The case of the disappearing AF647

[Jonkman, James]

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Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p excitation I can no longer detect it, though I know I'm exciting it.  Let me explain further:

Test slide:  I have fixed cultured cells labeled with DAPI and AF647-phallodin (coverslipped, Prolong gold).
Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)

1p with DAPI and AF647 - works great.
I start with 1p excitation (405nm and 633nm respectively), set up 2 sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice images.   Both channels look very nice, with AF647 requiring 8% laser power (at 633nm) and a modest gain setting of around 600V.  (Ok, those are meaningless numbers because the vendors don't bother to calibrate anything in real-world units, but for someone with a similar instrument it might be useful - sorry, pet peeve of mine!).  There is no appreaciable photobleaching with these settings.

2p with DAPI, PMTs in scanhead, PH wide open - works great.
Now I switch to 2p mode, first just to the exact same detectors (forget about NDDs for now).  I start with the DAPI channel by turning off the 405 laser, switching on the 2p laser at 780nm, and opening the pinhole wide open.  I keep the detector gain exactly as before, and now I slowly start increasing the 2p laser power until I achieve the exact same image as the previous 1p image.  Easy!  Again, no appreciable photobleaching with these settings for DAPI.  This makes sense: if I keep the detector the same and open the pinhole wide, there should exist a 2p excitation intensity that gives me more-or-less the exact same result as the 1p excitation.

2p with AF647, PMTs in scanhead, PH wide open - no emission!
Now I do the exact same thing with AF647.  I turn off the 633nm laser, turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I keep the detector gain exactly as it was for 1p excitation of AF647.  Now I slowly start increasing the 2p laser power... but I see nothing!  I can crank it up to 20% power or higher, being mindful of the fact that when you double the excitation, you get 4x the signal for 2p.  Now here's the real conundrum: if I pull it down to a more modest 10% power (which is still super high for our Discover laser), then move the stage to a fresh field of view, I see signal for just a single frame, and then it instantly and completely photobleaches.  In other words, I'm exciting tonnes of fluorescence, but just not detecting it.  In theory, I should be able to do exactly as I did for DAPI: if I leave the detector gain as I had it for 1p excitation, I should be able to change to 2p excitation and increase the laser power slowly until I get the exact same image as I had for 1p.  But something seems to be blocking the emission when I have the 2p laser engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR blocking filter when the 2p laser is scanning?  Our local application specialist is very knowledgeable but is not aware of any such thing.

Other things that I've considered/tried:
-  The 2p laser is coupled in with a "MBS 760+" dichroic, which should reflect wavelengths above 760nm and pass everything below it.  In fact, I already had this in place for the 1p images - it has virtually no effect on the 1p images so for consistency I just had it in from the beginning.  My other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws away some of the AF647 (as observed during 1p excitation) so I want to avoid it.

- I didn't try adjusting the GDD compensation, but again I know that I'm getting strong excitation, just not collecting it so this shouldn't affect anything.

- Maybe the focus is off?  But I tried being very careful with the laser power (cranking up the LUT to catch any hint of signal) and adusting the focus, but there is no better focal plane. In fact, when I move the stage to an adjacent position I can tell briefly that we're perfectly in focus, before it photobleaches.

- I tried the NDD detector.  We recently upgraded to a 4NDD module with 2 PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first element in the series).  This was my first time trying the NDDs with AF647.  We don't see anything at all - not even a hint of light.  There was a 690nm LP filter at the start of the NDD unit which I removed but it didn't help. So this is even more crazy - could there be a problem with both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647 disappearing!?

Thanks for your suggestions!
James

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



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[Benjamin Smith]

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*****

One thing I would try would be to see if I can detect second harmonic
generation (which can be MUCH brighter than 2P excitation).  To do this,
get some solid 2-nitro-4-methyl aniline and mount it on a slide (in my case
I just mixed it with some permount to make a permanent SHG reference
slide).  Mount the slide under the microscope, and see if you can see amber
light (575 nm) reflecting off the slide.  SHG is bright so you should see
the light even with a 10x objective and the room lights on.  This way you
can be certain that you are getting femtosecond pulses at 1150 nm onto the
sample. I would then see if I can image the sample with my detector.  If
you can't, that means that the issue may be in the detector path.  If you
can and you still can't see Alexa 647, that means you may have a filter
blocking in the emission range of Alexa 647.

I do know that non-descanned detectors require high OD IR blocking
(shortpass) filters to block the strong 2P laser reflections from getting
to the detectors.  While technically the detectors don't normally detect
nIR light, they definitely will saturate if exposed to the several
milliwatts of IR light that can reflect off the sample back into the
detector.  Additionally, laser reflections in the 680nm - 850nm range can
be especially damaging as this is in the detection range for most visible
wavelength photocathodes, so it would not surprise me at all if these
wavelengths are being blocked with a high OD filter.  The fact that you can
image DAPI at 720nm without seeing a strong laser reflection suggests such
a filter.

On Fri, Nov 13, 2020 at 12:26 PM Jonkman, James <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see
> Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p
> excitation I can no longer detect it, though I know I'm exciting it.  Let
> me explain further:
>
> Test slide:  I have fixed cultured cells labeled with DAPI and
> AF647-phallodin (coverslipped, Prolong gold).
> Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
> Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)
>
> 1p with DAPI and AF647 - works great.
> I start with 1p excitation (405nm and 633nm respectively), set up 2
> sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice
> images.   Both channels look very nice, with AF647 requiring 8% laser power
> (at 633nm) and a modest gain setting of around 600V.  (Ok, those are
> meaningless numbers because the vendors don't bother to calibrate anything
> in real-world units, but for someone with a similar instrument it might be
> useful - sorry, pet peeve of mine!).  There is no appreaciable
> photobleaching with these settings.
>
> 2p with DAPI, PMTs in scanhead, PH wide open - works great.
> Now I switch to 2p mode, first just to the exact same detectors (forget
> about NDDs for now).  I start with the DAPI channel by turning off the 405
> laser, switching on the 2p laser at 780nm, and opening the pinhole wide
> open.  I keep the detector gain exactly as before, and now I slowly start
> increasing the 2p laser power until I achieve the exact same image as the
> previous 1p image.  Easy!  Again, no appreciable photobleaching with these
> settings for DAPI.  This makes sense: if I keep the detector the same and
> open the pinhole wide, there should exist a 2p excitation intensity that
> gives me more-or-less the exact same result as the 1p excitation.
>
> 2p with AF647, PMTs in scanhead, PH wide open - no emission!
> Now I do the exact same thing with AF647.  I turn off the 633nm laser,
> turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I
> keep the detector gain exactly as it was for 1p excitation of AF647.  Now I
> slowly start increasing the 2p laser power... but I see nothing!  I can
> crank it up to 20% power or higher, being mindful of the fact that when you
> double the excitation, you get 4x the signal for 2p.  Now here's the real
> conundrum: if I pull it down to a more modest 10% power (which is still
> super high for our Discover laser), then move the stage to a fresh field of
> view, I see signal for just a single frame, and then it instantly and
> completely photobleaches.  In other words, I'm exciting tonnes of
> fluorescence, but just not detecting it.  In theory, I should be able to do
> exactly as I did for DAPI: if I leave the detector gain as I had it for 1p
> excitation, I should be able to change to 2p excitation and increase the
> laser power slowly until I get the exact same image as I had for 1p.  But
> something seems to be blocking the emission when I have the 2p laser
> engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR
> blocking filter when the 2p laser is scanning?  Our local application
> specialist is very knowledgeable but is not aware of any such thing.
>
> Other things that I've considered/tried:
> -  The 2p laser is coupled in with a "MBS 760+" dichroic, which should
> reflect wavelengths above 760nm and pass everything below it.  In fact, I
> already had this in place for the 1p images - it has virtually no effect on
> the 1p images so for consistency I just had it in from the beginning.  My
> other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws
> away some of the AF647 (as observed during 1p excitation) so I want to
> avoid it.
>
> - I didn't try adjusting the GDD compensation, but again I know that I'm
> getting strong excitation, just not collecting it so this shouldn't affect
> anything.
>
> - Maybe the focus is off?  But I tried being very careful with the laser
> power (cranking up the LUT to catch any hint of signal) and adusting the
> focus, but there is no better focal plane. In fact, when I move the stage
> to an adjacent position I can tell briefly that we're perfectly in focus,
> before it photobleaches.
>
> - I tried the NDD detector.  We recently upgraded to a 4NDD module with 2
> PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first
> element in the series).  This was my first time trying the NDDs with
> AF647.  We don't see anything at all - not even a hint of light.  There was
> a 690nm LP filter at the start of the NDD unit which I removed but it
> didn't help. So this is even more crazy - could there be a problem with
> both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647
> disappearing!?
>
> Thanks for your suggestions!
> James
>
> -----------------------------------------------
>    James Jonkman, Staff Scientist
>    Advanced Optical Microscopy Facility (AOMF)
>    and Wright Cell Imaging Facility (WCIF)
>    University Health Network
>    MaRS, PMCRT tower, 101 College St., Room 15-305
>    Toronto, ON, CANADA    M5G 1L7
>   [hidden email]<mailto:[hidden email]>  Tel: [phone]
>    www.aomf.ca<http://www.aomf.ca/>
>
>
>
> This e-mail may contain confidential and/or privileged information for the
> sole use of the intended recipient.
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--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

[Marc Reinig-2]

*****
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*****

Have you tried exciting the 647 at 1200 nm?

