Mounting media for STED

[xavier Sanjuan]

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Dear all,

For STED experiments we have always mounted our cells with ProLong Gold. It
is one of the recommended ones in the Guide to STED Sample Preparation
published by Leica (
https://webcdn.leica-microsystems.com/fileadmin/academy/2019/Quick_Guide_STED_Sample_Preparation/STED_Sample_Preparation_Guide_Online_20190705.pdf),
and it works well in our hands. As it hardens, it is well known it
squash/shrink the cells mainly in z dimension, thus affecting its shape. I
have never read or heard the squashing also affects at the molecular level
(i.e. changing molecular shape or distances between molecules), something
that would have a negative impact in our STED observations, but do you
think it could be the case? Is there any publication on this topic? And, is
there any alternative mounting media to avoid this (hypothetical) artifact
on the molecular structure of the sample?

Best,

Xavi.

___________________________________

*Xavier Sanjuan*
Advanced Light Microscopy Unit

Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88
08003 Barcelona - Spain
Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web:
http://www.crg.eu/en/core/programmes-groups/advanced-light-microscopy-unit

[Giang, William]

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Hi Xavier,

From this thread https://forum.microlist.org/t/hardening-mounting-media-that-doesnt-compress-sample/416 , it looks like ProLong Glass (cures to RI of 1.52) leads to less compression than ProLong Gold.

Have you tried non-hardening mounting media?

Before the sickness came, we were going to test SlowFade Glass vs ProLong Glass with SIM., but if someone's already done this with STED, I'd love to see the results!

Best,
Will


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of SANJUAN SAMARRA, XAVIER
Sent: Sunday, April 12, 2020 5:27 AM
To: [hidden email]
Subject: [External] Mounting media for STED

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Dear all,

For STED experiments we have always mounted our cells with ProLong Gold. It is one of the recommended ones in the Guide to STED Sample Preparation published by Leica ( https://webcdn.leica-microsystems.com/fileadmin/academy/2019/Quick_Guide_STED_Sample_Preparation/STED_Sample_Preparation_Guide_Online_20190705.pdf),
and it works well in our hands. As it hardens, it is well known it squash/shrink the cells mainly in z dimension, thus affecting its shape. I have never read or heard the squashing also affects at the molecular level (i.e. changing molecular shape or distances between molecules), something that would have a negative impact in our STED observations, but do you think it could be the case? Is there any publication on this topic? And, is there any alternative mounting media to avoid this (hypothetical) artifact on the molecular structure of the sample?

Best,

Xavi.

___________________________________

*Xavier Sanjuan*
Advanced Light Microscopy Unit

Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88
08003 Barcelona - Spain
Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web:
http://www.crg.eu/en/core/programmes-groups/advanced-light-microscopy-unit

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[Alison J. North]

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Dear Xavi,

The 3D squashing effect is something many of us have seen, but I am another person who keeps intending to publish a thorough comparison of the effects and never has time! And whether it has an effect at the molecular distance level is a good question.  You do not actually have to let the Prolong Gold harden.   If you seal around the coverslips with quick drying nail polish immediately after mounting, it doesn't harden and you retain more of the 3D information.  Sure, you don't end up with the higher refractive index of the cured mountant, but it's not as though the completely cured Prolong Gold has an r.i. matching that of regular immersion oil anyway.  We use non-hardened Prolong Gold/Diamond for 3D-SIM (using an OMX), and compensate for the r.i. mismatch using a different refractive index oil.  I haven't actually tried that with STED yet, and you need to be aware that the Cargille oils that we typically use for r.i. selection may induce some chromatic dispersion too, but you could give it a shot.  Otherwise, unless you have adaptive optics on your system, you will have to see whether your results are better or worse without curing - i.e. balancing the negative effects of spherical aberrations against squashing effects!

In the light sheet microscopy world, a lot of work has been done with r.i. matching, in particular using TDE-based mountants.  And I now notice however that Abberior has a kind of mounting medium on their webpage called Abberior TDE which comes in different types - r.i.s to match immersion oil, silicone oil or glycerol.  It says the TDE mountants are specifically designed for imaging thick specimens, which would imply to me that they minimize squashing artifacts, though I can't see that it explicitly says that.  I find the r.i. of uncured Prolong Gold to be pretty similar to that of Vectashield, which is glycerol-based (this is just by empirical testing, i.e. which r.i. oil matches best on the OMX).  So I wonder whether the Abberior TDE glycerol version would work well with uncured Prolong Gold?

Abberior guys (Christian, Mary Grace...?), does any of you know the answer to this?  Do the TDE mountants harden and squash the samples or not, and have you tried them in combination with uncured Prolong Gold?  (If not, please send me some and I will test them myself as soon as I'm back in the lab!).  Since Germany is a civilised country that actually believes in vacation days over Easter (unlike my current country of residence, grrr!) I am not expecting an immediate response, but I did want to ask the question before I forget....

Thanks for the question Xavi!
All the best,
Alison

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of SANJUAN SAMARRA, XAVIER <[hidden email]>
Sent: Sunday, April 12, 2020 5:27 AM
To: [hidden email] <[hidden email]>
Subject: Mounting media for STED

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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=TpmsRitFDyxjlgIZt1oYyXc2YzHQyMFQYmiMC4C8vs0&e=  and include the link in your posting.
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Dear all,

For STED experiments we have always mounted our cells with ProLong Gold. It
is one of the recommended ones in the Guide to STED Sample Preparation
published by Leica (
https://urldefense.proofpoint.com/v2/url?u=https-3A__webcdn.leica-2Dmicrosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5FPreparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pdf&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=Mk7C5-iSFnwD64vR5nG8EFEIBsqUOap4c080ALYtDm0&e= ),
and it works well in our hands. As it hardens, it is well known it
squash/shrink the cells mainly in z dimension, thus affecting its shape. I
have never read or heard the squashing also affects at the molecular level
(i.e. changing molecular shape or distances between molecules), something
that would have a negative impact in our STED observations, but do you
think it could be the case? Is there any publication on this topic? And, is
there any alternative mounting media to avoid this (hypothetical) artifact
on the molecular structure of the sample?

Best,

Xavi.

___________________________________

*Xavier Sanjuan*
Advanced Light Microscopy Unit

Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88
08003 Barcelona - Spain
Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web:
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=xEGAoB6Dl2fmc4iX1fuNb_NIHbU8DZeU4Wm0-ECz74Y&e=

[Douglas Richardson]

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Hi Allison,

This paper should answer all your questions on TDE including shrinkage,
dispersion, etc. https://onlinelibrary.wiley.com/doi/abs/10.1002/jemt.20396

Briefly, if you take your sample through a step-wise increase in TDE as
described in the paper, the shrinkage is minimal. We saw some great
improvements using TDE-based mounting medium over Prolong Gold for SIM (
https://hcbi.fas.harvard.edu/files/hcbidoug/files/hcbi_recommended_mounting_media_0.pdf).
This was before Prolong and Slow-fade Glass were available.

The drawbacks to TDE are the longer amount of time to prep the sample as
you step through different concentrations. Also, green and blue dyes
photobleach almost immediately in high TDE concentrations (those needed to
match oil immersion objectives), but are more stable when more water is
present (glycerol/silicone objectives).

The Abberior folks have told me that the high RI Prolong/Slow-fade Glass
products shift the emission spectrum of red and far-red dyes making STED
more difficult.  We had some samples ready to test when we got shutdown :(.

-Doug

On Mon, Apr 13, 2020 at 10:22 AM Alison J. North <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Xavi,
>
> The 3D squashing effect is something many of us have seen, but I am
> another person who keeps intending to publish a thorough comparison of the
> effects and never has time! And whether it has an effect at the molecular
> distance level is a good question.  You do not actually have to let the
> Prolong Gold harden.   If you seal around the coverslips with quick drying
> nail polish immediately after mounting, it doesn't harden and you retain
> more of the 3D information.  Sure, you don't end up with the higher
> refractive index of the cured mountant, but it's not as though the
> completely cured Prolong Gold has an r.i. matching that of regular
> immersion oil anyway.  We use non-hardened Prolong Gold/Diamond for 3D-SIM
> (using an OMX), and compensate for the r.i. mismatch using a different
> refractive index oil.  I haven't actually tried that with STED yet, and you
> need to be aware that the Cargille oils that we typically use for r.i.
> selection may induce some chromatic dispersion too, but you could give it a
> shot.  Otherwise, unless you have adaptive optics on your system, you will
> have to see whether your results are better or worse without curing - i.e.
> balancing the negative effects of spherical aberrations against squashing
> effects!
>
> In the light sheet microscopy world, a lot of work has been done with r.i.
> matching, in particular using TDE-based mountants.  And I now notice
> however that Abberior has a kind of mounting medium on their webpage called
> Abberior TDE which comes in different types - r.i.s to match immersion oil,
> silicone oil or glycerol.  It says the TDE mountants are specifically
> designed for imaging thick specimens, which would imply to me that they
> minimize squashing artifacts, though I can't see that it explicitly says
> that.  I find the r.i. of uncured Prolong Gold to be pretty similar to that
> of Vectashield, which is glycerol-based (this is just by empirical testing,
> i.e. which r.i. oil matches best on the OMX).  So I wonder whether the
> Abberior TDE glycerol version would work well with uncured Prolong Gold?
>
> Abberior guys (Christian, Mary Grace...?), does any of you know the answer
> to this?  Do the TDE mountants harden and squash the samples or not, and
> have you tried them in combination with uncured Prolong Gold?  (If not,
> please send me some and I will test them myself as soon as I'm back in the
> lab!).  Since Germany is a civilised country that actually believes in
> vacation days over Easter (unlike my current country of residence, grrr!) I
> am not expecting an immediate response, but I did want to ask the question
> before I forget....
>
> Thanks for the question Xavi!
> All the best,
> Alison
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of SANJUAN SAMARRA, XAVIER <[hidden email]>
> Sent: Sunday, April 12, 2020 5:27 AM
> To: [hidden email] <[hidden email]>
> Subject: Mounting media for STED
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=UT_HGylOCutPMdVi0EcPvL1f1TK7bxfzTOSTBCZm8e0&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=TpmsRitFDyxjlgIZt1oYyXc2YzHQyMFQYmiMC4C8vs0&e=
> and include the link in your posting.
> *****
>
> Dear all,
>
> For STED experiments we have always mounted our cells with ProLong Gold. It
> is one of the recommended ones in the Guide to STED Sample Preparation
> published by Leica (
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__webcdn.leica-2Dmicrosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5FPreparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pdf&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=Mk7C5-iSFnwD64vR5nG8EFEIBsqUOap4c080ALYtDm0&e=
> ),
> and it works well in our hands. As it hardens, it is well known it
> squash/shrink the cells mainly in z dimension, thus affecting its shape. I
> have never read or heard the squashing also affects at the molecular level
> (i.e. changing molecular shape or distances between molecules), something
> that would have a negative impact in our STED observations, but do you
> think it could be the case? Is there any publication on this topic? And, is
> there any alternative mounting media to avoid this (hypothetical) artifact
> on the molecular structure of the sample?
>
> Best,
>
> Xavi.
>
> ___________________________________
>
> *Xavier Sanjuan*
> Advanced Light Microscopy Unit
>
> Parc de Recerca Biomèdica de Barcelona
> Doctor Aiguader, 88
> 08003 Barcelona - Spain
> Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
> Fax: + 34 93 316 09 01
> E-mail: [hidden email]
> Web:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=xEGAoB6Dl2fmc4iX1fuNb_NIHbU8DZeU4Wm0-ECz74Y&e=
>

