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You are not alone Tim, and it is how the photochemistry generally works. I my experience setting any bulb/LED/laser at wavelength <550nm at high power for prolonged time disturbs functionality of live cardiac cells.
What James suggested may work for fixed cells exclusively. We should not forget that dyes typically are only minority of sample’s fluorophores and high laser intensity will be absorbed by other molecules and trigger a lot of chemistry and phototoxicity.
My 2 cents,
Arvydas
+++++++++++++++++++++++++++++
Arvydas Matiukas, Ph.D.
Director of Neuroscience Microscopy Core
Manager of NRB Shared Research Equipment
From: Tim F<mailto:[log in to unmask]>
Sent: Wednesday, April 27, 2022 2:32 PM
To: [log in to unmask]<mailto:[log in to unmask]>
Subject: Re: [External] Re: DAPI or (UV-excitable) Hoechsts - when to choose one over the other...
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Hi James and Alison, am I the only one who has seen the 405nm line bleach
other channels? This is purely my own experience, but I've tried to set up
multi-color FRAP where I bleach one channel with a second control channel
in the same ROI, and I've generally found that high-power lasers bleach
most stuff to the "red" of it. i.e., a 555nm laser bleaches Cy3 and Cy5
(though far-red fluors are generally hard to bleach), 488nm bleaches GFP,
Cy3, and Cy5, and 405nm bleaches everything. So I could bleach
mApple-tagged endosomes and counterstain with EGFP, but not vice versa.
That seems independent of actual fluorescence excitation by the laser, so I
don't know the physics. Maybe it was a fluke on my part? A phenomenon
specific to live imaging? I haven't seen anything in the literature.
Anyway I'd recommend testing the 405nm at high power to see if other
channels lose any signal.
All the best,
--TF
On Wed, Apr 27, 2022 at 1:57 PM Alison J. North <[log in to unmask]>
wrote:
> *****
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> *****
>
> Hi James,
>
> You make an excellent point. And, DAPI is actually pretty photostable -
> I can whack it on my OMX with the laser power set much higher than I would
> use for most other dyes, and since 3D-SIM requires 15 images per slice, we
> are really going to see photobleaching there if it were a major issue.
>
> Likewise, I find our users to be far too nervous about turning up the
> power on the puny 633 HeNe lasers on our older confocals. They simply
> don't realize that using an Argon laser 488 line at 2% is very different to
> setting the 633 HeNe laser at 2%. You can see them visibly panicking when
> I drag the 633 power up to 50% or higher for a weak (but photostable)
> sample. This, of course, is why our community is pushing hard for our
> commercial friends to implement real power values into the confocals,
> instead of a notoriously uninformative "%" number...
>
> Am I using this thread to shamelessly promote the efforts of QUAREP-LiMi
> and others to rectify this longstanding problem? Why YES, I certainly am!😁
>
> Alison
>
>
> ________________________________
> From: Confocal Microscopy List <[log in to unmask]> on
> behalf of Jonkman, James <[log in to unmask]>
> Sent: Wednesday, April 27, 2022 1:23 PM
> To: [log in to unmask] <[log in to unmask]>
> Subject: Re: [External] Re: DAPI or (UV-excitable) Hoechsts - when to
> choose one over the other... - MINOR CORRECTION
>
> Caution: External email
>
> *****
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> and include the link in your posting.
> *****
>
> Thanks for sharing this, Alison (funny typo and all!). And of course
> thanks to Jason, who is always willing to share his vast knowledge of
> probes with the community.
>
> There was a comment earlier in this thread about having to boost the DAPI
> or Hoechst concentration to account for the low excitation efficiency at
> 405nm. I don't know much about sample prep but that sounds to me like the
> wrong approach. You should consider instead boosting the laser power.
> We're all scared to set the lasers on our confocals above a few percent
> power, for fear of photobleaching. However, keep in mind that you don't do
> any photobleaching if the photons aren't absorbed. For a thin sample
> (cultured cells), since 405nm is less than 10% efficient at exciting DAPI,
> you can safely increase your laser power 10-fold (compared to a laser line
> that would excite at the peak of absorption) without any problem: the 90%
> of the photons that aren't absorbed pass harmlessly through the sample. So
> don't be shy with the DAPI laser power - many users are keeping this
> needlessly low.
>
> 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] Mobile: 647-990-8593
> https://urldefense.com/v3/__http://www.aomf.ca__;!!GobTDDpD7A!O7-RY_ijF7kqHnebsIwSYrzIq5GrHRoXDDbrZIv5EnBEiA8vRjs-M4K0g8EnM5RIpYHIA2CAeZPDMcKDG_3bWNCI8Q$<https://urldefense.com/v3/__http:/www.aomf.ca__;!!GobTDDpD7A!O7-RY_ijF7kqHnebsIwSYrzIq5GrHRoXDDbrZIv5EnBEiA8vRjs-M4K0g8EnM5RIpYHIA2CAeZPDMcKDG_3bWNCI8Q$> <https://urldefense.com/v3/__http://www.aomf.ca__;!!GobTDDpD7A!O7-RY_ijF7kqHnebsIwSYrzIq5GrHRoXDDbrZIv5EnBEiA8vRjs-M4K0g8EnM5RIpYHIA2CAeZPDMcKDG_3bWNCI8Q$ >
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]> On
> Behalf Of Alison J. North
> Sent: Wednesday, April 27, 2022 12:48 PM
> To: [log in to unmask]
> Subject: [External] Re: DAPI or (UV-excitable) Hoechsts - when to choose
> one over the other... - MINOR CORRECTION
>
> *****
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> [imgur[.]com] and include the link in your posting.
> *****
>
> Oops! Awkward typo below. Sorry everybody! Obviously I meant to write
> "IN CASE it appears to be self-promotion" not "unless"!!
> Slow down your fingers Alison, slow down...🤣
>
> ________________________________
> From: Confocal Microscopy List <[log in to unmask]> on
> behalf of Alison J. North <[log in to unmask]>
> Sent: Wednesday, April 27, 2022 12:42 PM
> To: [log in to unmask] <[log in to unmask]>
> Subject: DAPI or (UV-excitable) Hoechsts - when to choose one over the
> other...
>
> Caution: External email
>
> *****
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>
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> and include the link in your posting.