Marco

Marc R. Reinig
Sara Lab
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz

On Fri, Nov 13, 2020 at 1:14 PM Benjamin Smith
<[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.
> *****
>
> One thing I would try would be to see if I can detect second harmonic
> generation (which can be MUCH brighter than 2P excitation).  To do this,
> get some solid 2-nitro-4-methyl aniline and mount it on a slide (in my case
> I just mixed it with some permount to make a permanent SHG reference
> slide).  Mount the slide under the microscope, and see if you can see amber
> light (575 nm) reflecting off the slide.  SHG is bright so you should see
> the light even with a 10x objective and the room lights on.  This way you
> can be certain that you are getting femtosecond pulses at 1150 nm onto the
> sample. I would then see if I can image the sample with my detector.  If
> you can't, that means that the issue may be in the detector path.  If you
> can and you still can't see Alexa 647, that means you may have a filter
> blocking in the emission range of Alexa 647.
>
> I do know that non-descanned detectors require high OD IR blocking
> (shortpass) filters to block the strong 2P laser reflections from getting
> to the detectors.  While technically the detectors don't normally detect
> nIR light, they definitely will saturate if exposed to the several
> milliwatts of IR light that can reflect off the sample back into the
> detector.  Additionally, laser reflections in the 680nm - 850nm range can
> be especially damaging as this is in the detection range for most visible
> wavelength photocathodes, so it would not surprise me at all if these
> wavelengths are being blocked with a high OD filter.  The fact that you can
> image DAPI at 720nm without seeing a strong laser reflection suggests such
> a filter.
>
> On Fri, Nov 13, 2020 at 12:26 PM Jonkman, James <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your posting.
> > *****
> >
> > Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see
> > Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p
> > excitation I can no longer detect it, though I know I'm exciting it.  Let
> > me explain further:
> >
> > Test slide:  I have fixed cultured cells labeled with DAPI and
> > AF647-phallodin (coverslipped, Prolong gold).
> > Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
> > Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)
> >
> > 1p with DAPI and AF647 - works great.
> > I start with 1p excitation (405nm and 633nm respectively), set up 2
> > sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice
> > images.   Both channels look very nice, with AF647 requiring 8% laser power
> > (at 633nm) and a modest gain setting of around 600V.  (Ok, those are
> > meaningless numbers because the vendors don't bother to calibrate anything
> > in real-world units, but for someone with a similar instrument it might be
> > useful - sorry, pet peeve of mine!).  There is no appreaciable
> > photobleaching with these settings.
> >
> > 2p with DAPI, PMTs in scanhead, PH wide open - works great.
> > Now I switch to 2p mode, first just to the exact same detectors (forget
> > about NDDs for now).  I start with the DAPI channel by turning off the 405
> > laser, switching on the 2p laser at 780nm, and opening the pinhole wide
> > open.  I keep the detector gain exactly as before, and now I slowly start
> > increasing the 2p laser power until I achieve the exact same image as the
> > previous 1p image.  Easy!  Again, no appreciable photobleaching with these
> > settings for DAPI.  This makes sense: if I keep the detector the same and
> > open the pinhole wide, there should exist a 2p excitation intensity that
> > gives me more-or-less the exact same result as the 1p excitation.
> >
> > 2p with AF647, PMTs in scanhead, PH wide open - no emission!
> > Now I do the exact same thing with AF647.  I turn off the 633nm laser,
> > turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I
> > keep the detector gain exactly as it was for 1p excitation of AF647.  Now I
> > slowly start increasing the 2p laser power... but I see nothing!  I can
> > crank it up to 20% power or higher, being mindful of the fact that when you
> > double the excitation, you get 4x the signal for 2p.  Now here's the real
> > conundrum: if I pull it down to a more modest 10% power (which is still
> > super high for our Discover laser), then move the stage to a fresh field of
> > view, I see signal for just a single frame, and then it instantly and
> > completely photobleaches.  In other words, I'm exciting tonnes of
> > fluorescence, but just not detecting it.  In theory, I should be able to do
> > exactly as I did for DAPI: if I leave the detector gain as I had it for 1p
> > excitation, I should be able to change to 2p excitation and increase the
> > laser power slowly until I get the exact same image as I had for 1p.  But
> > something seems to be blocking the emission when I have the 2p laser
> > engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR
> > blocking filter when the 2p laser is scanning?  Our local application
> > specialist is very knowledgeable but is not aware of any such thing.
> >
> > Other things that I've considered/tried:
> > -  The 2p laser is coupled in with a "MBS 760+" dichroic, which should
> > reflect wavelengths above 760nm and pass everything below it.  In fact, I
> > already had this in place for the 1p images - it has virtually no effect on
> > the 1p images so for consistency I just had it in from the beginning.  My
> > other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws
> > away some of the AF647 (as observed during 1p excitation) so I want to
> > avoid it.
> >
> > - I didn't try adjusting the GDD compensation, but again I know that I'm
> > getting strong excitation, just not collecting it so this shouldn't affect
> > anything.
> >
> > - Maybe the focus is off?  But I tried being very careful with the laser
> > power (cranking up the LUT to catch any hint of signal) and adusting the
> > focus, but there is no better focal plane. In fact, when I move the stage
> > to an adjacent position I can tell briefly that we're perfectly in focus,
> > before it photobleaches.
> >
> > - I tried the NDD detector.  We recently upgraded to a 4NDD module with 2
> > PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first
> > element in the series).  This was my first time trying the NDDs with
> > AF647.  We don't see anything at all - not even a hint of light.  There was
> > a 690nm LP filter at the start of the NDD unit which I removed but it
> > didn't help. So this is even more crazy - could there be a problem with
> > both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647
> > disappearing!?
> >
> > Thanks for your suggestions!
> > James
> >
> > -----------------------------------------------
> >    James Jonkman, Staff Scientist
> >    Advanced Optical Microscopy Facility (AOMF)
> >    and Wright Cell Imaging Facility (WCIF)
> >    University Health Network
> >    MaRS, PMCRT tower, 101 College St., Room 15-305
> >    Toronto, ON, CANADA    M5G 1L7
> >   [hidden email]<mailto:[hidden email]>  Tel: [phone]
> >    www.aomf.ca<http://www.aomf.ca/>
> >
> >
> >
> > This e-mail may contain confidential and/or privileged information for the
> > sole use of the intended recipient.
> > Any review or distribution by anyone other than the person for whom it was
> > originally intended is strictly prohibited.
> > If you have received this e-mail in error, please contact the sender and
> > delete all copies.
> > Opinions, conclusions or other information contained in this e-mail may
> > not be that of the organization.
> >
> > If you feel you have received an email from UHN of a commercial nature and
> > would like to be removed from the sender's mailing list please do one of
> > the following:
> > (1) Follow any unsubscribe process the sender has included in their email
> > (2) Where no unsubscribe process has been included, reply to the sender
> > and type "unsubscribe" in the subject line. If you require additional
> > information please go to our UHN Newsletters and Mailing Lists page.
> > Please note that we are unable to automatically unsubscribe individuals
> > from all UHN mailing lists.
> >
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

[Cammer, Michael-2]

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

Have you tried exciting AF647 at around 800 nm?


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 Marc Reinig <[hidden email]>
Sent: Friday, November 13, 2020 4:27:13 PM
To: [hidden email]
Subject: Re: The case of the disappearing AF647

[EXTERNAL]

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*****

Have you tried exciting the 647 at 1200 nm?

Marco

Marc R. Reinig
Sara Lab
W. M. Keck Center for Adaptive Optical Microscopy
University of California Santa Cruz

On Fri, Nov 13, 2020 at 1:14 PM Benjamin Smith
<[hidden email]> wrote:

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=imGOvQ9jr7zOcEIiuq0Lovk5OCzCA_49N3RZxz7FFjg&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=2bebOGF4TOGhGwieKdThZQ_6kIu-oMrTdEFdmR7a95c&e=  and include the link in your posting.
> *****
>
> One thing I would try would be to see if I can detect second harmonic
> generation (which can be MUCH brighter than 2P excitation).  To do this,
> get some solid 2-nitro-4-methyl aniline and mount it on a slide (in my case
> I just mixed it with some permount to make a permanent SHG reference
> slide).  Mount the slide under the microscope, and see if you can see amber
> light (575 nm) reflecting off the slide.  SHG is bright so you should see
> the light even with a 10x objective and the room lights on.  This way you
> can be certain that you are getting femtosecond pulses at 1150 nm onto the
> sample. I would then see if I can image the sample with my detector.  If
> you can't, that means that the issue may be in the detector path.  If you
> can and you still can't see Alexa 647, that means you may have a filter
> blocking in the emission range of Alexa 647.
>
> I do know that non-descanned detectors require high OD IR blocking
> (shortpass) filters to block the strong 2P laser reflections from getting
> to the detectors.  While technically the detectors don't normally detect
> nIR light, they definitely will saturate if exposed to the several
> milliwatts of IR light that can reflect off the sample back into the
> detector.  Additionally, laser reflections in the 680nm - 850nm range can
> be especially damaging as this is in the detection range for most visible
> wavelength photocathodes, so it would not surprise me at all if these
> wavelengths are being blocked with a high OD filter.  The fact that you can
> image DAPI at 720nm without seeing a strong laser reflection suggests such
> a filter.
>
> On Fri, Nov 13, 2020 at 12:26 PM Jonkman, James <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=imGOvQ9jr7zOcEIiuq0Lovk5OCzCA_49N3RZxz7FFjg&e=
> > Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=2bebOGF4TOGhGwieKdThZQ_6kIu-oMrTdEFdmR7a95c&e=  and include the link in your posting.
> > *****
> >
> > Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see
> > Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p
> > excitation I can no longer detect it, though I know I'm exciting it.  Let
> > me explain further:
> >
> > Test slide:  I have fixed cultured cells labeled with DAPI and
> > AF647-phallodin (coverslipped, Prolong gold).
> > Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
> > Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)
> >
> > 1p with DAPI and AF647 - works great.
> > I start with 1p excitation (405nm and 633nm respectively), set up 2
> > sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice
> > images.   Both channels look very nice, with AF647 requiring 8% laser power
> > (at 633nm) and a modest gain setting of around 600V.  (Ok, those are
> > meaningless numbers because the vendors don't bother to calibrate anything
> > in real-world units, but for someone with a similar instrument it might be
> > useful - sorry, pet peeve of mine!).  There is no appreaciable
> > photobleaching with these settings.
> >
> > 2p with DAPI, PMTs in scanhead, PH wide open - works great.
> > Now I switch to 2p mode, first just to the exact same detectors (forget
> > about NDDs for now).  I start with the DAPI channel by turning off the 405
> > laser, switching on the 2p laser at 780nm, and opening the pinhole wide
> > open.  I keep the detector gain exactly as before, and now I slowly start
> > increasing the 2p laser power until I achieve the exact same image as the
> > previous 1p image.  Easy!  Again, no appreciable photobleaching with these
> > settings for DAPI.  This makes sense: if I keep the detector the same and
> > open the pinhole wide, there should exist a 2p excitation intensity that
> > gives me more-or-less the exact same result as the 1p excitation.
> >
> > 2p with AF647, PMTs in scanhead, PH wide open - no emission!
> > Now I do the exact same thing with AF647.  I turn off the 633nm laser,
> > turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I
> > keep the detector gain exactly as it was for 1p excitation of AF647.  Now I
> > slowly start increasing the 2p laser power... but I see nothing!  I can
> > crank it up to 20% power or higher, being mindful of the fact that when you
> > double the excitation, you get 4x the signal for 2p.  Now here's the real
> > conundrum: if I pull it down to a more modest 10% power (which is still
> > super high for our Discover laser), then move the stage to a fresh field of
> > view, I see signal for just a single frame, and then it instantly and
> > completely photobleaches.  In other words, I'm exciting tonnes of
> > fluorescence, but just not detecting it.  In theory, I should be able to do
> > exactly as I did for DAPI: if I leave the detector gain as I had it for 1p
> > excitation, I should be able to change to 2p excitation and increase the
> > laser power slowly until I get the exact same image as I had for 1p.  But
> > something seems to be blocking the emission when I have the 2p laser
> > engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR
> > blocking filter when the 2p laser is scanning?  Our local application
> > specialist is very knowledgeable but is not aware of any such thing.
> >
> > Other things that I've considered/tried:
> > -  The 2p laser is coupled in with a "MBS 760+" dichroic, which should
> > reflect wavelengths above 760nm and pass everything below it.  In fact, I
> > already had this in place for the 1p images - it has virtually no effect on
> > the 1p images so for consistency I just had it in from the beginning.  My
> > other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws
> > away some of the AF647 (as observed during 1p excitation) so I want to
> > avoid it.
> >
> > - I didn't try adjusting the GDD compensation, but again I know that I'm
> > getting strong excitation, just not collecting it so this shouldn't affect
> > anything.
> >
> > - Maybe the focus is off?  But I tried being very careful with the laser
> > power (cranking up the LUT to catch any hint of signal) and adusting the
> > focus, but there is no better focal plane. In fact, when I move the stage
> > to an adjacent position I can tell briefly that we're perfectly in focus,
> > before it photobleaches.
> >
> > - I tried the NDD detector.  We recently upgraded to a 4NDD module with 2
> > PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first
> > element in the series).  This was my first time trying the NDDs with
> > AF647.  We don't see anything at all - not even a hint of light.  There was
> > a 690nm LP filter at the start of the NDD unit which I removed but it
> > didn't help. So this is even more crazy - could there be a problem with
> > both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647
> > disappearing!?
> >
> > Thanks for your suggestions!
> > James
> >
> > -----------------------------------------------
> >    James Jonkman, Staff Scientist
> >    Advanced Optical Microscopy Facility (AOMF)
> >    and Wright Cell Imaging Facility (WCIF)
> >    University Health Network
> >    MaRS, PMCRT tower, 101 College St., Room 15-305
> >    Toronto, ON, CANADA    M5G 1L7
> >   [hidden email]<mailto:[hidden email]>  Tel: [phone]
> >    https://urldefense.proofpoint.com/v2/url?u=http-3A__www.aomf.ca&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=8viPswPYNCo9AZZpsx3Vb609-MXRxIABaCGEe657bFE&e= <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.aomf.ca_&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=DFRE7eEYousEaBseVJhsSZh2InWB6UcJl8Q1BcZNL7c&e= >
> >
> >
> >
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>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> https://urldefense.proofpoint.com/v2/url?u=https-3A__vision.berkeley.edu_faculty_core-2Dgrants-2Dnei_core-2Dgrant-2Dmicroscopic-2Dimaging_&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=Z86flGBIKFY_sNUw1CExsae69XHdlvq8WcTjzhBul8o&e=

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=================================

[Craig Brideau]

*****
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.
*****

As Marc and Michael suggest, try different wavelengths. It may be that
1150nm is doing something strange: In my own experience water and tissue
absorption gets complicated between 900 to 1300nm, so you may be hitting
some sort of absorption sweet spot and are thermally
shifting/cooking/bleaching your sample. The fact you see it for a single
frame and then it vanishes is telling.

