!!
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
>
> [hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Carol Heckman <[hidden email]>
> Sent: Monday, April 26, 2021 9:02 PM
> To: [hidden email]
> Subject: Re: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?
>
> [EXTERNAL]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!MXfaZl3l!JWKj7hLYJdK7cci5dKsFff3ry6VmzUy9DjlmxOHN7QCvwbXqdkWHqobavWDtXk9yB3Q9Cmw$
> Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!MXfaZl3l!JWKj7hLYJdK7cci5dKsFff3ry6VmzUy9DjlmxOHN7QCvwbXqdkWHqobavWDtXk9yU6nQ-HA$  and include the link in your posting.
> *****
>
> Unlabeled cells do fluoresce in the yellow-green range.   I have always thought it is because they have soluble molecules such as flavenoids in the cytoplasm.  If the cells are permeabilized, for example, to use them in immunofluorescence procedures, there is much less of this background.
> Carol Heckman
> Bowling Green State University
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Cromey, Douglas W - (dcromey) <[hidden email]>
> Sent: Monday, April 26, 2021 12:47 PM
> To: [hidden email] <[hidden email]>
> Subject: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=04%7C01%7Checkman%40BGSU.EDU%7Cf170296dad8f4744d55f08d908d3772c%7Ccdcb729d51064d7cb75ba30c455d5b0a%7C1%7C0%7C637550526594680955%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=dCTyUY%2F8ngiEn%2FVHtKCIEu74ht85UrmxHLeIHH%2FWxIA%3D&amp;reserved=0
> Post images on https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&amp;data=04%7C01%7Checkman%40BGSU.EDU%7Cf170296dad8f4744d55f08d908d3772c%7Ccdcb729d51064d7cb75ba30c455d5b0a%7C1%7C0%7C637550526594680955%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=2losqYJ9Z9qpo5yOu5KBsmNg4AM6XWpo5p1oBLSDCTg%3D&amp;reserved=0 and include the link in your posting.
> *****
>
> A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.
>
> My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
>
> office:  LSN 463        email: [hidden email]<mailto:[hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> UA Microscopy Alliance - https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopy.arizona.edu%2F&amp;data=04%7C01%7Checkman%40BGSU.EDU%7Cf170296dad8f4744d55f08d908d3772c%7Ccdcb729d51064d7cb75ba30c455d5b0a%7C1%7C0%7C637550526594680955%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=qV%2BaW43NPHbyhJnzTKGCzr2KRicX1uPf%2BxkC2%2Fnx3yg%3D&amp;reserved=0
> A collaborative effort to bring information about shared microscopy
> facilities to the University of Arizona and the community.
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

[Cammer, Michael-2]

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

We would very much appreciate troubleshooting help with the question of autofluorescence increasing after exposure to UV light.


However, not using UV is not an option as the lab needs to use 4 probes for the experiments.


Cheers-


Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

[hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/

Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of George McNamara <[hidden email]>
Sent: Tuesday, April 27, 2021 7:41:46 AM
To: [hidden email]
Subject: Re: additional autofluorescence in unlabeled cells - a biochemical mystery?

[EXTERNAL]

*****
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.
*****

The solution to all of these problems may be to change the solution: use
PBS (pH 7.4) or 80% glycerol : 20% PBS (pH 7.4) instead of whatever is
being used now (and gently stir with pipet tip - not shaken, not
vortexed) the Glycerol : PBS). Old mounting media often oxidize the
various "antifade" abbreviations/acronyms (DABCO, PPD, etc). Sure, PBS
may permit more rapid photobleaching of some fluorophores; mitigated by
efficient operation of the microscope.