> *****
>
> Dear confocal listserver friends,
>
> I have often wondered how to choose - or advise users - between Hoechst
> and DAPI, and seeing Jason Kilgore's interesting post just this morning, I
> reached out to him to ask him for his thoughts on when you would pick one
> over the other, and why - see below for entire thread. (Apologies for my
> ignorance if many of you knew all of this already!)
>
> I found his response so helpful that I asked him whether he would be OK
> with my posting it on the listserver - I know that our commercial
> colleagues are often worried about posting too much unless it appears to be
> self-promotion, but I really think that many of you would be as interested
> in his response as I was. So here it is below - and thanks again Jason for
> taking the time to answer my questions in such detail!
>
> All the best,
> Alison
>
> ________________________________
> From: Kilgore, Jason A. <[log in to unmask]>
> Sent: Wednesday, April 27, 2022 12:22 PM
> To: Alison J. North <[log in to unmask]>; [log in to unmask] <
> [log in to unmask]>
> Subject: RE: FYI - info on Hoechsts
>
> Caution: External email
>
> Thanks for writing.
>
>
>
> Yes, Hoechst 34580 isn’t as well known, but has gained more usage with
> flow cytometry since many flow cytometers lack a UV laser but often have
> 405.
>
>
>
> These three Hoechst dyes are all more permeant than DAPI. DAPI can get
> into live cells in some instances, and not with others, so it’s really
> “semi-permeant” (here in Tech support we sometimes get customers who are
> even using it as a “dead cell indicator”, like you would PI or SYTOX dyes,
> which would be a mistake due to its semi-permeant nature). So I only
> recommend DAPI for labeling nuclei in fixed cell/tissue samples. Since
> Hoechst dyes are more permeant, they also make a better choice for thicker
> tissue sections, organoids, and spheroids.
>
>
>
> [As an aside, Nuclear Yellow, which is also known as Hoechst S769121, is
> not cell permeant and has a very long Stokes shift, exciting in UV at 355
> but emitting up around 500 nm. Quite beautiful, really, but almost no one
> has the right filter pair.]
>
>
>
> Like DAPI, the Hoechst dyes are minor groove binders, and thus are highly
> selective (one might say almost “specific”) for dsDNA (nuclear as well as
> mitochondrial, but the mito signal is dim in comparison), and also have
> around a 10,000 fold increase in intensity upon binding. So all of these
> make for excellent nuclear labels over background and are exceedingly
> bright. (Most blue dyes have a very low excitation coefficient and thus are
> “dim”, compared to DAPI or Hoechst).
>
>
>
> Hoechst dyes do need a slightly higher concentration and label time. I
> tend to use DAPI at about 0.2 ug/mL for 5 minutes for most samples, but
> Hoechst dyes are around 0.4 ug/mL for about 10 minutes. All of these are
> very robust and can label for much longer with little or no background.
>
>
>
> Strangely, the full spectra for Hoechst 34580 is not easy to find online,
> but I have attached a scan to this email.
>
>
>
> DAPI and the two Hoechst dyes that are comparable in wavelength to it all
> share about the same bleedthrough potential, I’d say. Hoechst 34580, being
> of higher excitation and emission, is much more likely to bleed through
> into the green channel, which is the drawback of using it. As you’ll see in
> the attached scan, the emission curve is pretty broad.
>
>
>
> I hope this helps. Please let me know if you need further info. Cheers,
>
>
>
> Jason
>
>
>
> Jason A. Kilgore
>
> Supervisor, Cell Analysis Tech Support
>
> Life Sciences Solutions
>
> Thermo Fisher Scientific
>
>
>
> From: Alison J. North <[log in to unmask]>
> Sent: Wednesday, April 27, 2022 9:02 AM
> To: [log in to unmask] <[log in to unmask]>; Kilgore, Jason A. <
> [log in to unmask]>
> Subject: FYI - info on Hoechsts
>
>
>
> CAUTION: This email originated from outside of Thermo Fisher Scientific.
> If you believe it to be suspicious, report using the Report Phish button in
> Outlook or send to [log in to unmask]
>
>
>
> Hi Jason,
>
>
>
> Well well, thank you so much for posting this response! In all the time I
> have been working with DAPI and Hoechst, I didn't know about this type of
> Hoechst that excites better with a 405 line! I am including my staff on
> this e-mail in case they don't see your post (see below)...
>
>
>
> It does bring me to a question. What are your thoughts on DAPI vs.
> Hoechst in general? I know that Hoechst 33342 can be used to stain live
> cells, so that's an obvious reason to choose it on some occasions. But I
> have never been sure how to advise users otherwise. DAPI has a super long
> emission tail, which can cause bleed-through issues. Is Hoechst better in
> this regard? What about brightness? I'm just wondering if you have any
> general rules of thumb on how to choose between them?
>
>
>
> Thanks and all the best,
>
> Alison
>
>
>
> Alison J. North, Ph.D.
>
> Senior Director of the Bio-Imaging Resource Center and Research Associate
> Professor
>
> The Rockefeller University
>
> 1230 York Avenue
>
> New York
>
> NY 10065
>
> Tel. 212 327 7488 (office - direct line)
>
>
> https://urldefense.com/v3/__https://www.rockefeller.edu/bioimaging/__;!!CjcC7IQ!fal63iK-SNWJ_TOPGgddm759STJGWQGcIbl5S4BepjiCFEkNPU2JeCP2qXzWnEuw7Z54$<https://urldefense.com/v3/__https:/www.rockefeller.edu/bioimaging/__;!!CjcC7IQ!fal63iK-SNWJ_TOPGgddm759STJGWQGcIbl5S4BepjiCFEkNPU2JeCP2qXzWnEuw7Z54$>
> [rockefeller[.]edu]<
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> >
>
> RRID:SCR_017791
>
> ________________________________
>
> From: Confocal Microscopy List <[log in to unmask]<mailto:
> [log in to unmask]>> on behalf of Kilgore, Jason A. <
> [log in to unmask]<mailto:[log in to unmask]>>
> Sent: Wednesday, April 27, 2022 11:46 AM
> To: [log in to unmask]<mailto:
> [log in to unmask]> <[log in to unmask]
> <mailto:[log in to unmask]>>
> Subject: Re: Strange Hoechst signal solution
>
>
>
> Caution: External email
>
> *****
> To join or leave the confocal microscopy listserv or to change your email
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> and include the link in your posting.