[Jakub Chojnacki]

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Hi Xavi, hi Alison,

Regarding curing vs non-curing media I cannot comment reliably (I would
love to hear if Abberior has done any comparisons for STED?) but back in my
time at Oxford (around 2017) I know that there was a comparison done
between non-curing mounting media in terms of inducing shrinkage and
distortion artefacts in cells. I am not sure if that was ever published but
the 90% glycerol mounting medium was shown to be the best.

Personally, to avoid any potential issues with curing, I have just always
used non-curing mounting media only. Currently I use SlowFade Diamond for
STED and in my hands it has performed really well. It is glycerol based and
all the usual STED fluorophores appear to work well.

I have not tried SlowFade Glass yet as I presume this is intended for more
deep imaging samples.

Hope this helps,
Jakub


On Mon, 13 Apr 2020 at 16:22, Alison J. North <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Xavi,
>
> The 3D squashing effect is something many of us have seen, but I am
> another person who keeps intending to publish a thorough comparison of the
> effects and never has time! And whether it has an effect at the molecular
> distance level is a good question.  You do not actually have to let the
> Prolong Gold harden.   If you seal around the coverslips with quick drying
> nail polish immediately after mounting, it doesn't harden and you retain
> more of the 3D information.  Sure, you don't end up with the higher
> refractive index of the cured mountant, but it's not as though the
> completely cured Prolong Gold has an r.i. matching that of regular
> immersion oil anyway.  We use non-hardened Prolong Gold/Diamond for 3D-SIM
> (using an OMX), and compensate for the r.i. mismatch using a different
> refractive index oil.  I haven't actually tried that with STED yet, and you
> need to be aware that the Cargille oils that we typically use for r.i.
> selection may induce some chromatic dispersion too, but you could give it a
> shot.  Otherwise, unless you have adaptive optics on your system, you will
> have to see whether your results are better or worse without curing - i.e.
> balancing the negative effects of spherical aberrations against squashing
> effects!
>
> In the light sheet microscopy world, a lot of work has been done with r.i.
> matching, in particular using TDE-based mountants.  And I now notice
> however that Abberior has a kind of mounting medium on their webpage called
> Abberior TDE which comes in different types - r.i.s to match immersion oil,
> silicone oil or glycerol.  It says the TDE mountants are specifically
> designed for imaging thick specimens, which would imply to me that they
> minimize squashing artifacts, though I can't see that it explicitly says
> that.  I find the r.i. of uncured Prolong Gold to be pretty similar to that
> of Vectashield, which is glycerol-based (this is just by empirical testing,
> i.e. which r.i. oil matches best on the OMX).  So I wonder whether the
> Abberior TDE glycerol version would work well with uncured Prolong Gold?
>
> Abberior guys (Christian, Mary Grace...?), does any of you know the answer
> to this?  Do the TDE mountants harden and squash the samples or not, and
> have you tried them in combination with uncured Prolong Gold?  (If not,
> please send me some and I will test them myself as soon as I'm back in the
> lab!).  Since Germany is a civilised country that actually believes in
> vacation days over Easter (unlike my current country of residence, grrr!) I
> am not expecting an immediate response, but I did want to ask the question
> before I forget....
>
> Thanks for the question Xavi!
> All the best,
> Alison
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of SANJUAN SAMARRA, XAVIER <[hidden email]>
> Sent: Sunday, April 12, 2020 5:27 AM
> To: [hidden email] <[hidden email]>
> Subject: Mounting media for STED
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=UT_HGylOCutPMdVi0EcPvL1f1TK7bxfzTOSTBCZm8e0&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=TpmsRitFDyxjlgIZt1oYyXc2YzHQyMFQYmiMC4C8vs0&e=
> and include the link in your posting.
> *****
>
> Dear all,
>
> For STED experiments we have always mounted our cells with ProLong Gold. It
> is one of the recommended ones in the Guide to STED Sample Preparation
> published by Leica (
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__webcdn.leica-2Dmicrosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5FPreparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pdf&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=Mk7C5-iSFnwD64vR5nG8EFEIBsqUOap4c080ALYtDm0&e=
> ),
> and it works well in our hands. As it hardens, it is well known it
> squash/shrink the cells mainly in z dimension, thus affecting its shape. I
> have never read or heard the squashing also affects at the molecular level
> (i.e. changing molecular shape or distances between molecules), something
> that would have a negative impact in our STED observations, but do you
> think it could be the case? Is there any publication on this topic? And, is
> there any alternative mounting media to avoid this (hypothetical) artifact
> on the molecular structure of the sample?
>
> Best,
>
> Xavi.
>
> ___________________________________
>
> *Xavier Sanjuan*
> Advanced Light Microscopy Unit
>
> Parc de Recerca Biomèdica de Barcelona
> Doctor Aiguader, 88
> 08003 Barcelona - Spain
> Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
> Fax: + 34 93 316 09 01
> E-mail: [hidden email]
> Web:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=xEGAoB6Dl2fmc4iX1fuNb_NIHbU8DZeU4Wm0-ECz74Y&e=
>

--
Dr Jakub Chojnacki
IrsiCaixa AIDS Research Institute
Barcelona, Spain
[hidden email] <https://twitter.com/IrsiCaixa>

[Cammer, Michael-2]

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Are the samples getting physically flatter as the mounting media cure or are mismatches in refractive indexes (indices?) making depths appear different?


Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

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

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

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



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Jakub Chojnacki <[hidden email]>
Sent: Monday, April 13, 2020 10:57:06 AM
To: [hidden email]
Subject: Mounting media for STED

[EXTERNAL]

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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=q6DFX6_068IWH_gf-DOVSh-LB_rcnG6nV6pwtJ5niN4&s=WOHb_MKApWl0Uv-3JwlHupVLVdLYRlZuSc8BLiJB-do&e=  and include the link in your posting.
*****

Hi Xavi, hi Alison,

Regarding curing vs non-curing media I cannot comment reliably (I would
love to hear if Abberior has done any comparisons for STED?) but back in my
time at Oxford (around 2017) I know that there was a comparison done
between non-curing mounting media in terms of inducing shrinkage and
distortion artefacts in cells. I am not sure if that was ever published but
the 90% glycerol mounting medium was shown to be the best.

Personally, to avoid any potential issues with curing, I have just always
used non-curing mounting media only. Currently I use SlowFade Diamond for
STED and in my hands it has performed really well. It is glycerol based and
all the usual STED fluorophores appear to work well.

I have not tried SlowFade Glass yet as I presume this is intended for more
deep imaging samples.