In 2021 there should be very few users who really need DAPI as their DNA
counterstain. Consider using Zombie NIR (BioLegend, and a much cooler
name than DAPI ... need appropriate emission path for 750nm). Perhaps
ThermoFisher can un-retire To-Pro-5. Eliminating DAPI also enables using
the wavelengths for Brilliant Violet 421 (BD Biosciences, also available
from BioLegend and in one flavor [streptavidin] Jackson ImmunoResearch),
or SuperBright 436 (ThermoFisher) or SuperNova v428 (Beckman Coulter).
And then multiplex with tandems. I also note that ThremoFisher recently
acquired Phitonex, so various NovaFluors now available from them (40plex
by flow using NovaFluors, Brilliants, more have been posted online by
Phitonex, most fluorescence microcsopy experiments are stuck at 3plex +
DAPI). See PubMed 33376221 for a recent 65plex microscopy paper with ~10
fluorophores per iteration (Radtke ... Germain 2020 PNAS ... not open
access - can always email Ron Germain for the PDF (emails on that page).

Happy 2021,

George

On 4/26/2021 9:31 PM, Cammer, Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> When I first learned immunofluorescence, we added a small amount of glut to the PFA, and this caused autofluorescence.  Therefore, we bleached the cells (I think after Saponin extraction) with NaBH4 (which is not compatible with Bodipy).  After 4 washes, we would check the cells on a fluorescence microscope to assure that we had bleached them sufficiently.  I believe other labs similarly use NaIO4  &/or glycine.
>
>
> This discussion is particularly apropos to us now because a lab asked for my help with what I first thought was a simple autofluorescence problem, but appears to be something more difficult.  It also harkens to a discussion I saw elsewhere about imaging dapi after imaging green because the dapi may photoconvert.
>
>
> I was going to post a more detailed description of the problem, but the timing is right to post about it now.
>
>
> Rather than tell the story how we got to this point and discovered the problem, I will jump directly to the problem.
>
>
> Cell cultures that are fixed with fresh PFA in PBS and Triton extracted have a very faint background when excited with 488 nm light and similarly faint background with secondary ab or isotype labeling.  The background is weak enough to not need further bleaching before the specific labeling.  (So far, no problem.)
>
>
> The problem is that exposure to UV light (365 ex dapi filter with Hg lamp, 370 nm dapi filter with X-Cite LED, or 395nm or 405 nm LED or laser) causes the cells to emit brightly when subsequently excited at 488 nm.  Practically this means that any imaging of dapi or Hoechst prevents subsequent imaging of green.  This is dose dependent, so more exposure to UV means brighter green (no, we have not plotted a proper curve or looked for saturation, although we have a few data points).  This is not due to dapi photoconverting; we ran controls of completely unlabeled cells mounted both in Prolong Diamond and glycerol (where the response appears to be stronger).   They have seen this problem with a few cells types, so it's not something like a line mistakenly expressing a photoconvertible protein.
>
>
> Of course there are ways around this.  Stop using dapi as a convenient way to find cells.  Always take picture of green first with the last exposure being dapi.  Switch nucleic acid labels to another color.  But while these work when limiting the staining to three colors, this problem eliminates blue as a possible color.  By widefield fluorescence, tiling is ruled out because the exposed circle is larger than the rectangular camera FOV.