> *****
>
> ** vendor reply **
>
> Most people use Hoechst 33342, or sometimes 33258, both of which are like
> DAPI in wavelength, with a peak at 350 nm. But a different Hoechst dye,
> Hoechst 34580, excites higher with a peak at 392. It is more efficiently
> excited with 405 nm lasers. The protocol and binding characteristics for
> all three is the same.
>
> Jason
>
> Jason A. Kilgore
> Supervisor, Cell Analysis Tech Support
> Life Sciences Solutions
> Thermo Fisher Scientific
>
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]<mailto:
> [log in to unmask]>> On Behalf Of Jeremy Adler
> Sent: Tuesday, April 26, 2022 9:16 PM
> To: [log in to unmask]<mailto:
> [log in to unmask]>
> Subject: Re: Strange Hoechst signal solution
>
> CAUTION: This email originated from outside of Thermo Fisher Scientific.
> If you believe it to be suspicious, report using the Report Phish button in
> Outlook or send to [log in to unmask]<mailto:[log in to unmask]>.
>
>
> *****
> To join or leave the confocal microscopy listserv or to change your email
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> and include the link in your posting.
> *****
>
> The 405nm laser that almost every confocal has, is extremely inefficient
> at exciting Hoechst and related nuclear markers.
> Solution - use more Hoechst.
> More than the low concentrations from protocols optimized for widefield
> imaging with more effective excitation and no pinhole.
> A bonus is that you often find a very faint diffuse signal from the whole
> cell - sufficient to show its extent.
> .
> Clearly high, or even any, concentrations of a dye that intercalates DNA
> is less than ideal for live cells.
> An assumption is that the interference with normal cellular function may
> not be a problem short term live imaging experiments that start with the
> application of Hoechst.
> But does anyone know if this true and if so the "safe" timescale or is
> this merely wishful thinking?
>
> Jeremy
> ===============================================
> B i o V i s P l a t f o r m of Uppsala University
> Light & EM microscopy / FlowCytometry & Cell Sorting /
> Image Analysis ===============================================
> Jeremy Adler PhD - Senior research engineer
> Light, Confocal Microscopy, Image Analysis
> E-mail: [log in to unmask]<mailto:[log in to unmask]>
> 070-1679349
>
> Dag Hammarskjölds v 20
> 751 85 UPPSALA, SWEDEN
>
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> ===============================================
>
>
>
>
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]<mailto:
> [log in to unmask]>> On Behalf Of Tim F
> Sent: Wednesday, April 27, 2022 2:17 AM
> To: [log in to unmask]<mailto:
> [log in to unmask]>
> Subject: Re: Strange Hoechst signal
>
> *****
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>
> Agreed, but opening up the pinhole will mimic a 3D Gaussian blur, which is
> a helpful post-processing step that I often apply to confocal DNA images.
> You can usually spare some spatial precision in that channel and it
> counteracts the terrible noise properties that a 1AU-pinhole Hoechst image
> normally has. Doing it this way gets the same effect without needing to
> bounce the data files through Fiji. .
>
> Best,
>
>
> -T
>
> On Tue, Apr 26, 2022 at 7:41 PM Abby Dernburg PhD <[log in to unmask]
> <mailto:[log in to unmask]>>
> wrote:
>
> > *****
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> and include the link in your posting.
> > *****
> >
> > Hi Arvydas - the better signal in wide-field is primarily due to the
> > use of more suitable light sources - wide-field illumination is
> > usually done with LEDs (or, historically, with arc or xenon lamps)
> > that have very strong bands that match the excitation maxima of
> > Hoechst and DAPI much better than a 405 laser.
> >
> > > On Apr 26, 2022, at 12:04 PM, Arvydas Matiukas
> > > <[log in to unmask]<mailto:[log in to unmask]>>
> > wrote:
> > >
> > > *****
> > > To join or leave the confocal microscopy listserv or to change your
> > email address, go to:
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> > > gi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIGaQ&c=q6k2DsT
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> > > tx-ixR0dwX8MgNOaQyeufjTaYHIQt&s=A7UV86wU3OaC1AoGOCQot8wznhmVbouQwfoD
> > > 0IQblhM&e=
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> > d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-y
> > > QtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=OLlVQ3TGIQaIJvvW62eBuNhTaHbZhTDIB
> > > DFtx-ixR0dwX8MgNOaQyeufjTaYHIQt&s=XhXmcvi8rOwoVDrQv1VshgI63wIqSIY4t6
> > > MIxwouX98&e= > and include
> > the link in your posting.
> > > *****
> > >
> > >
> > >
> > > I am happy to summarize and forward many thanks for all the
> > comments/advises. They helped to convince my user that the confocal is
> > performing OK and concentrate on optimizing the settings. His main
> > concern really was accurately counting the cells (nuclei) in 30um thick
> slice.
> > Following the advice that nuclear signal looks better in widefield we
> > reimaged his slide at increased pinhole (63x, ~3AU) and this addressed
> > the issue. I also suggested to enable the z-correction of collected
> > signal but its effect was less pronounced. In summary, reimaging under
> > advised conditions allowed to improve the image quality and count
> > cells without re-mounting the sample.
> > >
> > > I forward the deepest appreciation of your expertise – you made his
> day!
> > >
> > > Arvydas
> > > +++++++++++++++++++++++++++++
> > > Arvydas Matiukas, Ph.D.
> > > Director of Neuroscience Microscopy Core Manager of NRB Shared
> > > Research Equipment
> > >
> > >
> > > From: Sylvie Le Guyader<mailto:[log in to unmask] <mailto:
> <mailto:[log in to unmask]:%0b>> [log in to unmask]
> <mailto:[log in to unmask]>>>
> > > Sent: Friday, April 22, 2022 3:24 AM
> > > To: [log in to unmask]<mailto:
> [log in to unmask]> <mailto:
> <mailto:%0b>> [log in to unmask]><mailto:
> [log in to unmask]<mailto:[log in to unmask]
> %3e%3cmailto:[log in to unmask]>.