Craig

On Fri, Nov 13, 2020 at 2:35 PM Cammer, Michael <
[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.
> *****
>
> Have you tried exciting AF647 at around 800 nm?
>
>
> 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 Marc Reinig <[hidden email]>
> Sent: Friday, November 13, 2020 4:27:13 PM
> To: [hidden email]
> Subject: Re: The case of the disappearing AF647
>
> [EXTERNAL]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=imGOvQ9jr7zOcEIiuq0Lovk5OCzCA_49N3RZxz7FFjg&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=2bebOGF4TOGhGwieKdThZQ_6kIu-oMrTdEFdmR7a95c&e=
> and include the link in your posting.
> *****
>
> Have you tried exciting the 647 at 1200 nm?
>
> Marco
>
> Marc R. Reinig
> Sara Lab
> W. M. Keck Center for Adaptive Optical Microscopy
> University of California Santa Cruz
>
> On Fri, Nov 13, 2020 at 1:14 PM Benjamin Smith
> <[hidden email]> wrote:
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=imGOvQ9jr7zOcEIiuq0Lovk5OCzCA_49N3RZxz7FFjg&e=
> > Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=2bebOGF4TOGhGwieKdThZQ_6kIu-oMrTdEFdmR7a95c&e=
> and include the link in your posting.
> > *****
> >
> > One thing I would try would be to see if I can detect second harmonic
> > generation (which can be MUCH brighter than 2P excitation).  To do this,
> > get some solid 2-nitro-4-methyl aniline and mount it on a slide (in my
> case
> > I just mixed it with some permount to make a permanent SHG reference
> > slide).  Mount the slide under the microscope, and see if you can see
> amber
> > light (575 nm) reflecting off the slide.  SHG is bright so you should see
> > the light even with a 10x objective and the room lights on.  This way you
> > can be certain that you are getting femtosecond pulses at 1150 nm onto
> the
> > sample. I would then see if I can image the sample with my detector.  If
> > you can't, that means that the issue may be in the detector path.  If you
> > can and you still can't see Alexa 647, that means you may have a filter
> > blocking in the emission range of Alexa 647.
> >
> > I do know that non-descanned detectors require high OD IR blocking
> > (shortpass) filters to block the strong 2P laser reflections from getting
> > to the detectors.  While technically the detectors don't normally detect
> > nIR light, they definitely will saturate if exposed to the several
> > milliwatts of IR light that can reflect off the sample back into the
> > detector.  Additionally, laser reflections in the 680nm - 850nm range can
> > be especially damaging as this is in the detection range for most visible
> > wavelength photocathodes, so it would not surprise me at all if these
> > wavelengths are being blocked with a high OD filter.  The fact that you
> can
> > image DAPI at 720nm without seeing a strong laser reflection suggests
> such
> > a filter.
> >
> > On Fri, Nov 13, 2020 at 12:26 PM Jonkman, James <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=imGOvQ9jr7zOcEIiuq0Lovk5OCzCA_49N3RZxz7FFjg&e=
> > > Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=HU1xmMDw8aWYe-4hbODKLiYoxC1sgweVW4bczHiHunY&s=2bebOGF4TOGhGwieKdThZQ_6kIu-oMrTdEFdmR7a95c&e=
> and include the link in your posting.
> > > *****
> > >
> > > Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to
> see
> > > Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p
> > > excitation I can no longer detect it, though I know I'm exciting it.
> Let
> > > me explain further:
> > >
> > > Test slide:  I have fixed cultured cells labeled with DAPI and
> > > AF647-phallodin (coverslipped, Prolong gold).
> > > Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p
> type)
> > > Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)
> > >
> > > 1p with DAPI and AF647 - works great.
> > > I start with 1p excitation (405nm and 633nm respectively), set up 2
> > > sequential tracks, 1AU pinhole, and adjust the internal PMTs to get
> nice
> > > images.   Both channels look very nice, with AF647 requiring 8% laser
> power
> > > (at 633nm) and a modest gain setting of around 600V.  (Ok, those are
> > > meaningless numbers because the vendors don't bother to calibrate
> anything
> > > in real-world units, but for someone with a similar instrument it
> might be
> > > useful - sorry, pet peeve of mine!).  There is no appreaciable
> > > photobleaching with these settings.
> > >
> > > 2p with DAPI, PMTs in scanhead, PH wide open - works great.
> > > Now I switch to 2p mode, first just to the exact same detectors (forget
> > > about NDDs for now).  I start with the DAPI channel by turning off the
> 405
> > > laser, switching on the 2p laser at 780nm, and opening the pinhole wide
> > > open.  I keep the detector gain exactly as before, and now I slowly
> start
> > > increasing the 2p laser power until I achieve the exact same image as
> the
> > > previous 1p image.  Easy!  Again, no appreciable photobleaching with
> these
> > > settings for DAPI.  This makes sense: if I keep the detector the same
> and
> > > open the pinhole wide, there should exist a 2p excitation intensity
> that
> > > gives me more-or-less the exact same result as the 1p excitation.
> > >
> > > 2p with AF647, PMTs in scanhead, PH wide open - no emission!
> > > Now I do the exact same thing with AF647.  I turn off the 633nm laser,
> > > turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I
> > > keep the detector gain exactly as it was for 1p excitation of AF647.
> Now I
> > > slowly start increasing the 2p laser power... but I see nothing!  I can
> > > crank it up to 20% power or higher, being mindful of the fact that
> when you
> > > double the excitation, you get 4x the signal for 2p.  Now here's the
> real
> > > conundrum: if I pull it down to a more modest 10% power (which is still
> > > super high for our Discover laser), then move the stage to a fresh
> field of
> > > view, I see signal for just a single frame, and then it instantly and
> > > completely photobleaches.  In other words, I'm exciting tonnes of
> > > fluorescence, but just not detecting it.  In theory, I should be able
> to do
> > > exactly as I did for DAPI: if I leave the detector gain as I had it
> for 1p
> > > excitation, I should be able to change to 2p excitation and increase
> the
> > > laser power slowly until I get the exact same image as I had for 1p.
> But
> > > something seems to be blocking the emission when I have the 2p laser
> > > engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR
> > > blocking filter when the 2p laser is scanning?  Our local application
> > > specialist is very knowledgeable but is not aware of any such thing.
> > >
> > > Other things that I've considered/tried:
> > > -  The 2p laser is coupled in with a "MBS 760+" dichroic, which should
> > > reflect wavelengths above 760nm and pass everything below it.  In
> fact, I
> > > already had this in place for the 1p images - it has virtually no
> effect on
> > > the 1p images so for consistency I just had it in from the beginning.
> My
> > > other option is an MBS 690+ beamsplitter, but the 690 beamsplitter
> throws
> > > away some of the AF647 (as observed during 1p excitation) so I want to
> > > avoid it.
> > >
> > > - I didn't try adjusting the GDD compensation, but again I know that
> I'm
> > > getting strong excitation, just not collecting it so this shouldn't
> affect
> > > anything.
> > >
> > > - Maybe the focus is off?  But I tried being very careful with the
> laser
> > > power (cranking up the LUT to catch any hint of signal) and adusting
> the
> > > focus, but there is no better focal plane. In fact, when I move the
> stage
> > > to an adjacent position I can tell briefly that we're perfectly in
> focus,
> > > before it photobleaches.
> > >
> > > - I tried the NDD detector.  We recently upgraded to a 4NDD module
> with 2
> > > PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first
> > > element in the series).  This was my first time trying the NDDs with
> > > AF647.  We don't see anything at all - not even a hint of light.
> There was
> > > a 690nm LP filter at the start of the NDD unit which I removed but it
> > > didn't help. So this is even more crazy - could there be a problem with
> > > both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647
> > > disappearing!?
> > >
> > > Thanks for your suggestions!
> > > James
> > >
> > > -----------------------------------------------
> > >    James Jonkman, Staff Scientist
> > >    Advanced Optical Microscopy Facility (AOMF)
> > >    and Wright Cell Imaging Facility (WCIF)
> > >    University Health Network
> > >    MaRS, PMCRT tower, 101 College St., Room 15-305
> > >    Toronto, ON, CANADA    M5G 1L7
> > >   [hidden email]<mailto:[hidden email]>  Tel: [phone]
> > >
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> > --
> > Benjamin E. Smith, Ph. D.
> > Imaging Specialist, Vision Science
> > University of California, Berkeley
> > 195 Life Sciences Addition
> > Berkeley, CA  94720-3200
> > Tel  [phone]
> > Fax [phone]
> > e-mail: [hidden email]
> >
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[Jurkevic, Aleksandr]

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We had good results with 2P excitation of AF647, ATTO647N and STAR 635P at 840-850 nm.


Alexander Jurkevich, PhD
Associate Director
Molecular Cytology Core
University of Missouri
120 Life Sciences Center
1201 E. Rollins St.
Columbia, MO 65211-7310

Phone:    [phone]
Fax:           [phone]
website  http://microscopy.missouri.edu



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael
Sent: Friday, November 13, 2020 3:35 PM
To: [hidden email]
Subject: Re: The case of the disappearing AF647

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Have you tried exciting AF647 at around 800 nm?


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 Marc Reinig <[hidden email]>
Sent: Friday, November 13, 2020 4:27:13 PM
To: [hidden email]
Subject: Re: The case of the disappearing AF647

[EXTERNAL]

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Have you tried exciting the 647 at 1200 nm?

Marco

Marc R. Reinig
Sara Lab
W. M. Keck Center for Adaptive Optical Microscopy University of California Santa Cruz

On Fri, Nov 13, 2020 at 1:14 PM Benjamin Smith <[hidden email]> wrote:

>
> *****
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> *****
>
> One thing I would try would be to see if I can detect second harmonic
> generation (which can be MUCH brighter than 2P excitation).  To do
> this, get some solid 2-nitro-4-methyl aniline and mount it on a slide
> (in my case I just mixed it with some permount to make a permanent SHG
> reference slide).  Mount the slide under the microscope, and see if
> you can see amber light (575 nm) reflecting off the slide.  SHG is
> bright so you should see the light even with a 10x objective and the
> room lights on.  This way you can be certain that you are getting
> femtosecond pulses at 1150 nm onto the sample. I would then see if I
> can image the sample with my detector.  If you can't, that means that
> the issue may be in the detector path.  If you can and you still can't
> see Alexa 647, that means you may have a filter blocking in the emission range of Alexa 647.
>
> I do know that non-descanned detectors require high OD IR blocking
> (shortpass) filters to block the strong 2P laser reflections from
> getting to the detectors.  While technically the detectors don't
> normally detect nIR light, they definitely will saturate if exposed to
> the several milliwatts of IR light that can reflect off the sample
> back into the detector.  Additionally, laser reflections in the 680nm
> - 850nm range can be especially damaging as this is in the detection
> range for most visible wavelength photocathodes, so it would not
> surprise me at all if these wavelengths are being blocked with a high
> OD filter.  The fact that you can image DAPI at 720nm without seeing a
> strong laser reflection suggests such a filter.
>
> On Fri, Nov 13, 2020 at 12:26 PM Jonkman, James <
> [hidden email]> wrote:
>
> > *****
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> > *****
> >
> > Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able
> > to see Alexa 647 on my Zeiss LSM710 in confocal mode, but when I
> > switch to 2p excitation I can no longer detect it, though I know I'm
> > exciting it.  Let me explain further:
> >
> > Test slide:  I have fixed cultured cells labeled with DAPI and
> > AF647-phallodin (coverslipped, Prolong gold).
> > Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p
> > type)
> > Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed
> > beam)
> >
> > 1p with DAPI and AF647 - works great.
> > I start with 1p excitation (405nm and 633nm respectively), set up 2
> > sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice
> > images.   Both channels look very nice, with AF647 requiring 8% laser power
> > (at 633nm) and a modest gain setting of around 600V.  (Ok, those are
> > meaningless numbers because the vendors don't bother to calibrate
> > anything in real-world units, but for someone with a similar
> > instrument it might be useful - sorry, pet peeve of mine!).  There
> > is no appreaciable photobleaching with these settings.
> >
> > 2p with DAPI, PMTs in scanhead, PH wide open - works great.
> > Now I switch to 2p mode, first just to the exact same detectors
> > (forget about NDDs for now).  I start with the DAPI channel by
> > turning off the 405 laser, switching on the 2p laser at 780nm, and
> > opening the pinhole wide open.  I keep the detector gain exactly as
> > before, and now I slowly start increasing the 2p laser power until I
> > achieve the exact same image as the previous 1p image.  Easy!  
> > Again, no appreciable photobleaching with these settings for DAPI.  
> > This makes sense: if I keep the detector the same and open the
> > pinhole wide, there should exist a 2p excitation intensity that gives me more-or-less the exact same result as the 1p excitation.
> >
> > 2p with AF647, PMTs in scanhead, PH wide open - no emission!
> > Now I do the exact same thing with AF647.  I turn off the 633nm
> > laser, turn on the 2p laser at, say, 1150nm and open the pinhole
> > wide open.  I keep the detector gain exactly as it was for 1p
> > excitation of AF647.  Now I slowly start increasing the 2p laser
> > power... but I see nothing!  I can crank it up to 20% power or
> > higher, being mindful of the fact that when you double the
> > excitation, you get 4x the signal for 2p.  Now here's the real
> > conundrum: if I pull it down to a more modest 10% power (which is
> > still super high for our Discover laser), then move the stage to a
> > fresh field of view, I see signal for just a single frame, and then
> > it instantly and completely photobleaches.  In other words, I'm
> > exciting tonnes of fluorescence, but just not detecting it.  In
> > theory, I should be able to do exactly as I did for DAPI: if I leave
> > the detector gain as I had it for 1p excitation, I should be able to
> > change to 2p excitation and increase the laser power slowly until I
> > get the exact same image as I had for 1p.  But something seems to be
> > blocking the emission when I have the 2p laser engaged.  Has anybody
> > else seen this problem?  Does Zeiss slip in an IR blocking filter
> > when the 2p laser is scanning?  Our local application specialist is very knowledgeable but is not aware of any such thing.
> >
> > Other things that I've considered/tried:
> > -  The 2p laser is coupled in with a "MBS 760+" dichroic, which
> > should reflect wavelengths above 760nm and pass everything below it.  
> > In fact, I already had this in place for the 1p images - it has
> > virtually no effect on the 1p images so for consistency I just had
> > it in from the beginning.  My other option is an MBS 690+
> > beamsplitter, but the 690 beamsplitter throws away some of the AF647
> > (as observed during 1p excitation) so I want to avoid it.
> >
> > - I didn't try adjusting the GDD compensation, but again I know that
> > I'm getting strong excitation, just not collecting it so this
> > shouldn't affect anything.
> >
> > - Maybe the focus is off?  But I tried being very careful with the
> > laser power (cranking up the LUT to catch any hint of signal) and
> > adusting the focus, but there is no better focal plane. In fact,
> > when I move the stage to an adjacent position I can tell briefly
> > that we're perfectly in focus, before it photobleaches.
> >
> > - I tried the NDD detector.  We recently upgraded to a 4NDD module
> > with 2 PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT
> > (first element in the series).  This was my first time trying the
> > NDDs with AF647.  We don't see anything at all - not even a hint of
> > light.  There was a 690nm LP filter at the start of the NDD unit
> > which I removed but it didn't help. So this is even more crazy -
> > could there be a problem with both detectors!?  DAPI on the NDD
> > looks fantastic.  But why is my AF647 disappearing!?
> >
> > Thanks for your suggestions!
> > James
> >
> > -----------------------------------------------
> >    James Jonkman, Staff Scientist
> >    Advanced Optical Microscopy Facility (AOMF)
> >    and Wright Cell Imaging Facility (WCIF)
> >    University Health Network
> >    MaRS, PMCRT tower, 101 College St., Room 15-305
> >    Toronto, ON, CANADA    M5G 1L7
> >   [hidden email]<mailto:[hidden email]>  Tel: [phone]
> >    
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.aomf.ca&d=Dw
> > IBaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2
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> > HiHunY&s=8viPswPYNCo9AZZpsx3Vb609-MXRxIABaCGEe657bFE&e=
> > <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.aomf.ca_&d=
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> > czHiHunY&s=DFRE7eEYousEaBseVJhsSZh2InWB6UcJl8Q1BcZNL7c&e= >
> >
> >
> >
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>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> https://urldefense.proofpoint.com/v2/url?u=https-3A__vision.berkeley.e
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Hi James,

is it possible the high laser powers during 2p excitation puts the dye into a long-lived dark state? Alexa Fluor 647 can be a tricky dye, which you can observe when trying to get good STED images (in 2 colors). AF 647 is bright and photostable and has a great STED efficiency, meaning you can get great images even for very low STED powers. But couple AF 647 with a second dye that requires a higher STED power (so most other red dyes), then the AF 647 signal disappears almost instantaneously. It is impossible to record in a line-interleaved mode, as you can literally watch the far-red signal disappear with the scanning laser, and requires instead two sequential tracks (AF 647 first). What happens is the 775 nm STED laser bumps the AF 647 into a long-lived dark state, from which it recovers after 30--60 minutes. The blinking and photoswitchable dark-state are a reason why AF 647 is frequently used in dSTORM imaging modalities.

(See here: https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.201904117)

I've never tried imaging AF 647 with any 2p signal, but the laser power intensities of a 2p-excitation and a STED laser should be fairly comparable (with the 2p pulses being much tighter, so I would expect a much more severe effect). How about first imaging with 1p, then trying to "bleach" away a square pattern using 2p, then waiting half an hour or longer before recording another 1p image. I have no idea what happens at those longer wavelengths, but this should be an easy test you could attempt.

Let me know what happens!

Have a great weekend,
Nicolai

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Nicolai T. Urban, Ph.D.
MPFI Imaging Center - Light Microscopy Core
Max Planck Florida Institute for Neuroscience
One Max Planck Way, Jupiter FL 33418






-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jonkman, James
Sent: Friday, 13 November 2020 15:16
To: [hidden email]
Subject: The case of the disappearing AF647

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Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p excitation I can no longer detect it, though I know I'm exciting it.  Let me explain further:

Test slide:  I have fixed cultured cells labeled with DAPI and AF647-phallodin (coverslipped, Prolong gold).
Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)

1p with DAPI and AF647 - works great.
I start with 1p excitation (405nm and 633nm respectively), set up 2 sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice images.   Both channels look very nice, with AF647 requiring 8% laser power (at 633nm) and a modest gain setting of around 600V.  (Ok, those are meaningless numbers because the vendors don't bother to calibrate anything in real-world units, but for someone with a similar instrument it might be useful - sorry, pet peeve of mine!).  There is no appreaciable photobleaching with these settings.

2p with DAPI, PMTs in scanhead, PH wide open - works great.
Now I switch to 2p mode, first just to the exact same detectors (forget about NDDs for now).  I start with the DAPI channel by turning off the 405 laser, switching on the 2p laser at 780nm, and opening the pinhole wide open.  I keep the detector gain exactly as before, and now I slowly start increasing the 2p laser power until I achieve the exact same image as the previous 1p image.  Easy!  Again, no appreciable photobleaching with these settings for DAPI.  This makes sense: if I keep the detector the same and open the pinhole wide, there should exist a 2p excitation intensity that gives me more-or-less the exact same result as the 1p excitation.

2p with AF647, PMTs in scanhead, PH wide open - no emission!
Now I do the exact same thing with AF647.  I turn off the 633nm laser, turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I keep the detector gain exactly as it was for 1p excitation of AF647.  Now I slowly start increasing the 2p laser power... but I see nothing!  I can crank it up to 20% power or higher, being mindful of the fact that when you double the excitation, you get 4x the signal for 2p.  Now here's the real conundrum: if I pull it down to a more modest 10% power (which is still super high for our Discover laser), then move the stage to a fresh field of view, I see signal for just a single frame, and then it instantly and completely photobleaches.  In other words, I'm exciting tonnes of fluorescence, but just not detecting it.  In theory, I should be able to do exactly as I did for DAPI: if I leave the detector gain as I had it for 1p excitation, I should be able to change to 2p excitation and increase the laser power slowly until I get the exact same image as I had for 1p.  But something seems to be blocking the emission when I have the 2p laser engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR blocking filter when the 2p laser is scanning?  Our local application specialist is very knowledgeable but is not aware of any such thing.

Other things that I've considered/tried:
-  The 2p laser is coupled in with a "MBS 760+" dichroic, which should reflect wavelengths above 760nm and pass everything below it.  In fact, I already had this in place for the 1p images - it has virtually no effect on the 1p images so for consistency I just had it in from the beginning.  My other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws away some of the AF647 (as observed during 1p excitation) so I want to avoid it.

- I didn't try adjusting the GDD compensation, but again I know that I'm getting strong excitation, just not collecting it so this shouldn't affect anything.

- Maybe the focus is off?  But I tried being very careful with the laser power (cranking up the LUT to catch any hint of signal) and adusting the focus, but there is no better focal plane. In fact, when I move the stage to an adjacent position I can tell briefly that we're perfectly in focus, before it photobleaches.

- I tried the NDD detector.  We recently upgraded to a 4NDD module with 2 PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first element in the series).  This was my first time trying the NDDs with AF647.  We don't see anything at all - not even a hint of light.  There was a 690nm LP filter at the start of the NDD unit which I removed but it didn't help. So this is even more crazy - could there be a problem with both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647 disappearing!?

Thanks for your suggestions!
James

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

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Dear James,

We also experience difficulty imaging AF647 with two-photon excitation. In case of bright labeling, we can image it only once, then it has been bleached for most of its part. Furthermore, we have to start imaging with the long wavelength first in case we do sequential imaging including shorter excitations such as dapi.

It might indeed be that AF647 is put in a dark state, however, I never saw recovery after multiphoton imaging of AF647. In your case, exciting with 780 first might have excited AF647 as well. Many red dyes absorb energy efficiently at much shorter wavelength. I remember a article from Drobishev et al., Nat. Meth 2011, stating that at these short wavelengths molecules are being excited in a higher than first electron state. The energy release of these higher states favor chemical alteration of the molecules, causing rapid bleaching.

Turning around the sequence and starting with AF647 excitation might give you an image, if the detectors are sensitive enough and if labeling density is sufficient.

Kind regards,
Gert-Jan




G.J. Bakker, PhD
Researcher / Multiphoton microscopy specialist
Dept. of Cell Biology (283) and Microscopic Imaging Centre
[hidden email]
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________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
Sent: Saturday, November 14, 2020 12:29 AM
To: [hidden email]
Subject: Re: The case of the disappearing AF647

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Hi James,

is it possible the high laser powers during 2p excitation puts the dye into a long-lived dark state? Alexa Fluor 647 can be a tricky dye, which you can observe when trying to get good STED images (in 2 colors). AF 647 is bright and photostable and has a great STED efficiency, meaning you can get great images even for very low STED powers. But couple AF 647 with a second dye that requires a higher STED power (so most other red dyes), then the AF 647 signal disappears almost instantaneously. It is impossible to record in a line-interleaved mode, as you can literally watch the far-red signal disappear with the scanning laser, and requires instead two sequential tracks (AF 647 first). What happens is the 775 nm STED laser bumps the AF 647 into a long-lived dark state, from which it recovers after 30--60 minutes. The blinking and photoswitchable dark-state are a reason why AF 647 is frequently used in dSTORM imaging modalities.

(See here: https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.201904117)

I've never tried imaging AF 647 with any 2p signal, but the laser power intensities of a 2p-excitation and a STED laser should be fairly comparable (with the 2p pulses being much tighter, so I would expect a much more severe effect). How about first imaging with 1p, then trying to "bleach" away a square pattern using 2p, then waiting half an hour or longer before recording another 1p image. I have no idea what happens at those longer wavelengths, but this should be an easy test you could attempt.

Let me know what happens!

Have a great weekend,
Nicolai

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Nicolai T. Urban, Ph.D.
MPFI Imaging Center - Light Microscopy Core
Max Planck Florida Institute for Neuroscience
One Max Planck Way, Jupiter FL 33418






-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jonkman, James
Sent: Friday, 13 November 2020 15:16
To: [hidden email]
Subject: The case of the disappearing AF647

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Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p excitation I can no longer detect it, though I know I'm exciting it.  Let me explain further:

Test slide:  I have fixed cultured cells labeled with DAPI and AF647-phallodin (coverslipped, Prolong gold).
Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)

1p with DAPI and AF647 - works great.
I start with 1p excitation (405nm and 633nm respectively), set up 2 sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice images.   Both channels look very nice, with AF647 requiring 8% laser power (at 633nm) and a modest gain setting of around 600V.  (Ok, those are meaningless numbers because the vendors don't bother to calibrate anything in real-world units, but for someone with a similar instrument it might be useful - sorry, pet peeve of mine!).  There is no appreaciable photobleaching with these settings.