>
>
> Have other people seen this problem?  Any ideas?
>
>
> Thank you!!
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
>
> [hidden email]<mailto:[hidden email]>  http://nyulmc.org/micros  http://microscopynotes.com/
>
> Voice direct only, no text or messages:  1-914-309-3270 and 1-646-501-0567
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Carol Heckman <[hidden email]>
> Sent: Monday, April 26, 2021 9:02 PM
> To: [hidden email]
> Subject: Re: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?
>
> [EXTERNAL]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!MXfaZl3l!JWKj7hLYJdK7cci5dKsFff3ry6VmzUy9DjlmxOHN7QCvwbXqdkWHqobavWDtXk9yB3Q9Cmw$
> Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!MXfaZl3l!JWKj7hLYJdK7cci5dKsFff3ry6VmzUy9DjlmxOHN7QCvwbXqdkWHqobavWDtXk9yU6nQ-HA$  and include the link in your posting.
> *****
>
> Unlabeled cells do fluoresce in the yellow-green range.   I have always thought it is because they have soluble molecules such as flavenoids in the cytoplasm.  If the cells are permeabilized, for example, to use them in immunofluorescence procedures, there is much less of this background.
> Carol Heckman
> Bowling Green State University
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Cromey, Douglas W - (dcromey) <[hidden email]>
> Sent: Monday, April 26, 2021 12:47 PM
> To: [hidden email] <[hidden email]>
> Subject: [EXTERNAL] widefield autofluorescence in unlabeled cells - a filter mystery?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=04%7C01%7Checkman%40BGSU.EDU%7Cf170296dad8f4744d55f08d908d3772c%7Ccdcb729d51064d7cb75ba30c455d5b0a%7C1%7C0%7C637550526594680955%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=dCTyUY%2F8ngiEn%2FVHtKCIEu74ht85UrmxHLeIHH%2FWxIA%3D&amp;reserved=0
> Post images on https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&amp;data=04%7C01%7Checkman%40BGSU.EDU%7Cf170296dad8f4744d55f08d908d3772c%7Ccdcb729d51064d7cb75ba30c455d5b0a%7C1%7C0%7C637550526594680955%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=2losqYJ9Z9qpo5yOu5KBsmNg4AM6XWpo5p1oBLSDCTg%3D&amp;reserved=0 and include the link in your posting.
> *****
>
> A colleague asked me to take a look at their widefield microscope. It is an inverted microscope with a 100W Hg source, excitation filter wheel, a couple of choices for dichroics in the microscope filter changer and a filter wheel in front of the camera. They are seeing unlabeled cells fluoresce green (FITC/GFP set) with an otherwise black background where there are no cells. The microscope is approximately 15 years old.
>
> My guess is that the excitation filters have failed (or are failing) after being on the receiving end of a 100W Hg lamp for all this time. Any other thoughts?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
>
> office:  LSN 463        email: [hidden email]<mailto:[hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> UA Microscopy Alliance - https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopy.arizona.edu%2F&amp;data=04%7C01%7Checkman%40BGSU.EDU%7Cf170296dad8f4744d55f08d908d3772c%7Ccdcb729d51064d7cb75ba30c455d5b0a%7C1%7C0%7C637550526594680955%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&amp;sdata=qV%2BaW43NPHbyhJnzTKGCzr2KRicX1uPf%2BxkC2%2Fnx3yg%3D&amp;reserved=0
> A collaborative effort to bring information about shared microscopy
> facilities to the University of Arizona and the community.
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

