Re: The case of the disappearing AF647 **tech support response**

[Roy Edward-2]

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Dear James,
These papers from 2016 and 2011 from Steffen Dietzel’s group might be informative and give another approach to the combination you’re trying to achieve.

Rehberg, Markus, et al. "Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy." PloS one 6.11 (2011): e28237.
https://doi.org/10.1371/journal.pone.0028237

Dietzel, Steffen, et al. "Multiphoton microscopy of nonfluorescent nanoparticles in vitro and in vivo." Small 12.24 (2016): 3245-3257.
https://onlinelibrary.wiley.com/doi/abs/10.1002/smll.201503766

The authors confirmed to me that DRAQ5 was excited with 1275nm for the 2-P visualisation of the nuclei alongside the THG in the Small 2016 paper.
Best regards,
Roy

Roy Edward
E [hidden email]<mailto:[hidden email]>

BioStatus Limited
56a Charnwood Road, Shepshed, Leicestershire LE12 9NP
T [phone] | F [phone] | W www.biostatus.com<http://www.biostatus.com>

----------------------------------------------------------------------

Date:    Thu, 19 Nov 2020 08:56:47 +0000
From:    Sylvie Le Guyader <[hidden email]<mailto:[hidden email]>>
Subject: Re: The case of the disappearing AF647

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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!

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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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]


This e-mail may contain confidential and/or privileged information for the sole use of the intended recipient.
Any review or distribution by anyone other than the person for whom it was originally intended is strictly prohibited.
If you have received this e-mail in error, please contact the sender and delete all copies.
Opinions, conclusions or other information contained in this e-mail may not be that of the organization.

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--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

------------------------------

Date:    Thu, 19 Nov 2020 15:08:31 +0000
From:    Pablo Hernandez Varas <[hidden email]>
Subject: Job openings at CFIM, University of Copenahgen

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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.
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Dear listers,


We are happy to announce 2 job openings at CFIM, a large bioimaging platform serving life scientists at the Health and Medical Sciences Faculty at University of Copenhagen, Denmark.


We are an international team of light and electron microscopists, and we are looking into expanding our skill set by hiring a full-time bioimage analyst (permanent employment) and a light microscopist with SPIM experience (5 year-position).


Application deadline is 9th December 2020. Please follow the links below for more details.


In case you are interested and wish to know more about the positions, please email Clara Prats, head of the light microscpy team ([hidden email]).


Best regards,

Pablo.



Link to the Light Microscopy applications specialist position:


https://jobportal.ku.dk/administrative-og-forskningsunderstoettende-stillinger/administration-it-og-service/?show=153008

[https://jobportal.ku.dk/billeder/jobportal_fb_1200x630.png]<https://jobportal.ku.dk/administrative-og-forskningsunderstoettende-stillinger/administration-it-og-service/?show=153008>

Light Microscopy applications specialist at CFIM (Core Facility for Integrated Microscopy)<https://jobportal.ku.dk/administrative-og-forskningsunderstoettende-stillinger/administration-it-og-service/?show=153008>
jobportal.ku.dk






Link to the Bioimage Analyst position:


https://jobportal.ku.dk/administrative-og-forskningsunderstoettende-stillinger/administration-it-og-service/?show=153002

[https://jobportal.ku.dk/billeder/jobportal_fb_1200x630.png]<https://jobportal.ku.dk/administrative-og-forskningsunderstoettende-stillinger/administration-it-og-service/?show=153002>

Bioimage Analyst at CFIM (Core Facility for Integrated Microscopy)<https://jobportal.ku.dk/administrative-og-forskningsunderstoettende-stillinger/administration-it-og-service/?show=153002>
jobportal.ku.dk




Pablo Hernández-Varas, PhD.
Applications specialist, CFIM
[hidden email]

https://cfim.ku.dk/

University of Copenhagen
Faculty Of Health Sciences
Department of Biomedical Sciences
Blegdamsvej 3B,<https://webmail.ku.dk/OWA/UrlBlockedError.aspx> Build 21.01
DK-2200 Copenhagen<https://webmail.ku.dk/OWA/UrlBlockedError.aspx>
Denmark<https://webmail.ku.dk/OWA/UrlBlockedError.aspx>

MOB [phone]<tel:+45%2050%2052%2088%2033>
FAX [phone]<tel:+45%2035%2032%2074%2018>

------------------------------

End of CONFOCALMICROSCOPY Digest - 18 Nov 2020 to 19 Nov 2020 (#2020-252)
*************************************************************************