*****
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 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: +46 (0) 73 733 5008
LCI website
Follow our microscopy blog!
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Jonkman, James
Sent: 19 November 2020 04:48
To: [log in to unmask]
Subject: Re: The case of the disappearing AF647
*****
To join, leave or search the confocal microscopy listserv, go to:
https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C3d6d6ee0f49c497c11c508d88c3e0823%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C1%7C637413546249452213%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=qjLf2fMj1TMjfNfZrEXq7rnel3h%2FVnhNvyzRij9ct2w%3D&reserved=0
Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C3d6d6ee0f49c497c11c508d88c3e0823%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C1%7C637413546249452213%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=n3aSaB3D7V4ODWUfrTFLRQMJT%2FS66WzkdhJy%2F7n1ddw%3D&reserved=0 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
[log in to unmask] Tel: 416-581-8593
https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.aomf.ca%2F&data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C3d6d6ee0f49c497c11c508d88c3e0823%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C1%7C637413546249462199%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=gpDiIkfuQ%2BqeleprUTg4VwrcEP1I85hGzRv%2Bgeac%2BuI%3D&reserved=0
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Sylvie Le Guyader
Sent: Wednesday, November 18, 2020 4:24 AM
To: [log in to unmask]
Subject: [External] Re: The case of the disappearing AF647
*****
To join, leave or search the confocal microscopy listserv, go to:
https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.com%2Fv3%2F__http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy__%3B!!CjcC7IQ!f1hkOBB_8U8tD6SmOKX4gptjolZaozJJO2DTwXtisC3gnkzTHIHknLJQRJC-uoRP8cHaIEgQ%24&data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C3d6d6ee0f49c497c11c508d88c3e0823%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637413546249462199%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=m%2BvYYx53AVH9783aGSVQMV87ulJ3c28XSsi9rE8UFQg%3D&reserved=0 [lists[.]umn[.]edu] Post images on https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.com%2Fv3%2F__http%3A%2F%2Fwww.imgur.com__%3B!!CjcC7IQ!f1hkOBB_8U8tD6SmOKX4gptjolZaozJJO2DTwXtisC3gnkzTHIHknLJQRJC-uoRP8azYXJrd%24&data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C3d6d6ee0f49c497c11c508d88c3e0823%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637413546249462199%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=k%2Fz%2BmKSrSiHtqmca2gd5%2BLJLB5iqoXs1sZrvy4dEQLo%3D&reserved=0 [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: +46 (0) 73 733 5008
LCI website
Follow our microscopy blog!
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Martin Spitaler
Sent: Tuesday, November 17, 2020 1:29 PM
To: [log in to unmask]
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: +49 (0)89 8578-3971
E-mail: [log in to unmask]
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Gert-Jan Bakker
Sent: Monday, November 16, 2020 8:40 AM
To: [log in to unmask]
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 [log in to unmask] T +31 (0)24 36 142 96 / T(lab) +31 (0)24 36 515 81
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 <[log in to unmask]> on behalf of [log in to unmask] <[log in to unmask]>
Sent: Saturday, November 14, 2020 12:29 AM
To: [log in to unmask]
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&data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C3d6d6ee0f49c497c11c508d88c3e0823%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637413546249462199%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=Jyuzw%2BGQXVTv%2BEodQwxjf7nxX8VV23Y9pcg30l7JlpQ%3D&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 <[log in to unmask]> On Behalf Of Jonkman, James
Sent: Friday, 13 November 2020 15:16
To: [log in to unmask]
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
[log in to unmask]<mailto:[log in to unmask]> Tel: 416-581-8593
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.
När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>.
Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.
|