> > EDU <mailto:[log in to unmask]>>
> > > Subject: [EXTERNAL] Re: Strange Hoechst signal
> > >
> > > *****
> > > To join or leave the confocal microscopy listserv or to change your
> > email address, go to:
> > >
> > https://urldefense.com/v3/__https://lists.umn.edu/cgi-bin/wa?SUBED1=co<https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=co>
> > <https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=co
> > >
> > nfocalmicroscopy&A=1__;!!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM
> > 5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu8EGzCB0$
> > <
> > https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=con
> > focalmicroscopy&A=1__;!!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM5
> > xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu8EGzCB0$>
> > <
> > https://urldefense.com/v3/__https://lists.umn.edu/cgi-bin/wa?SUBED1=co<https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=co>
> > <https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=co
> > >
> > nfocalmicroscopy&A=1__;!!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM
> > 5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu8EGzCB0$%3Chttps://
> > urldefense.com/v3/__https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3&data=05%7C01%7Cmatiukaa%40UPSTATE.EDU%7C2b709a412cc541f9476b08da287c3aac%7C5cf50a665e2641dd89f883cf73ffee98%7C0%7C0%7C637866811303863244%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000%7C%7C%7C&sdata=4dQASX8zbmbin9oVJ9TaHFCqy3B0Gg7akbWzNVcsiS8%3D&reserved=0
> > A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmic&d=DwIGaQ&c=q6k2Ds
> > TcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQ
> > QpV-yioYjTLyQ&m=OLlVQ3TGIQaIJvvW62eBuNhTaHbZhTDIBDFtx-ixR0dwX8MgNOaQye
> > ufjTaYHIQt&s=8HncakyUTXJP1et7aIyj5fFC-unsBUOt6LeFRdCPQt8&e=
> > roscopy&A=1__;!!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAO
> > ZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu8EGzCB0$%3E
> > >
> > > Post images on
> > https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!OmA2E2<https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2>
> > <https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2
> > >
> > _ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8
> > bv8S46KNu3xatZ37$
> > <
> > https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2_
> > ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8b
> > v8S46KNu3xatZ37$>
> > <
> > https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!OmA2E2<https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2>
> > <https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2
> > >
> > _ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8
> > bv8S46KNu3xatZ37$%3Chttps://urldefense.com/v3/__http:/www.imgur.com__;
> > !!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZ
> > lOissk7Zn1Q8zFCyFe8bv8S46KNu3xatZ37$%3E>
> > and include the link in your posting.
> > > *****
> > >
> > > Hi Arvydas
> > >
> > > Additionally to all that had already been said, the focus is soft in
> > > all
> > channels.
> > >
> > > If this is the best focus that can be achieved with this sample, it
> > could be due to a dirty objective or coverslip of course but I guess
> > you have checked.
> > >
> > > If these are ruled out, it is likely that your user seeds the cells
> > > on
> > chamber slides with a thick glass bottom, adding mounting medium then
> > a coverslip.
> > >
> > > The correct procedure is to have the sample directly against the
> > coverslip. Otherwise the mounting medium ends up between the objective
> > and the sample leading to a longer light path through a
> > potential/likely refractive index mismatch. Putting mounting medium
> > between the objective and the sample leads to low reproducibility,
> > since more or less of the pbs from staining is left behind and more or
> less medium is added.
> > >
> > > I suggest that your user compares his/her procedure with cells
> > > seeded on
> > #1. 5 coverslips either loose coverslips that can then be mounted on a
> > slide or chamber slides with a #1.5 coverslip at the bottom.
> > >
> > > Another point: if the objective has a ring, it must be adjusted.
> > > This
> > might salvage the sample. Air objectives would also be less sensitive
> > to RI mismatch than high NA objectives. By removing the aberrations
> > due to RI mismatch, you might get a better resolution with an
> > objective with a lower NA.
> > >
> > > Med vänlig hälsning / Best regards
> > >
> > > Sylvie
> > >
> > > @@@@@@@@@@@@@@@@@@@@@@@@
> > > Sylvie Le Guyader, PhD
> > > Live Cell Imaging Facility Manager
> > > Karolinska Institutet- Bionut Dpt
> > > Hälsovägen 7C,
> > > 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]<mailto:[log in to unmask]
> > > N.EDU>> On
> > Behalf Of Arvydas Matiukas
> > > Sent: 21 April 2022 18:30
> > > To:
> > > [log in to unmask]<mailto:[log in to unmask]
> > > .EDU>
> > > Subject: Re: Strange Hoechst signal
> > >
> > > *****
> > > To join or leave the confocal microscopy listserv or to change your
> > email address, go to:
> > >
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=https*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FSUBED1*3Dconfoc
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> > <
> > https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook.
> > com/?url=https*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FSUBED1*3Dconfoca
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> > ZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu6uc6okC$>
> > <
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=https*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FSUBED1*3Dconfoc
> > almicroscopy*26A*3D1&data=05*7C01*7Csylvie.le.guyader*40KI.SE*7Cb6
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> > 3Chttps://urldefense.com/v3/__https:/eur01.safelinks.protection.outloo
> > k.com/?url=https*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FSUBED1*3Dconfo
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> > %3E
> > >
> > > Post images on
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
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> > >
> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=05*7C01*7Csylvie.le.g
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> > KNu3Mq_piG$
> > <
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> > <
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=05*7C01*7Csylvie.le.g
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> > ction.outlook.com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=05*7C01*
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> > and include the link in your posting.
> > > *****
> > >
> > > Hello Microscopists,
> > >
> > > I am seeking your advice on behalf of my Core confocal user. He is
> > concerned about nuclear images produced by Hoechst labeling: a) some
> > areas show no detail structure (look like saturated, though 8 bit
> > signal levels are <200); b) high background level.
> > >
> > > I suspect that this may be related to the sample prep protocol as
> > > other
> > samples from the same and other labs have normal nuclear signal and
> > the confocal is performing within specs. Our core does not provide
> > sample prep service so labs are responsible themselves for this
> important step.
> > >
> > > I attach 3 color image, acquired with 20x objective on PMT detectors
> > > and
> > sample description.
> > >
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=https*3A*2F*2Fimgur.com*2Fa*2F8J2Sliq&data=05*7C01*7Csyl
> > vie.le.guyader*40KI.SE*7Cb6da3c5b0e0542d95f9d08da23b649e4
> > <
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=https*3A*2F*2Fimgur.com*2Fa*2F8J2Sliq&data=05*7C01*7Csyl
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> > >Oissk7Zn1Q8zFCyFe8bv8S46KNu9qYsMq_$<
> > https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook.
> > com/?url=https*3A*2F*2Fimgur.com*2Fa*2F8J2Sliq&data=05*7C01*7Csylv
> > ie.le.guyader*40KI.SE*7Cb6da3c5b0e0542d95f9d08da23b649e4
> > <
> > https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook.