Hope this helps,
Jakub


On Mon, 13 Apr 2020 at 16:22, Alison J. North <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=q6DFX6_068IWH_gf-DOVSh-LB_rcnG6nV6pwtJ5niN4&s=lM17LaE1IwRS-mA9pd3Ai5JD6TyQ0zCcelGicLFm8vg&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=q6DFX6_068IWH_gf-DOVSh-LB_rcnG6nV6pwtJ5niN4&s=WOHb_MKApWl0Uv-3JwlHupVLVdLYRlZuSc8BLiJB-do&e=  and include the link in your posting.
> *****
>
> Dear Xavi,
>
> The 3D squashing effect is something many of us have seen, but I am
> another person who keeps intending to publish a thorough comparison of the
> effects and never has time! And whether it has an effect at the molecular
> distance level is a good question.  You do not actually have to let the
> Prolong Gold harden.   If you seal around the coverslips with quick drying
> nail polish immediately after mounting, it doesn't harden and you retain
> more of the 3D information.  Sure, you don't end up with the higher
> refractive index of the cured mountant, but it's not as though the
> completely cured Prolong Gold has an r.i. matching that of regular
> immersion oil anyway.  We use non-hardened Prolong Gold/Diamond for 3D-SIM
> (using an OMX), and compensate for the r.i. mismatch using a different
> refractive index oil.  I haven't actually tried that with STED yet, and you
> need to be aware that the Cargille oils that we typically use for r.i.
> selection may induce some chromatic dispersion too, but you could give it a
> shot.  Otherwise, unless you have adaptive optics on your system, you will
> have to see whether your results are better or worse without curing - i.e.
> balancing the negative effects of spherical aberrations against squashing
> effects!
>
> In the light sheet microscopy world, a lot of work has been done with r.i.
> matching, in particular using TDE-based mountants.  And I now notice
> however that Abberior has a kind of mounting medium on their webpage called
> Abberior TDE which comes in different types - r.i.s to match immersion oil,
> silicone oil or glycerol.  It says the TDE mountants are specifically
> designed for imaging thick specimens, which would imply to me that they
> minimize squashing artifacts, though I can't see that it explicitly says
> that.  I find the r.i. of uncured Prolong Gold to be pretty similar to that
> of Vectashield, which is glycerol-based (this is just by empirical testing,
> i.e. which r.i. oil matches best on the OMX).  So I wonder whether the
> Abberior TDE glycerol version would work well with uncured Prolong Gold?
>
> Abberior guys (Christian, Mary Grace...?), does any of you know the answer
> to this?  Do the TDE mountants harden and squash the samples or not, and
> have you tried them in combination with uncured Prolong Gold?  (If not,
> please send me some and I will test them myself as soon as I'm back in the
> lab!).  Since Germany is a civilised country that actually believes in
> vacation days over Easter (unlike my current country of residence, grrr!) I
> am not expecting an immediate response, but I did want to ask the question
> before I forget....
>
> Thanks for the question Xavi!
> All the best,
> Alison
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of SANJUAN SAMARRA, XAVIER <[hidden email]>
> Sent: Sunday, April 12, 2020 5:27 AM
> To: [hidden email] <[hidden email]>
> Subject: Mounting media for STED
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=UT_HGylOCutPMdVi0EcPvL1f1TK7bxfzTOSTBCZm8e0&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=TpmsRitFDyxjlgIZt1oYyXc2YzHQyMFQYmiMC4C8vs0&e=
> and include the link in your posting.
> *****
>
> Dear all,
>
> For STED experiments we have always mounted our cells with ProLong Gold. It
> is one of the recommended ones in the Guide to STED Sample Preparation
> published by Leica (
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__webcdn.leica-2Dmicrosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5FPreparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pdf&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=Mk7C5-iSFnwD64vR5nG8EFEIBsqUOap4c080ALYtDm0&e=
> ),
> and it works well in our hands. As it hardens, it is well known it
> squash/shrink the cells mainly in z dimension, thus affecting its shape. I
> have never read or heard the squashing also affects at the molecular level
> (i.e. changing molecular shape or distances between molecules), something
> that would have a negative impact in our STED observations, but do you
> think it could be the case? Is there any publication on this topic? And, is
> there any alternative mounting media to avoid this (hypothetical) artifact
> on the molecular structure of the sample?
>
> Best,
>
> Xavi.
>
> ___________________________________
>
> *Xavier Sanjuan*
> Advanced Light Microscopy Unit
>
> Parc de Recerca Biomèdica de Barcelona
> Doctor Aiguader, 88
> 08003 Barcelona - Spain
> Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
> Fax: + 34 93 316 09 01
> E-mail: [hidden email]
> Web:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=xEGAoB6Dl2fmc4iX1fuNb_NIHbU8DZeU4Wm0-ECz74Y&e=
>

--
Dr Jakub Chojnacki
IrsiCaixa AIDS Research Institute
Barcelona, Spain
[hidden email] <https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_IrsiCaixa&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=q6DFX6_068IWH_gf-DOVSh-LB_rcnG6nV6pwtJ5niN4&s=YSEP0HSYOYttis3khPR-h2lLTn9ptOhBPh11T8ajmUQ&e= >

[Alison J. North]

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*****

Hi Jakub,

Thanks, this is useful!  So are you using the SlowFade Diamond with an oil immersion objective, without significant spherical aberration artifacts?   If so, then uncured Prolong media shouldn't be a problem either -  though it also depends on whether you are using an Abberior system - with AO - or a Leica system, right?  As far as I understand, the one drawback of the Slowfade reagents is that the samples don't last nearly as long - they should be imaged within a day or two - while I have kept Prolong mounted samples in the fridge for literally years without significant deterioration.  Do you find that your Slowfade samples go off quickly, or is it not as bad as I've been led to believe?

Best,
Alison

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Jakub Chojnacki <[hidden email]>
Sent: Monday, April 13, 2020 10:57 AM
To: [hidden email] <[hidden email]>
Subject: Mounting media for STED

*****
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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=y4feSNDZYBL-b53agvjgcCMQHpS7WDccK0ft7kytFUU&e=  and include the link in your posting.
*****

Hi Xavi, hi Alison,

Regarding curing vs non-curing media I cannot comment reliably (I would
love to hear if Abberior has done any comparisons for STED?) but back in my
time at Oxford (around 2017) I know that there was a comparison done
between non-curing mounting media in terms of inducing shrinkage and
distortion artefacts in cells. I am not sure if that was ever published but
the 90% glycerol mounting medium was shown to be the best.

Personally, to avoid any potential issues with curing, I have just always
used non-curing mounting media only. Currently I use SlowFade Diamond for
STED and in my hands it has performed really well. It is glycerol based and
all the usual STED fluorophores appear to work well.

I have not tried SlowFade Glass yet as I presume this is intended for more
deep imaging samples.

Hope this helps,
Jakub


On Mon, 13 Apr 2020 at 16:22, Alison J. North <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=WUlLvPIEiL3itAuXGle--4z0HjCGcgEdNAY2apbQdng&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=y4feSNDZYBL-b53agvjgcCMQHpS7WDccK0ft7kytFUU&e=  and include the link in your posting.
> *****
>
> Dear Xavi,
>
> The 3D squashing effect is something many of us have seen, but I am
> another person who keeps intending to publish a thorough comparison of the
> effects and never has time! And whether it has an effect at the molecular
> distance level is a good question.  You do not actually have to let the
> Prolong Gold harden.   If you seal around the coverslips with quick drying
> nail polish immediately after mounting, it doesn't harden and you retain
> more of the 3D information.  Sure, you don't end up with the higher
> refractive index of the cured mountant, but it's not as though the
> completely cured Prolong Gold has an r.i. matching that of regular
> immersion oil anyway.  We use non-hardened Prolong Gold/Diamond for 3D-SIM
> (using an OMX), and compensate for the r.i. mismatch using a different
> refractive index oil.  I haven't actually tried that with STED yet, and you
> need to be aware that the Cargille oils that we typically use for r.i.
> selection may induce some chromatic dispersion too, but you could give it a
> shot.  Otherwise, unless you have adaptive optics on your system, you will
> have to see whether your results are better or worse without curing - i.e.
> balancing the negative effects of spherical aberrations against squashing
> effects!
>
> In the light sheet microscopy world, a lot of work has been done with r.i.
> matching, in particular using TDE-based mountants.  And I now notice
> however that Abberior has a kind of mounting medium on their webpage called
> Abberior TDE which comes in different types - r.i.s to match immersion oil,
> silicone oil or glycerol.  It says the TDE mountants are specifically
> designed for imaging thick specimens, which would imply to me that they
> minimize squashing artifacts, though I can't see that it explicitly says
> that.  I find the r.i. of uncured Prolong Gold to be pretty similar to that
> of Vectashield, which is glycerol-based (this is just by empirical testing,
> i.e. which r.i. oil matches best on the OMX).  So I wonder whether the
> Abberior TDE glycerol version would work well with uncured Prolong Gold?
>
> Abberior guys (Christian, Mary Grace...?), does any of you know the answer
> to this?  Do the TDE mountants harden and squash the samples or not, and
> have you tried them in combination with uncured Prolong Gold?  (If not,
> please send me some and I will test them myself as soon as I'm back in the
> lab!).  Since Germany is a civilised country that actually believes in
> vacation days over Easter (unlike my current country of residence, grrr!) I
> am not expecting an immediate response, but I did want to ask the question
> before I forget....
>
> Thanks for the question Xavi!
> All the best,
> Alison
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of SANJUAN SAMARRA, XAVIER <[hidden email]>
> Sent: Sunday, April 12, 2020 5:27 AM
> To: [hidden email] <[hidden email]>
> Subject: Mounting media for STED
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=UT_HGylOCutPMdVi0EcPvL1f1TK7bxfzTOSTBCZm8e0&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=TpmsRitFDyxjlgIZt1oYyXc2YzHQyMFQYmiMC4C8vs0&e=
> and include the link in your posting.
> *****
>
> Dear all,
>
> For STED experiments we have always mounted our cells with ProLong Gold. It
> is one of the recommended ones in the Guide to STED Sample Preparation
> published by Leica (
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__webcdn.leica-2Dmicrosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5FPreparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pdf&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=Mk7C5-iSFnwD64vR5nG8EFEIBsqUOap4c080ALYtDm0&e=
> ),
> and it works well in our hands. As it hardens, it is well known it
> squash/shrink the cells mainly in z dimension, thus affecting its shape. I
> have never read or heard the squashing also affects at the molecular level
> (i.e. changing molecular shape or distances between molecules), something
> that would have a negative impact in our STED observations, but do you
> think it could be the case? Is there any publication on this topic? And, is
> there any alternative mounting media to avoid this (hypothetical) artifact
> on the molecular structure of the sample?
>
> Best,
>
> Xavi.
>
> ___________________________________
>
> *Xavier Sanjuan*
> Advanced Light Microscopy Unit
>
> Parc de Recerca Biomèdica de Barcelona
> Doctor Aiguader, 88
> 08003 Barcelona - Spain
> Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
> Fax: + 34 93 316 09 01
> E-mail: [hidden email]
> Web:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=xEGAoB6Dl2fmc4iX1fuNb_NIHbU8DZeU4Wm0-ECz74Y&e=
>

--
Dr Jakub Chojnacki
IrsiCaixa AIDS Research Institute
Barcelona, Spain
[hidden email] <https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_IrsiCaixa&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=tFbcrOQFFazDb77ZKbw7wObEomzccHJjB0Yn-hLvhDo&e= >

[Alison J. North]

*****
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.
*****

Hey Doug!!