2p with DAPI, PMTs in scanhead, PH wide open - works great.
Now I switch to 2p mode, first just to the exact same detectors (forget about NDDs for now).  I start with the DAPI channel by turning off the 405 laser, switching on the 2p laser at 780nm, and opening the pinhole wide open.  I keep the detector gain exactly as before, and now I slowly start increasing the 2p laser power until I achieve the exact same image as the previous 1p image.  Easy!  Again, no appreciable photobleaching with these settings for DAPI.  This makes sense: if I keep the detector the same and open the pinhole wide, there should exist a 2p excitation intensity that gives me more-or-less the exact same result as the 1p excitation.

2p with AF647, PMTs in scanhead, PH wide open - no emission!
Now I do the exact same thing with AF647.  I turn off the 633nm laser, turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I keep the detector gain exactly as it was for 1p excitation of AF647.  Now I slowly start increasing the 2p laser power... but I see nothing!  I can crank it up to 20% power or higher, being mindful of the fact that when you double the excitation, you get 4x the signal for 2p.  Now here's the real conundrum: if I pull it down to a more modest 10% power (which is still super high for our Discover laser), then move the stage to a fresh field of view, I see signal for just a single frame, and then it instantly and completely photobleaches.  In other words, I'm exciting tonnes of fluorescence, but just not detecting it.  In theory, I should be able to do exactly as I did for DAPI: if I leave the detector gain as I had it for 1p excitation, I should be able to change to 2p excitation and increase the laser power slowly until I get the exact same image as I had for 1p.  But something seems to be blocking the emission when I have the 2p laser engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR blocking filter when the 2p laser is scanning?  Our local application specialist is very knowledgeable but is not aware of any such thing.

Other things that I've considered/tried:
-  The 2p laser is coupled in with a "MBS 760+" dichroic, which should reflect wavelengths above 760nm and pass everything below it.  In fact, I already had this in place for the 1p images - it has virtually no effect on the 1p images so for consistency I just had it in from the beginning.  My other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws away some of the AF647 (as observed during 1p excitation) so I want to avoid it.

- I didn't try adjusting the GDD compensation, but again I know that I'm getting strong excitation, just not collecting it so this shouldn't affect anything.

- Maybe the focus is off?  But I tried being very careful with the laser power (cranking up the LUT to catch any hint of signal) and adusting the focus, but there is no better focal plane. In fact, when I move the stage to an adjacent position I can tell briefly that we're perfectly in focus, before it photobleaches.

- I tried the NDD detector.  We recently upgraded to a 4NDD module with 2 PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first element in the series).  This was my first time trying the NDDs with AF647.  We don't see anything at all - not even a hint of light.  There was a 690nm LP filter at the start of the NDD unit which I removed but it didn't help. So this is even more crazy - could there be a problem with both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647 disappearing!?

Thanks for your suggestions!
James

-----------------------------------------------
   James Jonkman, Staff Scientist
   Advanced Optical Microscopy Facility (AOMF)
   and Wright Cell Imaging Facility (WCIF)
   University Health Network
   MaRS, PMCRT tower, 101 College St., Room 15-305
   Toronto, ON, CANADA    M5G 1L7
  [hidden email]<mailto:[hidden email]>  Tel: [phone]
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[Martin Spitaler-2]

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Hi James,

   I think the answer to your question is simple, and you have already described it yourself: The fluorophores disappear after an instant. Nicolai might have explained part of the problem with the dark states, but you forgot an important difference between 1P and 2P excitation: The pulsing. The peak power is massive, which is fine in water as you know, you can do wonderful intravital imaging without major damage to the animal, because in water the heat dissipates instantly. But you are imaging in a fixed sample mounted in Prolong Gold sandwiched between two bits of glass. The heat can't go anywhere, and if you would have tried imaging the DAPI for a bit longer, you would also have observed nice little holes appearing in your Prolong, as if the nuclei would explode (which is exactly what they do). Probably Alx647 is just less heat-stable than DAPI, so it's instantly destroyed. Try the same in water, and it will all be fine.

Best wishes, and have fun trying out the nuclear explosions,

Martin

________________________________________
Martin Spitaler, PhD
Head of the
Imaging Facility
Max Planck Institute of Biochemistry
Am Klopferspitz 18
82152 Martinsried
Germany
Tel: [phone]
E-mail: [hidden email]
Website: https://www.biochem.mpg.de/de/imaging

[Sylvie Le Guyader]

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Hi Martin

This explains how I managed to burn holes in my convallaria samples a few years ago! Very nice to get an explanation! :)

Med vänlig hälsning / Best regards

Sylvie

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

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Martin Spitaler
Sent: 17 November 2020 19:29
To: [hidden email]
Subject: Re: The case of the disappearing AF647

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Hi James,

   I think the answer to your question is simple, and you have already described it yourself: The fluorophores disappear after an instant. Nicolai might have explained part of the problem with the dark states, but you forgot an important difference between 1P and 2P excitation: The pulsing. The peak power is massive, which is fine in water as you know, you can do wonderful intravital imaging without major damage to the animal, because in water the heat dissipates instantly. But you are imaging in a fixed sample mounted in Prolong Gold sandwiched between two bits of glass. The heat can't go anywhere, and if you would have tried imaging the DAPI for a bit longer, you would also have observed nice little holes appearing in your Prolong, as if the nuclei would explode (which is exactly what they do). Probably Alx647 is just less heat-stable than DAPI, so it's instantly destroyed. Try the same in water, and it will all be fine.

Best wishes, and have fun trying out the nuclear explosions,

Martin

________________________________________
Martin Spitaler, PhD
Head of the
Imaging Facility
Max Planck Institute of Biochemistry
Am Klopferspitz 18
82152 Martinsried
Germany
Tel: [phone]
E-mail: [hidden email]
Website: https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.biochem.mpg.de%2Fde%2Fimaging&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7Cf0d1099175164caa587408d88b26b4fe%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637412346275037852%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=UHBmbnMg2pZTwQKc5WFBlRKGKZPsnYgWssIxQha4b4g%3D&amp;reserved=0


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[Jonkman, James]

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Thank-you all for the excellent suggestions!  I've followed up on a number of them - keep reading below if you're interested.  Essentially AF647 doesn't turn out to be a very good far-red fluorphore in our hands, even with a better choice of wavelength and with better sample prep. Does anybody have a suggestion for 4-colour simultaneous 2p imaging, given just 2 laser lines: Fixed 1040nm and Tunable 680-1300nm (preferably without having to re-tune the laser in between channels)?  The user has a transgenic mouse expressing  TdTomato, but wants to perfuse antibody labels for 2 other proteins and add Hoechst or DAPI for good measure (that one is kind of optional), then excise an organ, slice, and image it live (eventually moving to a window chamber model).  I had suggested AF488 and AF647 to go along with the TdTomato and DAPI.  What do you suggest to replace my far-red choice?  And is it possible to excite 4 well-chosen fluorophores without having to change the tunable laser?

Here's what I found out over the last couple of days:

It's not a microscope problem
I can confirm that there are no extra filters swinging into place on my Zeiss LSM710 when I use the 2p laser.  To test this, I set up a regular confocal scan (633 Ex, 640-700 Em) and got a great AF647 image.  While scanning (live preview mode), I toggled on the 2p laser at 0% power: there is no change to the image.  As I continued to scan, I slowly turn up the 2p excitation power but I never see an increase in the AF647 emission:  instead at some point it starts to disappear.

Choice of wavelength
Thanks to several of you (Gert-Jan, Marco, Craig, Michael, Dan Stevens from Zeiss, I probably missed other!) for suggesting I try different wavelengths.  We had originally tried 800nm (Muetze, Biophysical Journal, 2012) and also 1150nm (Schuh, Kidney International, 2016).  The Spectra Database hosted at U Arizona shows a peak at 1240nm which I hadn't noticed before.  Switching to 1240nm gave me my best results.  I still can't get the image quite as bright as the 1p image, but I can get a half-decent image without completely photobleaching in a single scan.  Repeated scanning sees almost no photobleaching in 1p mode, but still rapid photobleaching in 2p mode as also described by Gert-Jan.  I've been looking up other references and Kobat et.al. (Kobat, Optics Express, 2009) show in Figure 7 that Alexa 680 gives much brighter signal than AF647 or Cy5. (best excited at 1280).  Ueki et. al. (Ueki, Nat Prot, 2020) tried exciting AF 594 and 647 both using 910nm Ex, but while AF594 was moderately bright, AF647 was "not detected".  Unfortunately overlap between TdTomato and AF594 is probably too severe.

Thermal Damage
Thanks very much to Martin for pointing out the mistake in my sample prep!  Instead of trouble-shooting on the user's fresh excised tissues, I just grabbed a slide we had sitting around with cultured cells and DAPI + AF647-phalloidin.  I guess I've just never tried 2p imaging before in such a thin sample, but I can confirm that also Molecular Probes Prepared Slide #1 gives you pretty severe thermal damage quite quickly.  My colleague Feng made me a fresh AF647-Tubulin sample with a bit of a spacer and plenty of PBS and I don't get the immediate thermal damage any more with 1240nm excitation.  Probably not nuclear fusion, but close!  :)

Dark state
Nicolai, I loved your suggestion that the AF647 was going into a dark state.  Would it recover...??!!  I tried it today using the better sample prep to avoid thermal damage (see previous paragraph), but alas after 30min I see the area as bleached out as before.  Nevertheless, I'm certain that you're on the right track: there is definitely some kind of photophysics happening here where a good proportion of the photons are being absorbed without leading to emission.  Maybe like blinking fluorophores are now used for STORM/PALM we might someday think of how to use this as a feature rather than thinking of it as a drawback!  But for now it just looks like AF647 isn't a very good far-red fluorophore for 2p excitation.

SHG, laser
Benjamin, great idea about making an SHG sample - I didn't know you could make one that was visible by eye!  I have ruled out any additional optics sneaking into the emission path (see above), but I haven't totally ruled out the possibility that my laser isn't behaving at these higher wavelengths. It is definitely giving me fs pulses from 700 to 800nm to give me great DAPI images.  The strong absorption I saw at 1150nm suggests a 2p behavior as these thin samples should otherwise be pretty transparent from 1000 to 1300nm, don't you think?

Cheers,
James

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




-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: Wednesday, November 18, 2020 4:24 AM
To: [hidden email]
Subject: [External] Re: The case of the disappearing AF647

*****
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*****

Hi Martin

This explains how I managed to burn holes in my convallaria samples a few years ago! Very nice to get an explanation! :)

Med vänlig hälsning / Best regards

Sylvie

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



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Martin Spitaler
Sent: Tuesday, November 17, 2020 1:29 PM
To: [hidden email]
Subject: [External] Re: The case of the disappearing AF647

*****
Hi James,

   I think the answer to your question is simple, and you have already described it yourself: The fluorophores disappear after an instant. Nicolai might have explained part of the problem with the dark states, but you forgot an important difference between 1P and 2P excitation: The pulsing. The peak power is massive, which is fine in water as you know, you can do wonderful intravital imaging without major damage to the animal, because in water the heat dissipates instantly. But you are imaging in a fixed sample mounted in Prolong Gold sandwiched between two bits of glass. The heat can't go anywhere, and if you would have tried imaging the DAPI for a bit longer, you would also have observed nice little holes appearing in your Prolong, as if the nuclei would explode (which is exactly what they do). Probably Alx647 is just less heat-stable than DAPI, so it's instantly destroyed. Try the same in water, and it will all be fine.

Best wishes, and have fun trying out the nuclear explosions,

Martin

________________________________________
Martin Spitaler, PhD
Head of the
Imaging Facility
Max Planck Institute of Biochemistry
Am Klopferspitz 18
82152 Martinsried
Germany
Tel: [phone]
E-mail: [hidden email]

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Gert-Jan Bakker
Sent: Monday, November 16, 2020 8:40 AM
To: [hidden email]
Subject: [External] Re: The case of the disappearing AF647

*****
Dear James,

We also experience difficulty imaging AF647 with two-photon excitation. In case of bright labeling, we can image it only once, then it has been bleached for most of its part. Furthermore, we have to start imaging with the long wavelength first in case we do sequential imaging including shorter excitations such as dapi.