> > com/?url=https*3A*2F*2Fimgur.com*2Fa*2F8J2Sliq&data=05*7C01*7Csylv
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> > >
> > >
> > > Cells are oligodendrocytes progenitor cells Stained by
> > Immunocytochemistry Pfa 4% fixed for 10 min then blocked with 30%
> > donkey serum in PBS.
> > > Primary antibody over night. Fridge
> > >
> > > 2nd ab 2 hours at room temperature
> > >
> > > Olig2 2 goat antibody 2nd ab 598
> > > Pdgfralpha rabbit ab 2nd ab is 488
> > > Hoechst applied at 1:10000 dilution.
> > >
> > > Any insights/comments how to improve nuclear signal are appreciated.
> > >
> > > Best,
> > > Arvydas
> > > **********************
> > > Director of Microscopy Core
> > > SUNY Upstate Medical University
> > > Syracuse, NY
> > > email:[log in to unmask] <mailto:[log in to unmask]>
> > >
> > >
> > > När du skickar e-post till Karolinska Institutet (KI) innebär detta
> > > att
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> ________________________________
> From: Confocal Microscopy List <[log in to unmask]> on
> behalf of Jonkman, James <[log in to unmask]>
> Sent: Wednesday, April 27, 2022 1:23 PM
> To: [log in to unmask] <[log in to unmask]>
> Subject: Re: [External] Re: DAPI or (UV-excitable) Hoechsts - when to
> choose one over the other... - MINOR CORRECTION
>
> Caution: External email
>
> *****
> To join or leave the confocal microscopy listserv or to change your email
> address, go to:
>
> https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3D0blSubCFgEihDKInlblRHcerrVPFw2juVwY06IqVKiU%26s%3DvHoC6oXeCjk3P4zmWX-qNyHoOvb3u7cKP_PCR683Nww%26e%3D&data=05%7C01%7Cmatiukaa%40UPSTATE.EDU%7C2b709a412cc541f9476b08da287c3aac%7C5cf50a665e2641dd89f883cf73ffee98%7C0%7C0%7C637866811303863244%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000%7C%7C%7C&sdata=Z3Ykue0jw8HdRNuAcw9laK7Qy2iOn4YaWYa0FSiZTDQ%3D&reserved=0
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> and include the link in your posting.
> *****
>
> Thanks for sharing this, Alison (funny typo and all!). And of course
> thanks to Jason, who is always willing to share his vast knowledge of
> probes with the community.
>
> There was a comment earlier in this thread about having to boost the DAPI
> or Hoechst concentration to account for the low excitation efficiency at
> 405nm. I don't know much about sample prep but that sounds to me like the
> wrong approach. You should consider instead boosting the laser power.
> We're all scared to set the lasers on our confocals above a few percent
> power, for fear of photobleaching. However, keep in mind that you don't do
> any photobleaching if the photons aren't absorbed. For a thin sample
> (cultured cells), since 405nm is less than 10% efficient at exciting DAPI,
> you can safely increase your laser power 10-fold (compared to a laser line
> that would excite at the peak of absorption) without any problem: the 90%
> of the photons that aren't absorbed pass harmlessly through the sample. So
> don't be shy with the DAPI laser power - many users are keeping this
> needlessly low.
>
> 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] Mobile: 647-990-8593
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>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]> On
> Behalf Of Alison J. North
> Sent: Wednesday, April 27, 2022 12:48 PM
> To: [log in to unmask]
> Subject: [External] Re: DAPI or (UV-excitable) Hoechsts - when to choose
> one over the other... - MINOR CORRECTION
>
> *****
> To join or leave the confocal microscopy listserv or to change your email
> address, go to:
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> [lists[.]umn[.]edu] Post images on
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> [imgur[.]com] and include the link in your posting.
> *****
>
> Oops! Awkward typo below. Sorry everybody! Obviously I meant to write
> "IN CASE it appears to be self-promotion" not "unless"!!
> Slow down your fingers Alison, slow down...🤣
>
> ________________________________
> From: Confocal Microscopy List <[log in to unmask]> on
> behalf of Alison J. North <[log in to unmask]>
> Sent: Wednesday, April 27, 2022 12:42 PM
> To: [log in to unmask] <[log in to unmask]>
> Subject: DAPI or (UV-excitable) Hoechsts - when to choose one over the
> other...
>
> Caution: External email
>
> *****
> To join or leave the confocal microscopy listserv or to change your email
> address, go to:
>
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> Post images on
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> and include the link in your posting.
> *****
>
> Dear confocal listserver friends,
>
> I have often wondered how to choose - or advise users - between Hoechst
> and DAPI, and seeing Jason Kilgore's interesting post just this morning, I
> reached out to him to ask him for his thoughts on when you would pick one
> over the other, and why - see below for entire thread. (Apologies for my
> ignorance if many of you knew all of this already!)
>
> I found his response so helpful that I asked him whether he would be OK
> with my posting it on the listserver - I know that our commercial
> colleagues are often worried about posting too much unless it appears to be
> self-promotion, but I really think that many of you would be as interested
> in his response as I was. So here it is below - and thanks again Jason for
> taking the time to answer my questions in such detail!
>
> All the best,
> Alison
>
> ________________________________
> From: Kilgore, Jason A. <[log in to unmask]>
> Sent: Wednesday, April 27, 2022 12:22 PM
> To: Alison J. North <[log in to unmask]>; [log in to unmask] <
> [log in to unmask]>
> Subject: RE: FYI - info on Hoechsts
>
> Caution: External email
>
> Thanks for writing.
>
>
>
> Yes, Hoechst 34580 isn’t as well known, but has gained more usage with
> flow cytometry since many flow cytometers lack a UV laser but often have
> 405.
>
>
>
> These three Hoechst dyes are all more permeant than DAPI. DAPI can get
> into live cells in some instances, and not with others, so it’s really
> “semi-permeant” (here in Tech support we sometimes get customers who are
> even using it as a “dead cell indicator”, like you would PI or SYTOX dyes,
> which would be a mistake due to its semi-permeant nature). So I only
> recommend DAPI for labeling nuclei in fixed cell/tissue samples. Since
> Hoechst dyes are more permeant, they also make a better choice for thicker
> tissue sections, organoids, and spheroids.