Nice to hear from you and super useful info.  The data comparing the TDE and PG is spectacular.
Now I definitely want to hear more info about the Abberior TDE mountants...
This is a great discussion!

Stay safe my friend...
🖖
Alison

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Douglas Richardson <[hidden email]>
Sent: Monday, April 13, 2020 10:52 AM
To: [hidden email] <[hidden email]>
Subject: Re: Mounting media for STED

*****
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Hi Allison,

This paper should answer all your questions on TDE including shrinkage,
dispersion, etc. https://urldefense.proofpoint.com/v2/url?u=https-3A__onlinelibrary.wiley.com_doi_abs_10.1002_jemt.20396&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=xdUgNozvQX5ZOs6rPEacTUTY9Jo9pGXGjb7Q2n4PQUU&s=1qaXvUg6xm-8G9OtHyg7XfYf0Utwi4Abm1X8e4YkRMc&e=

Briefly, if you take your sample through a step-wise increase in TDE as
described in the paper, the shrinkage is minimal. We saw some great
improvements using TDE-based mounting medium over Prolong Gold for SIM (
https://urldefense.proofpoint.com/v2/url?u=https-3A__hcbi.fas.harvard.edu_files_hcbidoug_files_hcbi-5Frecommended-5Fmounting-5Fmedia-5F0.pdf&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=xdUgNozvQX5ZOs6rPEacTUTY9Jo9pGXGjb7Q2n4PQUU&s=j9hGbXxvCh7VdoIzOdEneL5UzNc1dFvSX_jrclMbAKA&e= ).
This was before Prolong and Slow-fade Glass were available.

The drawbacks to TDE are the longer amount of time to prep the sample as
you step through different concentrations. Also, green and blue dyes
photobleach almost immediately in high TDE concentrations (those needed to
match oil immersion objectives), but are more stable when more water is
present (glycerol/silicone objectives).

The Abberior folks have told me that the high RI Prolong/Slow-fade Glass
products shift the emission spectrum of red and far-red dyes making STED
more difficult.  We had some samples ready to test when we got shutdown :(.

-Doug

On Mon, Apr 13, 2020 at 10:22 AM Alison J. North <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=xdUgNozvQX5ZOs6rPEacTUTY9Jo9pGXGjb7Q2n4PQUU&s=PDPLIGZx2_M88fY191-zoU_otHg31ifb9SXRQsGBxf8&e=  and include the link in your posting.
> *****
>
> Dear Xavi,
>
> The 3D squashing effect is something many of us have seen, but I am
> another person who keeps intending to publish a thorough comparison of the
> effects and never has time! And whether it has an effect at the molecular
> distance level is a good question.  You do not actually have to let the
> Prolong Gold harden.   If you seal around the coverslips with quick drying
> nail polish immediately after mounting, it doesn't harden and you retain
> more of the 3D information.  Sure, you don't end up with the higher
> refractive index of the cured mountant, but it's not as though the
> completely cured Prolong Gold has an r.i. matching that of regular
> immersion oil anyway.  We use non-hardened Prolong Gold/Diamond for 3D-SIM
> (using an OMX), and compensate for the r.i. mismatch using a different
> refractive index oil.  I haven't actually tried that with STED yet, and you
> need to be aware that the Cargille oils that we typically use for r.i.
> selection may induce some chromatic dispersion too, but you could give it a
> shot.  Otherwise, unless you have adaptive optics on your system, you will
> have to see whether your results are better or worse without curing - i.e.
> balancing the negative effects of spherical aberrations against squashing
> effects!
>
> In the light sheet microscopy world, a lot of work has been done with r.i.
> matching, in particular using TDE-based mountants.  And I now notice
> however that Abberior has a kind of mounting medium on their webpage called
> Abberior TDE which comes in different types - r.i.s to match immersion oil,
> silicone oil or glycerol.  It says the TDE mountants are specifically
> designed for imaging thick specimens, which would imply to me that they
> minimize squashing artifacts, though I can't see that it explicitly says
> that.  I find the r.i. of uncured Prolong Gold to be pretty similar to that
> of Vectashield, which is glycerol-based (this is just by empirical testing,
> i.e. which r.i. oil matches best on the OMX).  So I wonder whether the
> Abberior TDE glycerol version would work well with uncured Prolong Gold?
>
> Abberior guys (Christian, Mary Grace...?), does any of you know the answer
> to this?  Do the TDE mountants harden and squash the samples or not, and
> have you tried them in combination with uncured Prolong Gold?  (If not,
> please send me some and I will test them myself as soon as I'm back in the
> lab!).  Since Germany is a civilised country that actually believes in
> vacation days over Easter (unlike my current country of residence, grrr!) I
> am not expecting an immediate response, but I did want to ask the question
> before I forget....
>
> Thanks for the question Xavi!
> All the best,
> Alison
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of SANJUAN SAMARRA, XAVIER <[hidden email]>
> Sent: Sunday, April 12, 2020 5:27 AM
> To: [hidden email] <[hidden email]>
> Subject: Mounting media for STED
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=UT_HGylOCutPMdVi0EcPvL1f1TK7bxfzTOSTBCZm8e0&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=TpmsRitFDyxjlgIZt1oYyXc2YzHQyMFQYmiMC4C8vs0&e=
> and include the link in your posting.
> *****
>
> Dear all,
>
> For STED experiments we have always mounted our cells with ProLong Gold. It
> is one of the recommended ones in the Guide to STED Sample Preparation
> published by Leica (
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__webcdn.leica-2Dmicrosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5FPreparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pdf&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=Mk7C5-iSFnwD64vR5nG8EFEIBsqUOap4c080ALYtDm0&e=
> ),
> and it works well in our hands. As it hardens, it is well known it
> squash/shrink the cells mainly in z dimension, thus affecting its shape. I
> have never read or heard the squashing also affects at the molecular level
> (i.e. changing molecular shape or distances between molecules), something
> that would have a negative impact in our STED observations, but do you
> think it could be the case? Is there any publication on this topic? And, is
> there any alternative mounting media to avoid this (hypothetical) artifact
> on the molecular structure of the sample?
>
> Best,
>
> Xavi.
>
> ___________________________________
>
> *Xavier Sanjuan*
> Advanced Light Microscopy Unit
>
> Parc de Recerca Biomèdica de Barcelona
> Doctor Aiguader, 88
> 08003 Barcelona - Spain
> Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
> Fax: + 34 93 316 09 01
> E-mail: [hidden email]
> Web:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=xEGAoB6Dl2fmc4iX1fuNb_NIHbU8DZeU4Wm0-ECz74Y&e=
>

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*****

Hi Alison,

I had been in discussion with Abberior quite a while ago concerning mounting media for thick 3D samples, so we talked in length about the hardening vs non-hardening media. (As far as I recall) You are completely correct in assuming that the "3D-suitability" of specific mounting media (such as the Abberior TDE) are its non-hardening properties. I love working with Prolong Gold/Diamond, but it does shrink when hardening, so effectively squashes the tissue samples in the axial direction specifically. Obviously, this makes much more of a difference when imaging truly thick samples (such as brain slices) vs. monolayers or cells... but if you really need quantitative measurements of axial distance, then use non-hardening media. (So Slowfade, TDE, or similar). But then again, if you are imaging thick samples using a mounting media with the incorrect RI, then you are going to run into problems, anyway.

Concerning the Prolong Glass, Abberior had also warned me that they had observed significant spectral red-shifts of fluorescent dyes, yet I cannot confirm this in the samples I have been imaging. We prepared 3-colored green, red & far-red samples (STAR 488, AF 594, STAR RED) in either Mowiol, Prolong Gold or Prolong Glass, and imaged them side-by-side. We observed different brightness levels of the dyes for sure (but this might have to do with antibody concentration more than with the actual mounting media), but the STED efficiencies of all dyes were very similar, especially comparing Prolong Glass vs. Gold. If the spectral shifts were as pronounced as stated, the STED efficiencies (i.e. how much STED power do you need to achieve a specific resolution) should have also changed. This was not the case. I haven't recorded the spectra of the dyes, considering that the samples were extremely dense-labeled in 3 colors, but I expect it would prove my point.

I do not have extensive experience with non-hardened Prolong samples, can't comment there.

All the best from Florida
Nicolai



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Alison J. North
Sent: Monday, 13 April 2020 11:30
To: [hidden email]
Subject: Re: Mounting media for STED

*****
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*****

Hey Doug!!

Nice to hear from you and super useful info.  The data comparing the TDE and PG is spectacular.
Now I definitely want to hear more info about the Abberior TDE mountants...
This is a great discussion!