[Sathya Srinivasan]

*****
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.
*****

The article that Michael was mentioning about UV conversion of DAPI to
green emitting forms is:
"UV‐activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle
fluorescent dyes into blue‐excited, green‐emitting protonated forms".
Dominika Żurek‐Biesiada  Sylwia Kędracka‐Krok  Jurek W. Dobrucki
Volume83A, Issue5, May 2013, Pages 441-451
(https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.22260)

- There is a tech note on Leica website which deals with this topic:
https://www.leica-microsystems.com/science-lab/learn-how-to-remove-autofluorescence-from-your-confocal-images/

Fixatives, besides the mounting media, imaging dishes, culture media, etc.
also causes autofluorescence to name a few. Spectral imaging and unmixing
if available will be an option.

Just my two cents. Good luck!

Sathya Srinivasan


On Mon, Apr 26, 2021 at 6:32 PM Cammer, Michael <
[hidden email]> wrote:

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>
> When I first learned immunofluorescence, we added a small amount of glut
> to the PFA, and this caused autofluorescence.  Therefore, we bleached the
> cells (I think after Saponin extraction) with NaBH4 (which is not
> compatible with Bodipy).  After 4 washes, we would check the cells on a
> fluorescence microscope to assure that we had bleached them sufficiently.
> I believe other labs similarly use NaIO4  &/or glycine.
>
>
> This discussion is particularly apropos to us now because a lab asked for
> my help with what I first thought was a simple autofluorescence problem,
> but appears to be something more difficult.  It also harkens to a
> discussion I saw elsewhere about imaging dapi after imaging green because
> the dapi may photoconvert.
>
>
> I was going to post a more detailed description of the problem, but the
> timing is right to post about it now.
>
>
> Rather than tell the story how we got to this point and discovered the
> problem, I will jump directly to the problem.
>
>
> Cell cultures that are fixed with fresh PFA in PBS and Triton extracted
> have a very faint background when excited with 488 nm light and similarly
> faint background with secondary ab or isotype labeling.  The background is
> weak enough to not need further bleaching before the specific labeling.
> (So far, no problem.)
>
>
> The problem is that exposure to UV light (365 ex dapi filter with Hg lamp,
> 370 nm dapi filter with X-Cite LED, or 395nm or 405 nm LED or laser) causes
> the cells to emit brightly when subsequently excited at 488 nm.
> Practically this means that any imaging of dapi or Hoechst prevents
> subsequent imaging of green.  This is dose dependent, so more exposure to
> UV means brighter green (no, we have not plotted a proper curve or looked
> for saturation, although we have a few data points).  This is not due to
> dapi photoconverting; we ran controls of completely unlabeled cells mounted
> both in Prolong Diamond and glycerol (where the response appears to be
> stronger).   They have seen this problem with a few cells types, so it's
> not something like a line mistakenly expressing a photoconvertible protein.
>
>
> Of course there are ways around this.  Stop using dapi as a convenient way
> to find cells.  Always take picture of green first with the last exposure
> being dapi.  Switch nucleic acid labels to another color.  But while these
> work when limiting the staining to three colors, this problem eliminates
> blue as a possible color.  By widefield fluorescence, tiling is ruled out
> because the exposed circle is larger than the rectangular camera FOV.
>
>
> Have other people seen this problem?  Any ideas?
>
>
> Thank you!!
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
>
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
>
> [hidden email]<mailto:[hidden email]>
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>
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>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Carol Heckman <[hidden email]>
> Sent: Monday, April 26, 2021 9:02 PM
> To: [hidden email]
> Subject: Re: [EXTERNAL] widefield autofluorescence in unlabeled cells - a
> filter mystery?
>
> [EXTERNAL]
>
> *****
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> Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!MXfaZl3l!JWKj7hLYJdK7cci5dKsFff3ry6VmzUy9DjlmxOHN7QCvwbXqdkWHqobavWDtXk9yU6nQ-HA$
> and include the link in your posting.
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>
> Unlabeled cells do fluoresce in the yellow-green range.   I have always
> thought it is because they have soluble molecules such as flavenoids in the
> cytoplasm.  If the cells are permeabilized, for example, to use them in
> immunofluorescence procedures, there is much less of this background.
> Carol Heckman
> Bowling Green State University
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Cromey, Douglas W - (dcromey) <[hidden email]>
> Sent: Monday, April 26, 2021 12:47 PM
> To: [hidden email] <[hidden email]>
> Subject: [EXTERNAL] widefield autofluorescence in unlabeled cells - a
> filter mystery?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> A colleague asked me to take a look at their widefield microscope. It is
> an inverted microscope with a 100W Hg source, excitation filter wheel, a
> couple of choices for dichroics in the microscope filter changer and a
> filter wheel in front of the camera. They are seeing unlabeled cells
> fluoresce green (FITC/GFP set) with an otherwise black background where
> there are no cells. The microscope is approximately 15 years old.
>
> My guess is that the excitation filters have failed (or are failing) after
> being on the receiving end of a 100W Hg lamp for all this time. Any other
> thoughts?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
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