>
>
>
> [As an aside, Nuclear Yellow, which is also known as Hoechst S769121, is
> not cell permeant and has a very long Stokes shift, exciting in UV at 355
> but emitting up around 500 nm. Quite beautiful, really, but almost no one
> has the right filter pair.]
>
>
>
> Like DAPI, the Hoechst dyes are minor groove binders, and thus are highly
> selective (one might say almost “specific”) for dsDNA (nuclear as well as
> mitochondrial, but the mito signal is dim in comparison), and also have
> around a 10,000 fold increase in intensity upon binding. So all of these
> make for excellent nuclear labels over background and are exceedingly
> bright. (Most blue dyes have a very low excitation coefficient and thus are
> “dim”, compared to DAPI or Hoechst).
>
>
>
> Hoechst dyes do need a slightly higher concentration and label time. I
> tend to use DAPI at about 0.2 ug/mL for 5 minutes for most samples, but
> Hoechst dyes are around 0.4 ug/mL for about 10 minutes. All of these are
> very robust and can label for much longer with little or no background.
>
>
>
> Strangely, the full spectra for Hoechst 34580 is not easy to find online,
> but I have attached a scan to this email.
>
>
>
> DAPI and the two Hoechst dyes that are comparable in wavelength to it all
> share about the same bleedthrough potential, I’d say. Hoechst 34580, being
> of higher excitation and emission, is much more likely to bleed through
> into the green channel, which is the drawback of using it. As you’ll see in
> the attached scan, the emission curve is pretty broad.
>
>
>
> I hope this helps. Please let me know if you need further info. Cheers,
>
>
>
> Jason
>
>
>
> Jason A. Kilgore
>
> Supervisor, Cell Analysis Tech Support
>
> Life Sciences Solutions
>
> Thermo Fisher Scientific
>
>
>
> From: Alison J. North <[log in to unmask]>
> Sent: Wednesday, April 27, 2022 9:02 AM
> To: [log in to unmask] <[log in to unmask]>; Kilgore, Jason A. <
> [log in to unmask]>
> Subject: FYI - info on Hoechsts
>
>
>
> CAUTION: This email originated from outside of Thermo Fisher Scientific.
> If you believe it to be suspicious, report using the Report Phish button in
> Outlook or send to [log in to unmask]
>
>
>
> Hi Jason,
>
>
>
> Well well, thank you so much for posting this response! In all the time I
> have been working with DAPI and Hoechst, I didn't know about this type of
> Hoechst that excites better with a 405 line! I am including my staff on
> this e-mail in case they don't see your post (see below)...
>
>
>
> It does bring me to a question. What are your thoughts on DAPI vs.
> Hoechst in general? I know that Hoechst 33342 can be used to stain live
> cells, so that's an obvious reason to choose it on some occasions. But I
> have never been sure how to advise users otherwise. DAPI has a super long
> emission tail, which can cause bleed-through issues. Is Hoechst better in
> this regard? What about brightness? I'm just wondering if you have any
> general rules of thumb on how to choose between them?
>
>
>
> Thanks and all the best,
>
> Alison
>
>
>
> Alison J. North, Ph.D.
>
> Senior Director of the Bio-Imaging Resource Center and Research Associate
> Professor
>
> The Rockefeller University
>
> 1230 York Avenue
>
> New York
>
> NY 10065
>
> Tel. 212 327 7488 (office - direct line)
>
>
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> ________________________________
>
> From: Confocal Microscopy List <[log in to unmask]<mailto:
> [log in to unmask]>> on behalf of Kilgore, Jason A. <
> [log in to unmask]<mailto:[log in to unmask]>>
> Sent: Wednesday, April 27, 2022 11:46 AM
> To: [log in to unmask]<mailto:
> [log in to unmask]> <[log in to unmask]
> <mailto:[log in to unmask]>>
> Subject: Re: Strange Hoechst signal solution
>
>
>
> Caution: External email
>
> *****
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> *****
>
> ** vendor reply **
>
> Most people use Hoechst 33342, or sometimes 33258, both of which are like
> DAPI in wavelength, with a peak at 350 nm. But a different Hoechst dye,
> Hoechst 34580, excites higher with a peak at 392. It is more efficiently
> excited with 405 nm lasers. The protocol and binding characteristics for
> all three is the same.
>
> Jason
>
> Jason A. Kilgore
> Supervisor, Cell Analysis Tech Support
> Life Sciences Solutions
> Thermo Fisher Scientific
>
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]<mailto:
> [log in to unmask]>> On Behalf Of Jeremy Adler
> Sent: Tuesday, April 26, 2022 9:16 PM
> To: [log in to unmask]<mailto:
> [log in to unmask]>
> Subject: Re: Strange Hoechst signal solution
>
> CAUTION: This email originated from outside of Thermo Fisher Scientific.
> If you believe it to be suspicious, report using the Report Phish button in
> Outlook or send to [log in to unmask]<mailto:[log in to unmask]>.
>
>
> *****
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>
> The 405nm laser that almost every confocal has, is extremely inefficient
> at exciting Hoechst and related nuclear markers.
> Solution - use more Hoechst.
> More than the low concentrations from protocols optimized for widefield
> imaging with more effective excitation and no pinhole.
> A bonus is that you often find a very faint diffuse signal from the whole
> cell - sufficient to show its extent.
> .
> Clearly high, or even any, concentrations of a dye that intercalates DNA
> is less than ideal for live cells.
> An assumption is that the interference with normal cellular function may
> not be a problem short term live imaging experiments that start with the
> application of Hoechst.
> But does anyone know if this true and if so the "safe" timescale or is
> this merely wishful thinking?
>
> Jeremy
> ===============================================
> B i o V i s P l a t f o r m of Uppsala University
> Light & EM microscopy / FlowCytometry & Cell Sorting /
> Image Analysis ===============================================
> Jeremy Adler PhD - Senior research engineer
> Light, Confocal Microscopy, Image Analysis
> E-mail: [log in to unmask]<mailto:[log in to unmask]>
> 070-1679349
>
> Dag Hammarskjölds v 20
> 751 85 UPPSALA, SWEDEN
>
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> ===============================================
>
>
>
>
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]<mailto:
> [log in to unmask]>> On Behalf Of Tim F
> Sent: Wednesday, April 27, 2022 2:17 AM
> To: [log in to unmask]<mailto:
> [log in to unmask]>
> Subject: Re: Strange Hoechst signal
>
> *****
> To join or leave the confocal microscopy listserv or to change your email
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>
> Agreed, but opening up the pinhole will mimic a 3D Gaussian blur, which is
> a helpful post-processing step that I often apply to confocal DNA images.