Stay safe my friend...
🖖
Alison

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Douglas Richardson <[hidden email]>
Sent: Monday, April 13, 2020 10:52 AM
To: [hidden email] <[hidden email]>
Subject: Re: Mounting media for STED

*****
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*****

Hi Allison,

This paper should answer all your questions on TDE including shrinkage, dispersion, etc. https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__onlinelibrary.wiley.com_doi_abs_10.1002_jemt.20396%26d%3DDwIFaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DxdUgNozvQX5ZOs6rPEacTUTY9Jo9pGXGjb7Q2n4PQUU%26s%3D1qaXvUg6xm-8G9OtHyg7XfYf0Utwi4Abm1X8e4YkRMc%26e%3D&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C7e9db23f2b2d42cce57708d7dfbfb087%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C637223886782582595&amp;sdata=2qHjEMxu8rGUs%2BrovQBpC5en7%2BCmo7j5VjGCBFxtPGw%3D&amp;reserved=0

Briefly, if you take your sample through a step-wise increase in TDE as described in the paper, the shrinkage is minimal. We saw some great improvements using TDE-based mounting medium over Prolong Gold for SIM (
https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__hcbi.fas.harvard.edu_files_hcbidoug_files_hcbi-5Frecommended-5Fmounting-5Fmedia-5F0.pdf%26d%3DDwIFaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DxdUgNozvQX5ZOs6rPEacTUTY9Jo9pGXGjb7Q2n4PQUU%26s%3Dj9hGbXxvCh7VdoIzOdEneL5UzNc1dFvSX_jrclMbAKA%26e%3D&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C7e9db23f2b2d42cce57708d7dfbfb087%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C637223886782582595&amp;sdata=ALf1Jf5B9ze2zAPPGru0dgs1iemjrZ88N2T2UxFXNHQ%3D&amp;reserved=0 ).
This was before Prolong and Slow-fade Glass were available.

The drawbacks to TDE are the longer amount of time to prep the sample as you step through different concentrations. Also, green and blue dyes photobleach almost immediately in high TDE concentrations (those needed to match oil immersion objectives), but are more stable when more water is present (glycerol/silicone objectives).

The Abberior folks have told me that the high RI Prolong/Slow-fade Glass products shift the emission spectrum of red and far-red dyes making STED more difficult.  We had some samples ready to test when we got shutdown :(.

-Doug

On Mon, Apr 13, 2020 at 10:22 AM Alison J. North < [hidden email]> wrote:

> *****
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> *****
>
> Dear Xavi,
>
> The 3D squashing effect is something many of us have seen, but I am
> another person who keeps intending to publish a thorough comparison of
> the effects and never has time! And whether it has an effect at the
> molecular distance level is a good question.  You do not actually have to let the
> Prolong Gold harden.   If you seal around the coverslips with quick drying
> nail polish immediately after mounting, it doesn't harden and you
> retain more of the 3D information.  Sure, you don't end up with the
> higher refractive index of the cured mountant, but it's not as though
> the completely cured Prolong Gold has an r.i. matching that of regular
> immersion oil anyway.  We use non-hardened Prolong Gold/Diamond for
> 3D-SIM (using an OMX), and compensate for the r.i. mismatch using a
> different refractive index oil.  I haven't actually tried that with
> STED yet, and you need to be aware that the Cargille oils that we typically use for r.i.
> selection may induce some chromatic dispersion too, but you could give
> it a shot.  Otherwise, unless you have adaptive optics on your system,
> you will have to see whether your results are better or worse without curing - i.e.
> balancing the negative effects of spherical aberrations against
> squashing effects!
>
> In the light sheet microscopy world, a lot of work has been done with r.i.
> matching, in particular using TDE-based mountants.  And I now notice
> however that Abberior has a kind of mounting medium on their webpage
> called Abberior TDE which comes in different types - r.i.s to match
> immersion oil, silicone oil or glycerol.  It says the TDE mountants
> are specifically designed for imaging thick specimens, which would
> imply to me that they minimize squashing artifacts, though I can't see
> that it explicitly says that.  I find the r.i. of uncured Prolong Gold
> to be pretty similar to that of Vectashield, which is glycerol-based
> (this is just by empirical testing, i.e. which r.i. oil matches best
> on the OMX).  So I wonder whether the Abberior TDE glycerol version would work well with uncured Prolong Gold?
>
> Abberior guys (Christian, Mary Grace...?), does any of you know the
> answer to this?  Do the TDE mountants harden and squash the samples or
> not, and have you tried them in combination with uncured Prolong Gold?  
> (If not, please send me some and I will test them myself as soon as
> I'm back in the lab!).  Since Germany is a civilised country that
> actually believes in vacation days over Easter (unlike my current
> country of residence, grrr!) I am not expecting an immediate response,
> but I did want to ask the question before I forget....
>
> Thanks for the question Xavi!
> All the best,
> Alison
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of SANJUAN SAMARRA, XAVIER <[hidden email]>
> Sent: Sunday, April 12, 2020 5:27 AM
> To: [hidden email]
> <[hidden email]>
> Subject: Mounting media for STED
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> rved=0
> and include the link in your posting.
> *****
>
> Dear all,
>
> For STED experiments we have always mounted our cells with ProLong
> Gold. It is one of the recommended ones in the Guide to STED Sample
> Preparation published by Leica (
>
> https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Furld
> efense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__webcdn.leica-2Dmicros
> ystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5FPrep
> aration_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pdf%26
> d%3DDwIFaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx
> 0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3D0VemQzJmtronf8nlxMjnSQI
> f5Vh0elEu5nrOCJiomGc%26s%3DMk7C5-iSFnwD64vR5nG8EFEIBsqUOap4c080ALYtDm0
> %26e%3D&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C7e9db23f2b2d42cc
> e57708d7dfbfb087%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C63722388
> 6782592591&amp;sdata=TG%2BnO1rLRaxqu3ulfWnbev7rJfvOMsmZZfxk7igeDCo%3D&
> amp;reserved=0
> ),
> and it works well in our hands. As it hardens, it is well known it
> squash/shrink the cells mainly in z dimension, thus affecting its
> shape. I have never read or heard the squashing also affects at the
> molecular level (i.e. changing molecular shape or distances between
> molecules), something that would have a negative impact in our STED
> observations, but do you think it could be the case? Is there any
> publication on this topic? And, is there any alternative mounting
> media to avoid this (hypothetical) artifact on the molecular structure of the sample?
>
> Best,
>
> Xavi.
>
> ___________________________________
>
> *Xavier Sanjuan*
> Advanced Light Microscopy Unit
>
> Parc de Recerca Biomèdica de Barcelona Doctor Aiguader, 88
> 08003 Barcelona - Spain
> Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
> Fax: + 34 93 316 09 01
> E-mail: [hidden email]
> Web:
>
> https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Furld
> efense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit%26d%3DDwIFaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3D0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc%26s%3DxEGAoB6Dl2fmc4iX1fuNb_NIHbU8DZeU4Wm0-ECz74Y%26e%3D&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C7e9db23f2b2d42cce57708d7dfbfb087%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C637223886782592591&amp;sdata=lRp9R6WhsUfcA6CvmYR6eT2uHP6x6e8J9wqK9%2BTFh3U%3D&amp;reserved=0
>

[Jakub Chojnacki]

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Hi Alison,

Regarding SlowFade Diamond - I did not see any significant aberrations when
using it in with Leica's 100x/1.4 NA oil immersion STED objective. 2D and
3D STED but without any AO of course. However my samples were either
imaging cell compartments in cell monolayers or adhered individual virus
particles so not very deep imaging.

Regarding its stability one time, due to a conference, I had to image a
series fixed cell samples (3D STED) 2 days and 2 weeks after labelling and
I got great results in both session with no discernible signal degradation.
Samples were kept in the fridge over this time.
So, while as always this may be sample dependant, I think that SlowFade
Diamond can last longer than just few days.

Also thank you Doug and Nicolai for the info on Prolong/Slow-fade Glass -
very useful.