It might indeed be that AF647 is put in a dark state, however, I never saw recovery after multiphoton imaging of AF647. In your case, exciting with 780 first might have excited AF647 as well. Many red dyes absorb energy efficiently at much shorter wavelength. I remember a article from Drobishev et al., Nat. Meth 2011, stating that at these short wavelengths molecules are being excited in a higher than first electron state. The energy release of these higher states favor chemical alteration of the molecules, causing rapid bleaching.

Turning around the sequence and starting with AF647 excitation might give you an image, if the detectors are sensitive enough and if labeling density is sufficient.

Kind regards,
Gert-Jan

G.J. Bakker, PhD
Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email] T [phone] / T(lab) [phone]

Radboud university medical center
P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283)

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
Sent: Saturday, November 14, 2020 12:29 AM
To: [hidden email]
Subject: Re: The case of the disappearing AF647

*****
Hi James,

is it possible the high laser powers during 2p excitation puts the dye into a long-lived dark state? Alexa Fluor 647 can be a tricky dye, which you can observe when trying to get good STED images (in 2 colors). AF 647 is bright and photostable and has a great STED efficiency, meaning you can get great images even for very low STED powers. But couple AF 647 with a second dye that requires a higher STED power (so most other red dyes), then the AF 647 signal disappears almost instantaneously. It is impossible to record in a line-interleaved mode, as you can literally watch the far-red signal disappear with the scanning laser, and requires instead two sequential tracks (AF 647 first). What happens is the 775 nm STED laser bumps the AF 647 into a long-lived dark state, from which it recovers after 30--60 minutes. The blinking and photoswitchable dark-state are a reason why AF 647 is frequently used in dSTORM imaging modalities.

(See here: https://urldefense.com/v3/__https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.201904117__;!!CjcC7IQ!d-9v2BsLgb_KWO0Kt68-6wUkuyqmDBPt-jPEoJv4JYjvG8uEbl4yCm3zR84fqKZVcBYIft_m$ [chemistry-europe[.]onlinelibrary[.]wiley[.]com])

I've never tried imaging AF 647 with any 2p signal, but the laser power intensities of a 2p-excitation and a STED laser should be fairly comparable (with the 2p pulses being much tighter, so I would expect a much more severe effect). How about first imaging with 1p, then trying to "bleach" away a square pattern using 2p, then waiting half an hour or longer before recording another 1p image. I have no idea what happens at those longer wavelengths, but this should be an easy test you could attempt.

Let me know what happens!

Have a great weekend,
Nicolai

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Nicolai T. Urban, Ph.D.
MPFI Imaging Center - Light Microscopy Core Max Planck Florida Institute for Neuroscience One Max Planck Way, Jupiter FL 33418


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jonkman, James
Sent: Friday, 13 November 2020 15:16
To: [hidden email]
Subject: The case of the disappearing AF647

*****

Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p excitation I can no longer detect it, though I know I'm exciting it.  Let me explain further:

Test slide:  I have fixed cultured cells labeled with DAPI and AF647-phallodin (coverslipped, Prolong gold).
Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)

1p with DAPI and AF647 - works great.
I start with 1p excitation (405nm and 633nm respectively), set up 2 sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice images.   Both channels look very nice, with AF647 requiring 8% laser power (at 633nm) and a modest gain setting of around 600V.  (Ok, those are meaningless numbers because the vendors don't bother to calibrate anything in real-world units, but for someone with a similar instrument it might be useful - sorry, pet peeve of mine!).  There is no appreaciable photobleaching with these settings.

2p with DAPI, PMTs in scanhead, PH wide open - works great.
Now I switch to 2p mode, first just to the exact same detectors (forget about NDDs for now).  I start with the DAPI channel by turning off the 405 laser, switching on the 2p laser at 780nm, and opening the pinhole wide open.  I keep the detector gain exactly as before, and now I slowly start increasing the 2p laser power until I achieve the exact same image as the previous 1p image.  Easy!  Again, no appreciable photobleaching with these settings for DAPI.  This makes sense: if I keep the detector the same and open the pinhole wide, there should exist a 2p excitation intensity that gives me more-or-less the exact same result as the 1p excitation.

2p with AF647, PMTs in scanhead, PH wide open - no emission!
Now I do the exact same thing with AF647.  I turn off the 633nm laser, turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I keep the detector gain exactly as it was for 1p excitation of AF647.  Now I slowly start increasing the 2p laser power... but I see nothing!  I can crank it up to 20% power or higher, being mindful of the fact that when you double the excitation, you get 4x the signal for 2p.  Now here's the real conundrum: if I pull it down to a more modest 10% power (which is still super high for our Discover laser), then move the stage to a fresh field of view, I see signal for just a single frame, and then it instantly and completely photobleaches.  In other words, I'm exciting tonnes of fluorescence, but just not detecting it.  In theory, I should be able to do exactly as I did for DAPI: if I leave the detector gain as I had it for 1p excitation, I should be able to change to 2p excitation and increase the laser power slowly until I get the exact same image as I had for 1p.  But something seems to be blocking the emission when I have the 2p laser engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR blocking filter when the 2p laser is scanning?  Our local application specialist is very knowledgeable but is not aware of any such thing.

Other things that I've considered/tried:
-  The 2p laser is coupled in with a "MBS 760+" dichroic, which should reflect wavelengths above 760nm and pass everything below it.  In fact, I already had this in place for the 1p images - it has virtually no effect on the 1p images so for consistency I just had it in from the beginning.  My other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws away some of the AF647 (as observed during 1p excitation) so I want to avoid it.

- I didn't try adjusting the GDD compensation, but again I know that I'm getting strong excitation, just not collecting it so this shouldn't affect anything.

- Maybe the focus is off?  But I tried being very careful with the laser power (cranking up the LUT to catch any hint of signal) and adusting the focus, but there is no better focal plane. In fact, when I move the stage to an adjacent position I can tell briefly that we're perfectly in focus, before it photobleaches.

- I tried the NDD detector.  We recently upgraded to a 4NDD module with 2 PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first element in the series).  This was my first time trying the NDDs with AF647.  We don't see anything at all - not even a hint of light.  There was a 690nm LP filter at the start of the NDD unit which I removed but it didn't help. So this is even more crazy - could there be a problem with both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647 disappearing!?

Thanks for your suggestions!
James

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


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[Sylvie Le Guyader]

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

Hi James

I would not image the nucleus if it is not needed. Instead, I would collect second or third harmonics to get the tissue context for the fluorescent signal if it is needed. If possible I would acquire SGH/TGH together with a channel where the fluorescence signal looks very different from SGH/TGH pattern. This way you can collect 2 pieces of information in the same image but they can easily be separated during segmentation. This might make it easier to find a combination of excitation wavelengths for your 3 relevant signals + the tissue context.

Med vänlig hälsning / Best regards

Sylvie

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

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jonkman, James
Sent: 19 November 2020 04:48
To: [hidden email]
Subject: Re: The case of the disappearing AF647

*****
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*****

Thank-you all for the excellent suggestions!  I've followed up on a number of them - keep reading below if you're interested.  Essentially AF647 doesn't turn out to be a very good far-red fluorphore in our hands, even with a better choice of wavelength and with better sample prep. Does anybody have a suggestion for 4-colour simultaneous 2p imaging, given just 2 laser lines: Fixed 1040nm and Tunable 680-1300nm (preferably without having to re-tune the laser in between channels)?  The user has a transgenic mouse expressing  TdTomato, but wants to perfuse antibody labels for 2 other proteins and add Hoechst or DAPI for good measure (that one is kind of optional), then excise an organ, slice, and image it live (eventually moving to a window chamber model).  I had suggested AF488 and AF647 to go along with the TdTomato and DAPI.  What do you suggest to replace my far-red choice?  And is it possible to excite 4 well-chosen fluorophores without having to change the tunable laser?

Here's what I found out over the last couple of days:

It's not a microscope problem
I can confirm that there are no extra filters swinging into place on my Zeiss LSM710 when I use the 2p laser.  To test this, I set up a regular confocal scan (633 Ex, 640-700 Em) and got a great AF647 image.  While scanning (live preview mode), I toggled on the 2p laser at 0% power: there is no change to the image.  As I continued to scan, I slowly turn up the 2p excitation power but I never see an increase in the AF647 emission:  instead at some point it starts to disappear.

Choice of wavelength
Thanks to several of you (Gert-Jan, Marco, Craig, Michael, Dan Stevens from Zeiss, I probably missed other!) for suggesting I try different wavelengths.  We had originally tried 800nm (Muetze, Biophysical Journal, 2012) and also 1150nm (Schuh, Kidney International, 2016).  The Spectra Database hosted at U Arizona shows a peak at 1240nm which I hadn't noticed before.  Switching to 1240nm gave me my best results.  I still can't get the image quite as bright as the 1p image, but I can get a half-decent image without completely photobleaching in a single scan.  Repeated scanning sees almost no photobleaching in 1p mode, but still rapid photobleaching in 2p mode as also described by Gert-Jan.  I've been looking up other references and Kobat et.al. (Kobat, Optics Express, 2009) show in Figure 7 that Alexa 680 gives much brighter signal than AF647 or Cy5. (best excited at 1280).  Ueki et. al. (Ueki, Nat Prot, 2020) tried exciting AF 594 and 647 both using 910nm Ex, but while AF594 was moderately bright, AF647 was "not detected".  Unfortunately overlap between TdTomato and AF594 is probably too severe.

Thermal Damage
Thanks very much to Martin for pointing out the mistake in my sample prep!  Instead of trouble-shooting on the user's fresh excised tissues, I just grabbed a slide we had sitting around with cultured cells and DAPI + AF647-phalloidin.  I guess I've just never tried 2p imaging before in such a thin sample, but I can confirm that also Molecular Probes Prepared Slide #1 gives you pretty severe thermal damage quite quickly.  My colleague Feng made me a fresh AF647-Tubulin sample with a bit of a spacer and plenty of PBS and I don't get the immediate thermal damage any more with 1240nm excitation.  Probably not nuclear fusion, but close!  :)

Dark state
Nicolai, I loved your suggestion that the AF647 was going into a dark state.  Would it recover...??!!  I tried it today using the better sample prep to avoid thermal damage (see previous paragraph), but alas after 30min I see the area as bleached out as before.  Nevertheless, I'm certain that you're on the right track: there is definitely some kind of photophysics happening here where a good proportion of the photons are being absorbed without leading to emission.  Maybe like blinking fluorophores are now used for STORM/PALM we might someday think of how to use this as a feature rather than thinking of it as a drawback!  But for now it just looks like AF647 isn't a very good far-red fluorophore for 2p excitation.

SHG, laser
Benjamin, great idea about making an SHG sample - I didn't know you could make one that was visible by eye!  I have ruled out any additional optics sneaking into the emission path (see above), but I haven't totally ruled out the possibility that my laser isn't behaving at these higher wavelengths. It is definitely giving me fs pulses from 700 to 800nm to give me great DAPI images.  The strong absorption I saw at 1150nm suggests a 2p behavior as these thin samples should otherwise be pretty transparent from 1000 to 1300nm, don't you think?

Cheers,
James

-----------------------------------------------
   James Jonkman, Staff Scientist
   Advanced Optical Microscopy Facility (AOMF)
   and Wright Cell Imaging Facility (WCIF)
   University Health Network
   MaRS, PMCRT tower, 101 College St., Room 15-305
   Toronto, ON, CANADA    M5G 1L7
  [hidden email]  Tel: [phone]
   https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.aomf.ca%2F&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C3d6d6ee0f49c497c11c508d88c3e0823%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C1%7C637413546249462199%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=gpDiIkfuQ%2BqeleprUTg4VwrcEP1I85hGzRv%2Bgeac%2BuI%3D&amp;reserved=0




-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
Sent: Wednesday, November 18, 2020 4:24 AM
To: [hidden email]
Subject: [External] Re: The case of the disappearing AF647

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

Hi Martin

This explains how I managed to burn holes in my convallaria samples a few years ago! Very nice to get an explanation! :)

Med vänlig hälsning / Best regards

Sylvie

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



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Martin Spitaler
Sent: Tuesday, November 17, 2020 1:29 PM
To: [hidden email]
Subject: [External] Re: The case of the disappearing AF647

*****
Hi James,

   I think the answer to your question is simple, and you have already described it yourself: The fluorophores disappear after an instant. Nicolai might have explained part of the problem with the dark states, but you forgot an important difference between 1P and 2P excitation: The pulsing. The peak power is massive, which is fine in water as you know, you can do wonderful intravital imaging without major damage to the animal, because in water the heat dissipates instantly. But you are imaging in a fixed sample mounted in Prolong Gold sandwiched between two bits of glass. The heat can't go anywhere, and if you would have tried imaging the DAPI for a bit longer, you would also have observed nice little holes appearing in your Prolong, as if the nuclei would explode (which is exactly what they do). Probably Alx647 is just less heat-stable than DAPI, so it's instantly destroyed. Try the same in water, and it will all be fine.