> You can usually spare some spatial precision in that channel and it
> counteracts the terrible noise properties that a 1AU-pinhole Hoechst image
> normally has. Doing it this way gets the same effect without needing to
> bounce the data files through Fiji. .
>
> Best,
>
>
> -T
>
> On Tue, Apr 26, 2022 at 7:41 PM Abby Dernburg PhD <[log in to unmask]
> <mailto:[log in to unmask]>>
> wrote:
>
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> > *****
> >
> > Hi Arvydas - the better signal in wide-field is primarily due to the
> > use of more suitable light sources - wide-field illumination is
> > usually done with LEDs (or, historically, with arc or xenon lamps)
> > that have very strong bands that match the excitation maxima of
> > Hoechst and DAPI much better than a 405 laser.
> >
> > > On Apr 26, 2022, at 12:04 PM, Arvydas Matiukas
> > > <[log in to unmask]<mailto:[log in to unmask]>>
> > wrote:
> > >
> > > *****
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> > > 0IQblhM&e=
> > > <https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com_%26&data=05%7C01%7Cmatiukaa%40UPSTATE.EDU%7C2b709a412cc541f9476b08da287c3aac%7C5cf50a665e2641dd89f883cf73ffee98%7C0%7C0%7C637866811303863244%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000%7C%7C%7C&sdata=pfUgXjIfkSfsdnW0zOW7nh4htMU1algSegScBRQ4xck%3D&reserved=0
> <https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com_%26%250b&data=05%7C01%7Cmatiukaa%40UPSTATE.EDU%7C2b709a412cc541f9476b08da287c3aac%7C5cf50a665e2641dd89f883cf73ffee98%7C0%7C0%7C637866811303863244%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000%7C%7C%7C&sdata=DUayIUDv05JRpZN9JwJ7K%2FBqo0YjfD8SYZtJeiDOm3E%3D&reserved=0>>
> > d=DwIGaQ&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-y
> > > QtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=OLlVQ3TGIQaIJvvW62eBuNhTaHbZhTDIB
> > > DFtx-ixR0dwX8MgNOaQyeufjTaYHIQt&s=XhXmcvi8rOwoVDrQv1VshgI63wIqSIY4t6
> > > MIxwouX98&e= > and include
> > the link in your posting.
> > > *****
> > >
> > >
> > >
> > > I am happy to summarize and forward many thanks for all the
> > comments/advises. They helped to convince my user that the confocal is
> > performing OK and concentrate on optimizing the settings. His main
> > concern really was accurately counting the cells (nuclei) in 30um thick
> slice.
> > Following the advice that nuclear signal looks better in widefield we
> > reimaged his slide at increased pinhole (63x, ~3AU) and this addressed
> > the issue. I also suggested to enable the z-correction of collected
> > signal but its effect was less pronounced. In summary, reimaging under
> > advised conditions allowed to improve the image quality and count
> > cells without re-mounting the sample.
> > >
> > > I forward the deepest appreciation of your expertise – you made his
> day!
> > >
> > > Arvydas
> > > +++++++++++++++++++++++++++++
> > > Arvydas Matiukas, Ph.D.
> > > Director of Neuroscience Microscopy Core Manager of NRB Shared
> > > Research Equipment
> > >
> > >
> > > From: Sylvie Le Guyader<mailto:[log in to unmask] <mailto:
> <mailto:[log in to unmask]:%0b>> [log in to unmask]
> <mailto:[log in to unmask]>>>
> > > Sent: Friday, April 22, 2022 3:24 AM
> > > To: [log in to unmask]<mailto:
> [log in to unmask]> <mailto:
> <mailto:%0b>> [log in to unmask]><mailto:
> [log in to unmask]<mailto:[log in to unmask]
> %3e%3cmailto:[log in to unmask]>.
> > EDU <mailto:[log in to unmask]>>
> > > Subject: [EXTERNAL] Re: Strange Hoechst signal
> > >
> > > *****
> > > To join or leave the confocal microscopy listserv or to change your
> > email address, go to:
> > >
> > https://urldefense.com/v3/__https://lists.umn.edu/cgi-bin/wa?SUBED1=co<https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=co>
> > <https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=co
> > >
> > nfocalmicroscopy&A=1__;!!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM
> > 5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu8EGzCB0$
> > <
> > https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=con
> > focalmicroscopy&A=1__;!!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM5
> > xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu8EGzCB0$>
> > <
> > https://urldefense.com/v3/__https://lists.umn.edu/cgi-bin/wa?SUBED1=co<https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=co>
> > <https://urldefense.com/v3/__https:/lists.umn.edu/cgi-bin/wa?SUBED1=co
> > >
> > nfocalmicroscopy&A=1__;!!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM
> > 5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu8EGzCB0$%3Chttps://
> > urldefense.com/v3/__https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3&data=05%7C01%7Cmatiukaa%40UPSTATE.EDU%7C2b709a412cc541f9476b08da287c3aac%7C5cf50a665e2641dd89f883cf73ffee98%7C0%7C0%7C637866811303863244%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000%7C%7C%7C&sdata=4dQASX8zbmbin9oVJ9TaHFCqy3B0Gg7akbWzNVcsiS8%3D&reserved=0
> > A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmic&d=DwIGaQ&c=q6k2Ds
> > TcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQ
> > QpV-yioYjTLyQ&m=OLlVQ3TGIQaIJvvW62eBuNhTaHbZhTDIBDFtx-ixR0dwX8MgNOaQye
> > ufjTaYHIQt&s=8HncakyUTXJP1et7aIyj5fFC-unsBUOt6LeFRdCPQt8&e=
> > roscopy&A=1__;!!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAO
> > ZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu8EGzCB0$%3E
> > >
> > > Post images on
> > https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!OmA2E2<https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2>
> > <https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2
> > >
> > _ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8
> > bv8S46KNu3xatZ37$
> > <
> > https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2_
> > ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8b
> > v8S46KNu3xatZ37$>
> > <
> > https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!OmA2E2<https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2>
> > <https://urldefense.com/v3/__http:/www.imgur.com__;!!GobTDDpD7A!OmA2E2
> > >
> > _ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8
> > bv8S46KNu3xatZ37$%3Chttps://urldefense.com/v3/__http:/www.imgur.com__;
> > !!GobTDDpD7A!OmA2E2_ZIAHradNsHgFwA_iMPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZ
> > lOissk7Zn1Q8zFCyFe8bv8S46KNu3xatZ37$%3E>
> > and include the link in your posting.