Regards,
Jakub

On Mon, 13 Apr 2020 at 17:15, Alison J. North <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Jakub,
>
> Thanks, this is useful!  So are you using the SlowFade Diamond with an oil
> immersion objective, without significant spherical aberration artifacts?
>  If so, then uncured Prolong media shouldn't be a problem either -  though
> it also depends on whether you are using an Abberior system - with AO - or
> a Leica system, right?  As far as I understand, the one drawback of the
> Slowfade reagents is that the samples don't last nearly as long - they
> should be imaged within a day or two - while I have kept Prolong mounted
> samples in the fridge for literally years without significant
> deterioration.  Do you find that your Slowfade samples go off quickly, or
> is it not as bad as I've been led to believe?
>
> Best,
> Alison
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Jakub Chojnacki <[hidden email]>
> Sent: Monday, April 13, 2020 10:57 AM
> To: [hidden email] <[hidden email]>
> Subject: Mounting media for STED
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=WUlLvPIEiL3itAuXGle--4z0HjCGcgEdNAY2apbQdng&e=
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> and include the link in your posting.
> *****
>
> Hi Xavi, hi Alison,
>
> Regarding curing vs non-curing media I cannot comment reliably (I would
> love to hear if Abberior has done any comparisons for STED?) but back in my
> time at Oxford (around 2017) I know that there was a comparison done
> between non-curing mounting media in terms of inducing shrinkage and
> distortion artefacts in cells. I am not sure if that was ever published but
> the 90% glycerol mounting medium was shown to be the best.
>
> Personally, to avoid any potential issues with curing, I have just always
> used non-curing mounting media only. Currently I use SlowFade Diamond for
> STED and in my hands it has performed really well. It is glycerol based and
> all the usual STED fluorophores appear to work well.
>
> I have not tried SlowFade Glass yet as I presume this is intended for more
> deep imaging samples.
>
> Hope this helps,
> Jakub
>
>
> On Mon, 13 Apr 2020 at 16:22, Alison J. North <[hidden email]
> >
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=WUlLvPIEiL3itAuXGle--4z0HjCGcgEdNAY2apbQdng&e=
> > Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=y4feSNDZYBL-b53agvjgcCMQHpS7WDccK0ft7kytFUU&e=
> and include the link in your posting.
> > *****
> >
> > Dear Xavi,
> >
> > The 3D squashing effect is something many of us have seen, but I am
> > another person who keeps intending to publish a thorough comparison of
> the
> > effects and never has time! And whether it has an effect at the molecular
> > distance level is a good question.  You do not actually have to let the
> > Prolong Gold harden.   If you seal around the coverslips with quick
> drying
> > nail polish immediately after mounting, it doesn't harden and you retain
> > more of the 3D information.  Sure, you don't end up with the higher
> > refractive index of the cured mountant, but it's not as though the
> > completely cured Prolong Gold has an r.i. matching that of regular
> > immersion oil anyway.  We use non-hardened Prolong Gold/Diamond for
> 3D-SIM
> > (using an OMX), and compensate for the r.i. mismatch using a different
> > refractive index oil.  I haven't actually tried that with STED yet, and
> you
> > need to be aware that the Cargille oils that we typically use for r.i.
> > selection may induce some chromatic dispersion too, but you could give
> it a
> > shot.  Otherwise, unless you have adaptive optics on your system, you
> will
> > have to see whether your results are better or worse without curing -
> i.e.
> > balancing the negative effects of spherical aberrations against squashing
> > effects!
> >
> > In the light sheet microscopy world, a lot of work has been done with
> r.i.
> > matching, in particular using TDE-based mountants.  And I now notice
> > however that Abberior has a kind of mounting medium on their webpage
> called
> > Abberior TDE which comes in different types - r.i.s to match immersion
> oil,
> > silicone oil or glycerol.  It says the TDE mountants are specifically
> > designed for imaging thick specimens, which would imply to me that they
> > minimize squashing artifacts, though I can't see that it explicitly says
> > that.  I find the r.i. of uncured Prolong Gold to be pretty similar to
> that
> > of Vectashield, which is glycerol-based (this is just by empirical
> testing,
> > i.e. which r.i. oil matches best on the OMX).  So I wonder whether the
> > Abberior TDE glycerol version would work well with uncured Prolong Gold?
> >
> > Abberior guys (Christian, Mary Grace...?), does any of you know the
> answer
> > to this?  Do the TDE mountants harden and squash the samples or not, and
> > have you tried them in combination with uncured Prolong Gold?  (If not,
> > please send me some and I will test them myself as soon as I'm back in
> the
> > lab!).  Since Germany is a civilised country that actually believes in
> > vacation days over Easter (unlike my current country of residence,
> grrr!) I
> > am not expecting an immediate response, but I did want to ask the
> question
> > before I forget....
> >
> > Thanks for the question Xavi!
> > All the best,
> > Alison
> >
> > ________________________________
> > From: Confocal Microscopy List <[hidden email]> on
> > behalf of SANJUAN SAMARRA, XAVIER <[hidden email]>
> > Sent: Sunday, April 12, 2020 5:27 AM
> > To: [hidden email] <[hidden email]>
> > Subject: Mounting media for STED
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=UT_HGylOCutPMdVi0EcPvL1f1TK7bxfzTOSTBCZm8e0&e=
> > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=TpmsRitFDyxjlgIZt1oYyXc2YzHQyMFQYmiMC4C8vs0&e=
> > and include the link in your posting.
> > *****
> >
> > Dear all,
> >
> > For STED experiments we have always mounted our cells with ProLong Gold.
> It
> > is one of the recommended ones in the Guide to STED Sample Preparation
> > published by Leica (
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__webcdn.leica-2Dmicrosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5FPreparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pdf&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=Mk7C5-iSFnwD64vR5nG8EFEIBsqUOap4c080ALYtDm0&e=
> > ),
> > and it works well in our hands. As it hardens, it is well known it
> > squash/shrink the cells mainly in z dimension, thus affecting its shape.
> I
> > have never read or heard the squashing also affects at the molecular
> level
> > (i.e. changing molecular shape or distances between molecules), something
> > that would have a negative impact in our STED observations, but do you
> > think it could be the case? Is there any publication on this topic? And,
> is
> > there any alternative mounting media to avoid this (hypothetical)
> artifact
> > on the molecular structure of the sample?
> >
> > Best,
> >
> > Xavi.
> >
> > ___________________________________
> >
> > *Xavier Sanjuan*
> > Advanced Light Microscopy Unit
> >
> > Parc de Recerca Biomèdica de Barcelona
> > Doctor Aiguader, 88
> > 08003 Barcelona - Spain
> > Tel: + 34 93 316 0195 (ext 1195 dins el PRBB)
> > Fax: + 34 93 316 09 01
> > E-mail: [hidden email]
> > Web:
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.crg.eu_en_core_programmes-2Dgroups_advanced-2Dlight-2Dmicroscopy-2Dunit&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=xEGAoB6Dl2fmc4iX1fuNb_NIHbU8DZeU4Wm0-ECz74Y&e=
> >
>
>
> --
> Dr Jakub Chojnacki
> IrsiCaixa AIDS Research Institute
> Barcelona, Spain
> [hidden email] <
> https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_IrsiCaixa&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=tFbcrOQFFazDb77ZKbw7wObEomzccHJjB0Yn-hLvhDo&e=
> >
>

--
Dr Jakub Chojnacki
IrsiCaixa AIDS Research Institute
Barcelona, Spain
[hidden email] <https://twitter.com/IrsiCaixa>

[Arvydas Matiukas]

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Hello Microscopists,


What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.


Thanks,
Arvydas


Director of Microscopy Core

[Csúcs Gábor-3]

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Dear Arvydas,

Personally, I have expected a little bit more. Unfortunately (they certainly did it on purpose) Leica did not show any comparison of the new HyDs compare to the SP8 ones and the same is true for the WLL. However based on the currently available (very preliminary) data I would have two remarks to your points:
1) For me it seems that the new HyDs will bring real improvement to the red (NIR) range, for the blue/green range they will be very similar to the current (SP8) ones.
2) I believe that you are mistaken concerning the 350 nm for the WLL. The Leica homepage states 440 nm. This is of course still an improvement compared to the currently available 470 nm one, but again not a full game changer as for example on our current system we also have and additional 440 nm pulsed laser, so for us there would be very little improvement between the old and the new system. Unless the new WLL is so much stronger than the old one that you do not need any more additional lasers for FRAP - but personally I doubt this.....

Greetings Gabor

P.S. Of course - if you can afford, it is better to buy a technical more up to date system - it is clear that the Stellaris offers improvements compared to an SP8.


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Arvydas Matiukas
Sent: Tuesday, April 14, 2020 2:40 PM
To: [hidden email]
Subject: Re: New Leica Stellaris vs SP8

*****
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Hello Microscopists,


What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.


Thanks,
Arvydas


Director of Microscopy Core

[Ralf Palmisano]

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Hi Director Arvydas of LEICA Microscopy Core,

This mail rang so many bells while reading. It is this sort of masked
commercial mailings that I don't like to see here.
I was wondering why this initial mail was subjected a re-post. In fact
it was not are-post. It is a sort of undercover commercial post.
Why are you hiding that this is the initial post?
Why are you hiding that you are director of Leica microscopy core.
Why do make me search online who "Director of Microscopy Core" is?

I don't think that Leica supports this type of misguiding this
community, by pretending to be sort of a fair balanced user and starting
a promotional tour.

Don't get me wrong. This is not about Leica pro or con. This is about
your rather shabby behaviour here.
Don't get me wrong. If you would have made yourself known to be a Leica
sponsored facility manager and asked for the communities opion, well
fair enough, then everys would be aware what one is stepping into.

Leica Microsystems deserve much better than people like you coming in
such questionable disguise (yes, that's a Shakespeare quote). Apart from
it, Leica would certainly have phrased this commercial way better than
you, Sir, ever will be able, too.

I sincerely apologise if you think this was personal, it is not and I
believe this sort of posts always prove to be of disservice for the
respective company.

Have a nice day,

Ralph Palmisano
Head Optical Imaging Centre Erlangen
Central Institute
Friedrich-Alexander University of Erlangen-Nuremberg

Am 14/04/2020 um 14:40 schrieb Arvydas Matiukas:

> *****
> 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.
> *****
>
> Hello Microscopists,
>
>
> What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.
>
>
> Thanks,
> Arvydas
>
>
> Director of Microscopy Core

--
Ralph Palmisano
Head - Optical Imaging Centre Erlangen

Fellow Royal Microscopical Society
Member Royal Society of Medicine

Speaker Scientific Advisory Board "German BioImaging"
Board of Directors Core Technologies for Life Science

Cauerstr. 3
91058 Erlangen, Germany

[phone] (Office)
[phone] (Secretary)

www.oice.uni-erlangen.de

[Rosemary White]

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Hi Arvydas et al.,

I agree with Gabor that the extended emission detection into the near-IR of the new HyDs will be useful, as is the extended range of the WLL - the "old" WLL spans 470-670 nm, the new model 440-790 nm.

We have a now quite old SP8 with WLL (and 405) and HyDs, and the flexibility offered by the WLL is a winner. Especially useful for plant tissues with diverse autofluorescence and a wide range of stains, etc. We'd find single-line or even multiple-line lasers limiting now.

We routinely gate our HyDs to remove reflection artefacts, curious as to the other features of the new HyDs.