Best wishes, and have fun trying out the nuclear explosions,

Martin

________________________________________
Martin Spitaler, PhD
Head of the
Imaging Facility
Max Planck Institute of Biochemistry
Am Klopferspitz 18
82152 Martinsried
Germany
Tel: [phone]
E-mail: [hidden email]

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Gert-Jan Bakker
Sent: Monday, November 16, 2020 8:40 AM
To: [hidden email]
Subject: [External] Re: The case of the disappearing AF647

*****
Dear James,

We also experience difficulty imaging AF647 with two-photon excitation. In case of bright labeling, we can image it only once, then it has been bleached for most of its part. Furthermore, we have to start imaging with the long wavelength first in case we do sequential imaging including shorter excitations such as dapi.

It might indeed be that AF647 is put in a dark state, however, I never saw recovery after multiphoton imaging of AF647. In your case, exciting with 780 first might have excited AF647 as well. Many red dyes absorb energy efficiently at much shorter wavelength. I remember a article from Drobishev et al., Nat. Meth 2011, stating that at these short wavelengths molecules are being excited in a higher than first electron state. The energy release of these higher states favor chemical alteration of the molecules, causing rapid bleaching.

Turning around the sequence and starting with AF647 excitation might give you an image, if the detectors are sensitive enough and if labeling density is sufficient.

Kind regards,
Gert-Jan

G.J. Bakker, PhD
Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email] T [phone] / T(lab) [phone]

Radboud university medical center
P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283)

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
Sent: Saturday, November 14, 2020 12:29 AM
To: [hidden email]
Subject: Re: The case of the disappearing AF647

*****
Hi James,

is it possible the high laser powers during 2p excitation puts the dye into a long-lived dark state? Alexa Fluor 647 can be a tricky dye, which you can observe when trying to get good STED images (in 2 colors). AF 647 is bright and photostable and has a great STED efficiency, meaning you can get great images even for very low STED powers. But couple AF 647 with a second dye that requires a higher STED power (so most other red dyes), then the AF 647 signal disappears almost instantaneously. It is impossible to record in a line-interleaved mode, as you can literally watch the far-red signal disappear with the scanning laser, and requires instead two sequential tracks (AF 647 first). What happens is the 775 nm STED laser bumps the AF 647 into a long-lived dark state, from which it recovers after 30--60 minutes. The blinking and photoswitchable dark-state are a reason why AF 647 is frequently used in dSTORM imaging modalities.

(See here: https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.com%2Fv3%2F__https%3A%2F%2Fchemistry-europe.onlinelibrary.wiley.com%2Fdoi%2F10.1002%2Fchem.201904117__%3B!!CjcC7IQ!d-9v2BsLgb_KWO0Kt68-6wUkuyqmDBPt-jPEoJv4JYjvG8uEbl4yCm3zR84fqKZVcBYIft_m%24&amp;data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C3d6d6ee0f49c497c11c508d88c3e0823%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637413546249462199%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&amp;sdata=Jyuzw%2BGQXVTv%2BEodQwxjf7nxX8VV23Y9pcg30l7JlpQ%3D&amp;reserved=0 [chemistry-europe[.]onlinelibrary[.]wiley[.]com])

I've never tried imaging AF 647 with any 2p signal, but the laser power intensities of a 2p-excitation and a STED laser should be fairly comparable (with the 2p pulses being much tighter, so I would expect a much more severe effect). How about first imaging with 1p, then trying to "bleach" away a square pattern using 2p, then waiting half an hour or longer before recording another 1p image. I have no idea what happens at those longer wavelengths, but this should be an easy test you could attempt.

Let me know what happens!

Have a great weekend,
Nicolai

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Nicolai T. Urban, Ph.D.
MPFI Imaging Center - Light Microscopy Core Max Planck Florida Institute for Neuroscience One Max Planck Way, Jupiter FL 33418


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jonkman, James
Sent: Friday, 13 November 2020 15:16
To: [hidden email]
Subject: The case of the disappearing AF647

*****

Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p excitation I can no longer detect it, though I know I'm exciting it.  Let me explain further:

Test slide:  I have fixed cultured cells labeled with DAPI and AF647-phallodin (coverslipped, Prolong gold).
Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)

1p with DAPI and AF647 - works great.
I start with 1p excitation (405nm and 633nm respectively), set up 2 sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice images.   Both channels look very nice, with AF647 requiring 8% laser power (at 633nm) and a modest gain setting of around 600V.  (Ok, those are meaningless numbers because the vendors don't bother to calibrate anything in real-world units, but for someone with a similar instrument it might be useful - sorry, pet peeve of mine!).  There is no appreaciable photobleaching with these settings.

2p with DAPI, PMTs in scanhead, PH wide open - works great.
Now I switch to 2p mode, first just to the exact same detectors (forget about NDDs for now).  I start with the DAPI channel by turning off the 405 laser, switching on the 2p laser at 780nm, and opening the pinhole wide open.  I keep the detector gain exactly as before, and now I slowly start increasing the 2p laser power until I achieve the exact same image as the previous 1p image.  Easy!  Again, no appreciable photobleaching with these settings for DAPI.  This makes sense: if I keep the detector the same and open the pinhole wide, there should exist a 2p excitation intensity that gives me more-or-less the exact same result as the 1p excitation.

2p with AF647, PMTs in scanhead, PH wide open - no emission!
Now I do the exact same thing with AF647.  I turn off the 633nm laser, turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I keep the detector gain exactly as it was for 1p excitation of AF647.  Now I slowly start increasing the 2p laser power... but I see nothing!  I can crank it up to 20% power or higher, being mindful of the fact that when you double the excitation, you get 4x the signal for 2p.  Now here's the real conundrum: if I pull it down to a more modest 10% power (which is still super high for our Discover laser), then move the stage to a fresh field of view, I see signal for just a single frame, and then it instantly and completely photobleaches.  In other words, I'm exciting tonnes of fluorescence, but just not detecting it.  In theory, I should be able to do exactly as I did for DAPI: if I leave the detector gain as I had it for 1p excitation, I should be able to change to 2p excitation and increase the laser power slowly until I get the exact same image as I had for 1p.  But something seems to be blocking the emission when I have the 2p laser engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR blocking filter when the 2p laser is scanning?  Our local application specialist is very knowledgeable but is not aware of any such thing.

Other things that I've considered/tried:
-  The 2p laser is coupled in with a "MBS 760+" dichroic, which should reflect wavelengths above 760nm and pass everything below it.  In fact, I already had this in place for the 1p images - it has virtually no effect on the 1p images so for consistency I just had it in from the beginning.  My other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws away some of the AF647 (as observed during 1p excitation) so I want to avoid it.

- I didn't try adjusting the GDD compensation, but again I know that I'm getting strong excitation, just not collecting it so this shouldn't affect anything.

- Maybe the focus is off?  But I tried being very careful with the laser power (cranking up the LUT to catch any hint of signal) and adusting the focus, but there is no better focal plane. In fact, when I move the stage to an adjacent position I can tell briefly that we're perfectly in focus, before it photobleaches.

- I tried the NDD detector.  We recently upgraded to a 4NDD module with 2 PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first element in the series).  This was my first time trying the NDDs with AF647.  We don't see anything at all - not even a hint of light.  There was a 690nm LP filter at the start of the NDD unit which I removed but it didn't help. So this is even more crazy - could there be a problem with both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647 disappearing!?

Thanks for your suggestions!
James

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


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[Steffen Dietzel]

*****
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.
*****

James,

thanks for the nice description.

Atto647N and Aberrior Star 635P are excellent STED dyes, depleted with a
pulsed 775. Although pulses are quite different for 2P excitation and
depletion, this might be a start. You possibly could get Dapi with the
same wavelenght with 3P excitation. Or you use SIR-DNA or SIR-700 DNA
for counterstain.

According to
https://www.sciencedirect.com/science/article/pii/S000634951200063X 
Figure 2, Alexa 633 should be a lot brighter than A647 and excitable
with 800 nm (probably S2-state) as is A488.

Good luck.

Steffen

Am 19.11.2020 um 04:48 schrieb Jonkman, James:

> *****
> 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.
> *****
>
> Thank-you all for the excellent suggestions!  I've followed up on a number of them - keep reading below if you're interested.  Essentially AF647 doesn't turn out to be a very good far-red fluorphore in our hands, even with a better choice of wavelength and with better sample prep. Does anybody have a suggestion for 4-colour simultaneous 2p imaging, given just 2 laser lines: Fixed 1040nm and Tunable 680-1300nm (preferably without having to re-tune the laser in between channels)?  The user has a transgenic mouse expressing  TdTomato, but wants to perfuse antibody labels for 2 other proteins and add Hoechst or DAPI for good measure (that one is kind of optional), then excise an organ, slice, and image it live (eventually moving to a window chamber model).  I had suggested AF488 and AF647 to go along with the TdTomato and DAPI.  What do you suggest to replace my far-red choice?  And is it possible to excite 4 well-chosen fluorophores without having to change the tunable laser?
>
> Here's what I found out over the last couple of days:
>
> It's not a microscope problem
> I can confirm that there are no extra filters swinging into place on my Zeiss LSM710 when I use the 2p laser.  To test this, I set up a regular confocal scan (633 Ex, 640-700 Em) and got a great AF647 image.  While scanning (live preview mode), I toggled on the 2p laser at 0% power: there is no change to the image.  As I continued to scan, I slowly turn up the 2p excitation power but I never see an increase in the AF647 emission:  instead at some point it starts to disappear.
>
> Choice of wavelength
> Thanks to several of you (Gert-Jan, Marco, Craig, Michael, Dan Stevens from Zeiss, I probably missed other!) for suggesting I try different wavelengths.  We had originally tried 800nm (Muetze, Biophysical Journal, 2012) and also 1150nm (Schuh, Kidney International, 2016).  The Spectra Database hosted at U Arizona shows a peak at 1240nm which I hadn't noticed before.  Switching to 1240nm gave me my best results.  I still can't get the image quite as bright as the 1p image, but I can get a half-decent image without completely photobleaching in a single scan.  Repeated scanning sees almost no photobleaching in 1p mode, but still rapid photobleaching in 2p mode as also described by Gert-Jan.  I've been looking up other references and Kobat et.al. (Kobat, Optics Express, 2009) show in Figure 7 that Alexa 680 gives much brighter signal than AF647 or Cy5. (best excited at 1280).  Ueki et. al. (Ueki, Nat Prot, 2020) tried exciting AF 594 and 647 both using 910nm Ex, but while AF594 was moderately bright, AF647 was "not detected".  Unfortunately overlap between TdTomato and AF594 is probably too severe.
>
> Thermal Damage
> Thanks very much to Martin for pointing out the mistake in my sample prep!  Instead of trouble-shooting on the user's fresh excised tissues, I just grabbed a slide we had sitting around with cultured cells and DAPI + AF647-phalloidin.  I guess I've just never tried 2p imaging before in such a thin sample, but I can confirm that also Molecular Probes Prepared Slide #1 gives you pretty severe thermal damage quite quickly.  My colleague Feng made me a fresh AF647-Tubulin sample with a bit of a spacer and plenty of PBS and I don't get the immediate thermal damage any more with 1240nm excitation.  Probably not nuclear fusion, but close!  :)
>
> Dark state
> Nicolai, I loved your suggestion that the AF647 was going into a dark state.  Would it recover...??!!  I tried it today using the better sample prep to avoid thermal damage (see previous paragraph), but alas after 30min I see the area as bleached out as before.  Nevertheless, I'm certain that you're on the right track: there is definitely some kind of photophysics happening here where a good proportion of the photons are being absorbed without leading to emission.  Maybe like blinking fluorophores are now used for STORM/PALM we might someday think of how to use this as a feature rather than thinking of it as a drawback!  But for now it just looks like AF647 isn't a very good far-red fluorophore for 2p excitation.
>
> SHG, laser
> Benjamin, great idea about making an SHG sample - I didn't know you could make one that was visible by eye!  I have ruled out any additional optics sneaking into the emission path (see above), but I haven't totally ruled out the possibility that my laser isn't behaving at these higher wavelengths. It is definitely giving me fs pulses from 700 to 800nm to give me great DAPI images.  The strong absorption I saw at 1150nm suggests a 2p behavior as these thin samples should otherwise be pretty transparent from 1000 to 1300nm, don't you think?
>
> Cheers,
> James
>
> -----------------------------------------------
>     James Jonkman, Staff Scientist
>     Advanced Optical Microscopy Facility (AOMF)
>     and Wright Cell Imaging Facility (WCIF)
>     University Health Network
>     MaRS, PMCRT tower, 101 College St., Room 15-305
>     Toronto, ON, CANADA    M5G 1L7
>    [hidden email]  Tel: [phone]
>     www.aomf.ca
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader
> Sent: Wednesday, November 18, 2020 4:24 AM
> To: [hidden email]
> Subject: [External] Re: The case of the disappearing AF647
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!f1hkOBB_8U8tD6SmOKX4gptjolZaozJJO2DTwXtisC3gnkzTHIHknLJQRJC-uoRP8cHaIEgQ$ [lists[.]umn[.]edu] Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!f1hkOBB_8U8tD6SmOKX4gptjolZaozJJO2DTwXtisC3gnkzTHIHknLJQRJC-uoRP8azYXJrd$ [imgur[.]com] and include the link in your posting.
> *****
>
> Hi Martin
>
> This explains how I managed to burn holes in my convallaria samples a few years ago! Very nice to get an explanation! :)
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Blickagången 16,
> Room 7362 (lab)/7840 (office)
> 14157 Huddinge, Sweden
> mobile: [phone]
> LCI website
> Follow our microscopy blog!
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Martin Spitaler
> Sent: Tuesday, November 17, 2020 1:29 PM
> To: [hidden email]
> Subject: [External] Re: The case of the disappearing AF647
>
> *****
> Hi James,
>
>     I think the answer to your question is simple, and you have already described it yourself: The fluorophores disappear after an instant. Nicolai might have explained part of the problem with the dark states, but you forgot an important difference between 1P and 2P excitation: The pulsing. The peak power is massive, which is fine in water as you know, you can do wonderful intravital imaging without major damage to the animal, because in water the heat dissipates instantly. But you are imaging in a fixed sample mounted in Prolong Gold sandwiched between two bits of glass. The heat can't go anywhere, and if you would have tried imaging the DAPI for a bit longer, you would also have observed nice little holes appearing in your Prolong, as if the nuclei would explode (which is exactly what they do). Probably Alx647 is just less heat-stable than DAPI, so it's instantly destroyed. Try the same in water, and it will all be fine.
>
> Best wishes, and have fun trying out the nuclear explosions,
>
> Martin
>
> ________________________________________
> Martin Spitaler, PhD
> Head of the
> Imaging Facility
> Max Planck Institute of Biochemistry
> Am Klopferspitz 18
> 82152 Martinsried
> Germany
> Tel: [phone]
> E-mail: [hidden email]
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Gert-Jan Bakker
> Sent: Monday, November 16, 2020 8:40 AM
> To: [hidden email]
> Subject: [External] Re: The case of the disappearing AF647
>
> *****
> Dear James,
>
> We also experience difficulty imaging AF647 with two-photon excitation. In case of bright labeling, we can image it only once, then it has been bleached for most of its part. Furthermore, we have to start imaging with the long wavelength first in case we do sequential imaging including shorter excitations such as dapi.
>
> It might indeed be that AF647 is put in a dark state, however, I never saw recovery after multiphoton imaging of AF647. In your case, exciting with 780 first might have excited AF647 as well. Many red dyes absorb energy efficiently at much shorter wavelength. I remember a article from Drobishev et al., Nat. Meth 2011, stating that at these short wavelengths molecules are being excited in a higher than first electron state. The energy release of these higher states favor chemical alteration of the molecules, causing rapid bleaching.
>
> Turning around the sequence and starting with AF647 excitation might give you an image, if the detectors are sensitive enough and if labeling density is sufficient.
>
> Kind regards,
> Gert-Jan
>
> G.J. Bakker, PhD
> Researcher / Multiphoton microscopy specialist Dept. of Cell Biology (283) and Microscopic Imaging Centre [hidden email] T [phone] / T(lab) [phone]
>
> Radboud university medical center
> P.O.Box 9101, 6500 HB Nijmegen (283), The Netherlands Visiting address: Geert Grooteplein 28 (route 283)
>
> ________________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
> Sent: Saturday, November 14, 2020 12:29 AM
> To: [hidden email]
> Subject: Re: The case of the disappearing AF647
>
> *****
> Hi James,
>
> is it possible the high laser powers during 2p excitation puts the dye into a long-lived dark state? Alexa Fluor 647 can be a tricky dye, which you can observe when trying to get good STED images (in 2 colors). AF 647 is bright and photostable and has a great STED efficiency, meaning you can get great images even for very low STED powers. But couple AF 647 with a second dye that requires a higher STED power (so most other red dyes), then the AF 647 signal disappears almost instantaneously. It is impossible to record in a line-interleaved mode, as you can literally watch the far-red signal disappear with the scanning laser, and requires instead two sequential tracks (AF 647 first). What happens is the 775 nm STED laser bumps the AF 647 into a long-lived dark state, from which it recovers after 30--60 minutes. The blinking and photoswitchable dark-state are a reason why AF 647 is frequently used in dSTORM imaging modalities.
>
> (See here: https://urldefense.com/v3/__https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.201904117__;!!CjcC7IQ!d-9v2BsLgb_KWO0Kt68-6wUkuyqmDBPt-jPEoJv4JYjvG8uEbl4yCm3zR84fqKZVcBYIft_m$ [chemistry-europe[.]onlinelibrary[.]wiley[.]com])
>
> I've never tried imaging AF 647 with any 2p signal, but the laser power intensities of a 2p-excitation and a STED laser should be fairly comparable (with the 2p pulses being much tighter, so I would expect a much more severe effect). How about first imaging with 1p, then trying to "bleach" away a square pattern using 2p, then waiting half an hour or longer before recording another 1p image. I have no idea what happens at those longer wavelengths, but this should be an easy test you could attempt.
>
> Let me know what happens!
>
> Have a great weekend,
> Nicolai
>
> Nicolai T. Urban, Ph.D.
> MPFI Imaging Center - Light Microscopy Core Max Planck Florida Institute for Neuroscience One Max Planck Way, Jupiter FL 33418
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Jonkman, James
> Sent: Friday, 13 November 2020 15:16
> To: [hidden email]
> Subject: The case of the disappearing AF647
>
> *****
>
> Hi, all.  I wonder if anyone can help me solve a puzzle.  I'm able to see Alexa 647 on my Zeiss LSM710 in confocal mode, but when I switch to 2p excitation I can no longer detect it, though I know I'm exciting it.  Let me explain further:
>
> Test slide:  I have fixed cultured cells labeled with DAPI and AF647-phallodin (coverslipped, Prolong gold).
> Objective lens:  20x/1.0NA water immersion (expensive, intravital 2p type)
> Laser: Coherent Discovery (680-1300nm tunable, and 1040nm fixed beam)
>
> 1p with DAPI and AF647 - works great.
> I start with 1p excitation (405nm and 633nm respectively), set up 2 sequential tracks, 1AU pinhole, and adjust the internal PMTs to get nice images.   Both channels look very nice, with AF647 requiring 8% laser power (at 633nm) and a modest gain setting of around 600V.  (Ok, those are meaningless numbers because the vendors don't bother to calibrate anything in real-world units, but for someone with a similar instrument it might be useful - sorry, pet peeve of mine!).  There is no appreaciable photobleaching with these settings.
>
> 2p with DAPI, PMTs in scanhead, PH wide open - works great.
> Now I switch to 2p mode, first just to the exact same detectors (forget about NDDs for now).  I start with the DAPI channel by turning off the 405 laser, switching on the 2p laser at 780nm, and opening the pinhole wide open.  I keep the detector gain exactly as before, and now I slowly start increasing the 2p laser power until I achieve the exact same image as the previous 1p image.  Easy!  Again, no appreciable photobleaching with these settings for DAPI.  This makes sense: if I keep the detector the same and open the pinhole wide, there should exist a 2p excitation intensity that gives me more-or-less the exact same result as the 1p excitation.
>
> 2p with AF647, PMTs in scanhead, PH wide open - no emission!
> Now I do the exact same thing with AF647.  I turn off the 633nm laser, turn on the 2p laser at, say, 1150nm and open the pinhole wide open.  I keep the detector gain exactly as it was for 1p excitation of AF647.  Now I slowly start increasing the 2p laser power... but I see nothing!  I can crank it up to 20% power or higher, being mindful of the fact that when you double the excitation, you get 4x the signal for 2p.  Now here's the real conundrum: if I pull it down to a more modest 10% power (which is still super high for our Discover laser), then move the stage to a fresh field of view, I see signal for just a single frame, and then it instantly and completely photobleaches.  In other words, I'm exciting tonnes of fluorescence, but just not detecting it.  In theory, I should be able to do exactly as I did for DAPI: if I leave the detector gain as I had it for 1p excitation, I should be able to change to 2p excitation and increase the laser power slowly until I get the exact same image as I had for 1p.  But something seems to be blocking the emission when I have the 2p laser engaged.  Has anybody else seen this problem?  Does Zeiss slip in an IR blocking filter when the 2p laser is scanning?  Our local application specialist is very knowledgeable but is not aware of any such thing.
>
> Other things that I've considered/tried:
> -  The 2p laser is coupled in with a "MBS 760+" dichroic, which should reflect wavelengths above 760nm and pass everything below it.  In fact, I already had this in place for the 1p images - it has virtually no effect on the 1p images so for consistency I just had it in from the beginning.  My other option is an MBS 690+ beamsplitter, but the 690 beamsplitter throws away some of the AF647 (as observed during 1p excitation) so I want to avoid it.
>
> - I didn't try adjusting the GDD compensation, but again I know that I'm getting strong excitation, just not collecting it so this shouldn't affect anything.
>
> - Maybe the focus is off?  But I tried being very careful with the laser power (cranking up the LUT to catch any hint of signal) and adusting the focus, but there is no better focal plane. In fact, when I move the stage to an adjacent position I can tell briefly that we're perfectly in focus, before it photobleaches.
>
> - I tried the NDD detector.  We recently upgraded to a 4NDD module with 2 PMTs and 2 GaAsPs, which has a BP 645-710nm IR+ cube on a PMT (first element in the series).  This was my first time trying the NDDs with AF647.  We don't see anything at all - not even a hint of light.  There was a 690nm LP filter at the start of the NDD unit which I removed but it didn't help. So this is even more crazy - could there be a problem with both detectors!?  DAPI on the NDD looks fantastic.  But why is my AF647 disappearing!?
>
> Thanks for your suggestions!
> James
>
> -----------------------------------------------
>     James Jonkman, Staff Scientist
>     Advanced Optical Microscopy Facility (AOMF)
>     and Wright Cell Imaging Facility (WCIF)
>     University Health Network
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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
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[Glyn Nelson]

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Hi James,

I have the same laser setup and have done 4 colour as follows:
Scan line sequentially with 1040 and ~920nm.

For the 1040, capture a narrow 520/5 or 520/10 BP for SHG and your choice of red filter for tdTomato.
For 920, you can capture 2 photon of EGFP or AF488 (I think, I was using EGFP) with ~525/50 filter, and you can also grab 3pi excitation of Hoechst with a 460/80 filter.  I think AF488 and Hoechst can both be excited at about 780nm too.

The problem with this setup is that if you used such a DNA counterstain, it will bleed through into the green, and may even be picked up with the 1040 excitation, so usually requires some unmixing.  I agree with an earlier post that leaving it out is usually better, and the SHG signal can give enough cellular resolution of the tissue.

Glyn