> > > *****
> > >
> > > Hi Arvydas
> > >
> > > Additionally to all that had already been said, the focus is soft in
> > > all
> > channels.
> > >
> > > If this is the best focus that can be achieved with this sample, it
> > could be due to a dirty objective or coverslip of course but I guess
> > you have checked.
> > >
> > > If these are ruled out, it is likely that your user seeds the cells
> > > on
> > chamber slides with a thick glass bottom, adding mounting medium then
> > a coverslip.
> > >
> > > The correct procedure is to have the sample directly against the
> > coverslip. Otherwise the mounting medium ends up between the objective
> > and the sample leading to a longer light path through a
> > potential/likely refractive index mismatch. Putting mounting medium
> > between the objective and the sample leads to low reproducibility,
> > since more or less of the pbs from staining is left behind and more or
> less medium is added.
> > >
> > > I suggest that your user compares his/her procedure with cells
> > > seeded on
> > #1. 5 coverslips either loose coverslips that can then be mounted on a
> > slide or chamber slides with a #1.5 coverslip at the bottom.
> > >
> > > Another point: if the objective has a ring, it must be adjusted.
> > > This
> > might salvage the sample. Air objectives would also be less sensitive
> > to RI mismatch than high NA objectives. By removing the aberrations
> > due to RI mismatch, you might get a better resolution with an
> > objective with a lower NA.
> > >
> > > Med vänlig hälsning / Best regards
> > >
> > > Sylvie
> > >
> > > @@@@@@@@@@@@@@@@@@@@@@@@
> > > Sylvie Le Guyader, PhD
> > > Live Cell Imaging Facility Manager
> > > Karolinska Institutet- Bionut Dpt
> > > Hälsovägen 7C,
> > > 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]<mailto:[log in to unmask]
> > > N.EDU>> On
> > Behalf Of Arvydas Matiukas
> > > Sent: 21 April 2022 18:30
> > > To:
> > > [log in to unmask]<mailto:[log in to unmask]
> > > .EDU>
> > > Subject: Re: Strange Hoechst signal
> > >
> > > *****
> > > To join or leave the confocal microscopy listserv or to change your
> > email address, go to:
> > >
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=https*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FSUBED1*3Dconfoc
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> > PZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu6uc6okC$
> > <
> > https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook.
> > com/?url=https*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FSUBED1*3Dconfoca
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> > ZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu6uc6okC$>
> > <
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=https*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FSUBED1*3Dconfoc
> > almicroscopy*26A*3D1&data=05*7C01*7Csylvie.le.guyader*40KI.SE*7Cb6
> > da3c5b0e0542d95f9d08da23b649e4*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0*
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> > 3Chttps://urldefense.com/v3/__https:/eur01.safelinks.protection.outloo
> > k.com/?url=https*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FSUBED1*3Dconfo
> > calmicroscopy*26A*3D1&data=05*7C01*7Csylvie.le.guyader*40KI.SE*7Cb
> > 6da3c5b0e0542d95f9d08da23b649e4*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0
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> > MPZEFZrhDNM5xcxQ0LAOZP-k16WJzL9wkZlOissk7Zn1Q8zFCyFe8bv8S46KNu6uc6okC$
> > %3E
> > >
> > > Post images on
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=05*7C01*7Csylvie.le.g
> > uyader*40KI.SE*7Cb6da3c5b0e0542d95f9d08da23b649e4*7Cbff7eef1cf4b4f32be
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> > KNu3Mq_piG$
> > <
> > https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook.
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> > Nu3Mq_piG$>
> > <
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=05*7C01*7Csylvie.le.g
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> > ction.outlook.com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=05*7C01*
> > 7Csylvie.le.guyader*40KI.SE*7Cb6da3c5b0e0542d95f9d08da23b649e4*7Cbff7e
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> > zFCyFe8bv8S46KNu3Mq_piG$%3E>
> > and include the link in your posting.
> > > *****
> > >
> > > Hello Microscopists,
> > >
> > > I am seeking your advice on behalf of my Core confocal user. He is
> > concerned about nuclear images produced by Hoechst labeling: a) some
> > areas show no detail structure (look like saturated, though 8 bit
> > signal levels are <200); b) high background level.
> > >
> > > I suspect that this may be related to the sample prep protocol as
> > > other
> > samples from the same and other labs have normal nuclear signal and
> > the confocal is performing within specs. Our core does not provide
> > sample prep service so labs are responsible themselves for this
> important step.
> > >
> > > I attach 3 color image, acquired with 20x objective on PMT detectors
> > > and
> > sample description.
> > >
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=https*3A*2F*2Fimgur.com*2Fa*2F8J2Sliq&data=05*7C01*7Csyl
> > vie.le.guyader*40KI.SE*7Cb6da3c5b0e0542d95f9d08da23b649e4
> > <
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook<https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook>
> > <https://urldefense.com/v3/__https:/eur01.safelinks.protection.outlook
> > >
> > .com/?url=https*3A*2F*2Fimgur.com*2Fa*2F8J2Sliq&data=05*7C01*7Csyl
> > vie.le.guyader*40KI.SE*7Cb6da3c5b0e0542d95f9d08da23b649e4
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> > >
> > >
> > > Cells are oligodendrocytes progenitor cells Stained by
> > Immunocytochemistry Pfa 4% fixed for 10 min then blocked with 30%
> > donkey serum in PBS.
> > > Primary antibody over night. Fridge
> > >
> > > 2nd ab 2 hours at room temperature
> > >
> > > Olig2 2 goat antibody 2nd ab 598
> > > Pdgfralpha rabbit ab 2nd ab is 488
> > > Hoechst applied at 1:10000 dilution.
> > >
> > > Any insights/comments how to improve nuclear signal are appreciated.
> > >
> > > Best,
> > > Arvydas
> > > **********************
> > > Director of Microscopy Core
> > > SUNY Upstate Medical University
> > > Syracuse, NY
> > > email:[log in to unmask] <mailto:[log in to unmask]>
> > >
> > >
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