The Stellaris doesn't sound quite revolutionary but if it's as versatile as the SP8 it will be a good workhorse instrument. My only gripe may be that they've probably changed the GUI utterly (haven't seen it in the flesh), can't leave well enough alone....

cheers,
Rosemary

Visiting scientist
RSB, ANU
Canberra
Australia

M: 61-0468966713
E: [hidden email]
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Csucs Gabor [[hidden email]]
Sent: Tuesday, 14 April 2020 11:40 PM
To: [hidden email]
Subject: Re: New Leica Stellaris vs SP8

*****
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Dear Arvydas,

Personally, I have expected a little bit more. Unfortunately (they certainly did it on purpose) Leica did not show any comparison of the new HyDs compare to the SP8 ones and the same is true for the WLL. However based on the currently available (very preliminary) data I would have two remarks to your points:
1) For me it seems that the new HyDs will bring real improvement to the red (NIR) range, for the blue/green range they will be very similar to the current (SP8) ones.
2) I believe that you are mistaken concerning the 350 nm for the WLL. The Leica homepage states 440 nm. This is of course still an improvement compared to the currently available 470 nm one, but again not a full game changer as for example on our current system we also have and additional 440 nm pulsed laser, so for us there would be very little improvement between the old and the new system. Unless the new WLL is so much stronger than the old one that you do not need any more additional lasers for FRAP - but personally I doubt this.....

Greetings                       Gabor

P.S. Of course - if you can afford, it is better to buy a technical more up to date system - it is clear that the Stellaris offers improvements compared to an SP8.


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Arvydas Matiukas
Sent: Tuesday, April 14, 2020 2:40 PM
To: [hidden email]
Subject: Re: New Leica Stellaris vs SP8

*****
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Hello Microscopists,


What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.


Thanks,
Arvydas


Director of Microscopy Core

[Giang, William]

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Hi Gabor,

I was also annoyed at not showing QE curves comparing new HyDs, current HyDs, typical GaAsP PMT, and multi-alkali PMTs. (Also pet peeve: why was this plot made to be 3D?)

I'm going to add that Leica has made the old HyD's QE spectrum available via PDF on page 9 at [0] and WebPlotDigitizer [1] is a great tool for extracting data from plots like that.

Their orange-red bin spans from 560-720nm, and I doubt the sensitivity is flat in that region for a uniform 39% PDE.

I'm not affiliated with Leica in any way besides there being two SP8s in our core facility.

[0] https://downloads.leica-microsystems.com/Leica%20TCS%20SP8/Brochures/Leica%20TCS%20SP8%20HyD-Brochure_EN.pdf
[1] https://automeris.io/WebPlotDigitizer/

Best,
Will

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Csucs Gabor
Sent: Tuesday, April 14, 2020 9:40 AM
To: [hidden email]
Subject: [External] Re: New Leica Stellaris vs SP8

*****
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Dear Arvydas,

Personally, I have expected a little bit more. Unfortunately (they certainly did it on purpose) Leica did not show any comparison of the new HyDs compare to the SP8 ones and the same is true for the WLL. However based on the currently available (very preliminary) data I would have two remarks to your points:
1) For me it seems that the new HyDs will bring real improvement to the red (NIR) range, for the blue/green range they will be very similar to the current (SP8) ones.
2) I believe that you are mistaken concerning the 350 nm for the WLL. The Leica homepage states 440 nm. This is of course still an improvement compared to the currently available 470 nm one, but again not a full game changer as for example on our current system we also have and additional 440 nm pulsed laser, so for us there would be very little improvement between the old and the new system. Unless the new WLL is so much stronger than the old one that you do not need any more additional lasers for FRAP - but personally I doubt this.....

GreetingsGabor

P.S. Of course - if you can afford, it is better to buy a technical more up to date system - it is clear that the Stellaris offers improvements compared to an SP8.


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Arvydas Matiukas
Sent: Tuesday, April 14, 2020 2:40 PM
To: [hidden email]
Subject: Re: New Leica Stellaris vs SP8

*****
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*****

Hello Microscopists,


What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.


Thanks,
Arvydas


Director of Microscopy Core

________________________________

This e-mail message (including any attachments) is for the sole use of
the intended recipient(s) and may contain confidential and privileged
information. If the reader of this message is not the intended
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or copying of this message (including any attachments) is strictly
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If you have received this message in error, please contact
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original message (including attachments).

[Arvydas Matiukas]

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Hi Gabor,

Thanks for your comments. I meant 350nm wavelength range from 440nm to 790nm.

Our local Leica rep announced that they will de doimg in depth Stellaris presentation on Apr 28th: Leica must be preparing detail specs and features right now.

Best ,
Arvydas



>>> "Giang, William" <[hidden email]> 04/14/20 10:14 AM >>>
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Hi Gabor,

I was also annoyed at not showing QE curves comparing new HyDs, current HyDs, typical GaAsP PMT, and multi-alkali PMTs. (Also pet peeve: why was this plot made to be 3D?)

I'm going to add that Leica has made the old HyD's QE spectrum available via PDF on page 9 at [0] and WebPlotDigitizer [1] is a great tool for extracting data from plots like that.

Their orange-red bin spans from 560-720nm, and I doubt the sensitivity is flat in that region for a uniform 39% PDE.

I'm not affiliated with Leica in any way besides there being two SP8s in our core facility.

[0] https://urldefense.com/v3/__https://downloads.leica-microsystems.com/Leica*20TCS*20SP8/Brochures/Leica*20TCS*20SP8*20HyD-Brochure_EN.pdf__;JSUlJSU!!GobTDDpD7A!bZSmnDwX7nTxDvpzy0LcgTFFsPEIGXSphCi8g4UAv3cBwR5tmK0pW2q56AinxL0vDg$ 
[1] https://urldefense.com/v3/__https://automeris.io/WebPlotDigitizer/__;!!GobTDDpD7A!bZSmnDwX7nTxDvpzy0LcgTFFsPEIGXSphCi8g4UAv3cBwR5tmK0pW2q56AiSIgAsSA$ 

Best,
Will

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Csucs Gabor
Sent: Tuesday, April 14, 2020 9:40 AM
To: [hidden email]
Subject: [External] Re: New Leica Stellaris vs SP8

*****
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Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!bZSmnDwX7nTxDvpzy0LcgTFFsPEIGXSphCi8g4UAv3cBwR5tmK0pW2q56AhI029OfA$  and include the link in your posting.
*****

Dear Arvydas,

Personally, I have expected a little bit more. Unfortunately (they certainly did it on purpose) Leica did not show any comparison of the new HyDs compare to the SP8 ones and the same is true for the WLL. However based on the currently available (very preliminary) data I would have two remarks to your points:
1) For me it seems that the new HyDs will bring real improvement to the red (NIR) range, for the blue/green range they will be very similar to the current (SP8) ones.
2) I believe that you are mistaken concerning the 350 nm for the WLL. The Leica homepage states 440 nm. This is of course still an improvement compared to the currently available 470 nm one, but again not a full game changer as for example on our current system we also have and additional 440 nm pulsed laser, so for us there would be very little improvement between the old and the new system. Unless the new WLL is so much stronger than the old one that you do not need any more additional lasers for FRAP - but personally I doubt this.....

GreetingsGabor

P.S. Of course - if you can afford, it is better to buy a technical more up to date system - it is clear that the Stellaris offers improvements compared to an SP8.


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Arvydas Matiukas
Sent: Tuesday, April 14, 2020 2:40 PM
To: [hidden email]
Subject: Re: New Leica Stellaris vs SP8

*****
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Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!GobTDDpD7A!bZSmnDwX7nTxDvpzy0LcgTFFsPEIGXSphCi8g4UAv3cBwR5tmK0pW2q56AhI029OfA$  and include the link in your posting.
*****

Hello Microscopists,


What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.


Thanks,
Arvydas


Director of Microscopy Core

________________________________

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the intended recipient(s) and may contain confidential and privileged
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If you have received this message in error, please contact
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original message (including attachments).

[Christoph Ruediger Bauer]

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Dear Ralph,
I think you got this one wrong. By curiosity I googled Arvydas facility and found this: "Core: Advanced Fluorescent Imaging Core Core Director: Arvydas Matiukas, PhD". From looking at his site (https://www.upstate.edu/research/facilities/confocal.php) it seems clear to me that this is not a Leica facility and his list of core facility instruments starts actually with two Zeiss confocals.
I think your conclusions concerning a commercial mailing were a bit fast.
Best wishes,
Christoph


BIOIMAGING CENTER
University of Geneva - Science II
Room 245
30, Quai Ernest Ansermet
CH - 1211 Genève 4

Dr. Christoph R. Bauer
Managing Director
Tel.: + 41 22 3796632
Fax: + 41 22 3796868
email: [hidden email]
website: http://bioimaging.unige.ch/



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Ralf Palmisano
Sent: 14 April 2020 15:41
To: [hidden email]
Subject: Re: New Leica Stellaris vs SP8

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Hi Director Arvydas of LEICA Microscopy Core,

This mail rang so many bells while reading. It is this sort of masked commercial mailings that I don't like to see here.
I was wondering why this initial mail was subjected a re-post. In fact it was not are-post. It is a sort of undercover commercial post.
Why are you hiding that this is the initial post?
Why are you hiding that you are director of Leica microscopy core.
Why do make me search online who "Director of Microscopy Core" is?

I don't think that Leica supports this type of misguiding this community, by pretending to be sort of a fair balanced user and starting a promotional tour.

Don't get me wrong. This is not about Leica pro or con. This is about your rather shabby behaviour here.
Don't get me wrong. If you would have made yourself known to be a Leica sponsored facility manager and asked for the communities opion, well fair enough, then everys would be aware what one is stepping into.

Leica Microsystems deserve much better than people like you coming in such questionable disguise (yes, that's a Shakespeare quote). Apart from it, Leica would certainly have phrased this commercial way better than you, Sir, ever will be able, too.

I sincerely apologise if you think this was personal, it is not and I believe this sort of posts always prove to be of disservice for the respective company.

Have a nice day,

Ralph Palmisano
Head Optical Imaging Centre Erlangen
Central Institute
Friedrich-Alexander University of Erlangen-Nuremberg

Am 14/04/2020 um 14:40 schrieb Arvydas Matiukas:

> *****
> 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.
> *****
>
> Hello Microscopists,
>
>
> What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.
>
>
> Thanks,
> Arvydas
>
>
> Director of Microscopy Core

--
Ralph Palmisano
Head - Optical Imaging Centre Erlangen

Fellow Royal Microscopical Society
Member Royal Society of Medicine

Speaker Scientific Advisory Board "German BioImaging"
Board of Directors Core Technologies for Life Science

Cauerstr. 3
91058 Erlangen, Germany

[phone] (Office)
[phone] (Secretary)

www.oice.uni-erlangen.de

[Zdenek Svindrych-2]

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Hi all,
yes, it would be great to see some photocathode specs from Leica.
Looking at what's already available from Hamamatsu (
https://www.becker-hickl.com/wp-content/uploads/2018/11/HPM-100-series-cathode-spectral-sensitivity-2019-02-13-aj.png
),
the red enhanced GaAsP -42 material suffers too much in the visible range
quantum efficiency. Interestingly, Hamamatsu only publishes the -40 and -50
spectral sensitivities of their hybrid photodetectors.
But there are promising materials in the intensifier field (
https://images.slideplayer.com/20/5946449/slides/slide_3.jpg ), where the
trade-off between -74 and -73 cathode is not huge.
We may have to wait for Leica to publish more quantitative comparisons
(both the HyDs and the WLLs). I hope they'll let us "Get Closer to the
Truth" :-).
@Rosemary, yes, gating is a must with WLL (at least with SP8), the AOBS has
poor rejection on it's own.
Best, zdenek


On Tue, Apr 14, 2020 at 10:14 AM Giang, William <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Gabor,
>
> I was also annoyed at not showing QE curves comparing new HyDs, current
> HyDs, typical GaAsP PMT, and multi-alkali PMTs. (Also pet peeve: why was
> this plot made to be 3D?)
>
> I'm going to add that Leica has made the old HyD's QE spectrum available
> via PDF on page 9 at [0] and WebPlotDigitizer [1] is a great tool for
> extracting data from plots like that.
>
> Their orange-red bin spans from 560-720nm, and I doubt the sensitivity is
> flat in that region for a uniform 39% PDE.
>
> I'm not affiliated with Leica in any way besides there being two SP8s in
> our core facility.
>
> [0]
> https://downloads.leica-microsystems.com/Leica%20TCS%20SP8/Brochures/Leica%20TCS%20SP8%20HyD-Brochure_EN.pdf
> [1] https://automeris.io/WebPlotDigitizer/
>
> Best,
> Will
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Csucs Gabor
> Sent: Tuesday, April 14, 2020 9:40 AM
> To: [hidden email]
> Subject: [External] Re: New Leica Stellaris vs SP8
>
> *****
> 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.
> *****
>
> Dear Arvydas,
>
> Personally, I have expected a little bit more. Unfortunately (they
> certainly did it on purpose) Leica did not show any comparison of the new
> HyDs compare to the SP8 ones and the same is true for the WLL. However
> based on the currently available (very preliminary) data I would have two
> remarks to your points:
> 1) For me it seems that the new HyDs will bring real improvement to the
> red (NIR) range, for the blue/green range they will be very similar to the
> current (SP8) ones.
> 2) I believe that you are mistaken concerning the 350 nm for the WLL. The
> Leica homepage states 440 nm. This is of course still an improvement
> compared to the currently available 470 nm one, but again not a full game
> changer as for example on our current system we also have and additional
> 440 nm pulsed laser, so for us there would be very little improvement
> between the old and the new system. Unless the new WLL is so much stronger
> than the old one that you do not need any more additional lasers for FRAP -
> but personally I doubt this.....
>
> GreetingsGabor
>
> P.S. Of course - if you can afford, it is better to buy a technical more
> up to date system - it is clear that the Stellaris offers improvements
> compared to an SP8.
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Arvydas Matiukas
> Sent: Tuesday, April 14, 2020 2:40 PM
> To: [hidden email]
> Subject: Re: New Leica Stellaris vs SP8
>
> *****
> 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.
> *****
>
> Hello Microscopists,
>
>
> What do you think of the last week presented Leica Stellaris new
> generation of confocal/STED microscopes? I was planning to buy SP8 but now
> I think it is worth to wait till Stellaris is available. Second generation
> WLL with 350 nm range and most sensitiive new HyD detectors are worth it.
>
>
> Thanks,
> Arvydas
>
>
> Director of Microscopy Core
>
> ________________________________
>
> This e-mail message (including any attachments) is for the sole use of
> the intended recipient(s) and may contain confidential and privileged
> information. If the reader of this message is not the intended
> recipient, you are hereby notified that any dissemination, distribution
> or copying of this message (including any attachments) is strictly
> prohibited.
>
> If you have received this message in error, please contact
> the sender by reply e-mail message and destroy all copies of the
> original message (including attachments).
>

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

[Ben Abrams-2]

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I agree with Christoph on this and am glad to see the reply discussion
focusing on the technical aspects of the hardware.  I think the bigger
threat to our community here is not covert commercial promotion, but
spreading the idea that our community members should not be able to ask
questions freely, for fear of an appearance of a conflict of interest.
-Ben

On 4/14/20 7:50 AM, Christoph Ruediger Bauer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Ralph,
> I think you got this one wrong. By curiosity I googled Arvydas facility and found this: "Core: Advanced Fluorescent Imaging Core Core Director: Arvydas Matiukas, PhD". From looking at his site (https://www.upstate.edu/research/facilities/confocal.php) it seems clear to me that this is not a Leica facility and his list of core facility instruments starts actually with two Zeiss confocals.
> I think your conclusions concerning a commercial mailing were a bit fast.
> Best wishes,
> Christoph
>
>
> BIOIMAGING CENTER
> University of Geneva - Science II
> Room 245
> 30, Quai Ernest Ansermet
> CH - 1211 Genève 4
>
> Dr. Christoph R. Bauer
> Managing Director
> Tel.: + 41 22 3796632
> Fax: + 41 22 3796868
> email: [hidden email]
> website: http://bioimaging.unige.ch/
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Ralf Palmisano
> Sent: 14 April 2020 15:41
> To: [hidden email]
> Subject: Re: New Leica Stellaris vs SP8
>
> *****
> 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 Director Arvydas of LEICA Microscopy Core,
>
> This mail rang so many bells while reading. It is this sort of masked commercial mailings that I don't like to see here.
> I was wondering why this initial mail was subjected a re-post. In fact it was not are-post. It is a sort of undercover commercial post.
> Why are you hiding that this is the initial post?
> Why are you hiding that you are director of Leica microscopy core.
> Why do make me search online who "Director of Microscopy Core" is?
>
> I don't think that Leica supports this type of misguiding this community, by pretending to be sort of a fair balanced user and starting a promotional tour.
>
> Don't get me wrong. This is not about Leica pro or con. This is about your rather shabby behaviour here.
> Don't get me wrong. If you would have made yourself known to be a Leica sponsored facility manager and asked for the communities opion, well fair enough, then everys would be aware what one is stepping into.
>
> Leica Microsystems deserve much better than people like you coming in such questionable disguise (yes, that's a Shakespeare quote). Apart from it, Leica would certainly have phrased this commercial way better than you, Sir, ever will be able, too.
>
> I sincerely apologise if you think this was personal, it is not and I believe this sort of posts always prove to be of disservice for the respective company.
>
> Have a nice day,
>
> Ralph Palmisano
> Head Optical Imaging Centre Erlangen
> Central Institute
> Friedrich-Alexander University of Erlangen-Nuremberg
>
> Am 14/04/2020 um 14:40 schrieb Arvydas Matiukas:
>> *****
>> 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.
>> *****
>>
>> Hello Microscopists,
>>
>>
>> What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.
>>
>>
>> Thanks,
>> Arvydas
>>
>>
>> Director of Microscopy Core
> --
> Ralph Palmisano
> Head - Optical Imaging Centre Erlangen
>
> Fellow Royal Microscopical Society
> Member Royal Society of Medicine
>
> Speaker Scientific Advisory Board "German BioImaging"
> Board of Directors Core Technologies for Life Science
>
> Cauerstr. 3
> 91058 Erlangen, Germany
>
> [phone] (Office)
> [phone] (Secretary)
>
> www.oice.uni-erlangen.de

--
Benjamin Abrams,  Ph.D.
Director
UCSC Life Sciences Microscopy Center

University of California, Santa Cruz
1156 High Street
150 Sinsheimer labs
Santa Cruz, CA 95064

Office/voicemail: [phone]
Mobile: [phone]

http://stemcell.soe.ucsc.edu/facilities/microscopy

Training Request Form:
http://goo.gl/forms/M9jd1ZHyohTnj0xk1

[Vitaly Boyko-2]

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 I asked for 440-750 nm, and they did 440-790 for the WLL. At least Leica DOES listen!!! ... compared to others, including "fake" multiplex by the "neighbor"... Stellaris will be very useful for what we are currently doing at our facility!!! Cheers,VitalyP.S. Sarcastic joke: If even Vitaly likes it, it is a big step forward!!!

    On Tuesday, April 14, 2020, 11:03:54 AM EDT, Christoph Ruediger Bauer <[hidden email]> wrote:  
 
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Dear Ralph,
I think you got this one wrong. By curiosity I googled Arvydas facility and found this: "Core: Advanced Fluorescent Imaging Core Core Director: Arvydas Matiukas, PhD". From looking at his site (https://www.upstate.edu/research/facilities/confocal.php) it seems clear to me that this is not a Leica facility and his list of core facility instruments starts actually with two Zeiss confocals.
I think your conclusions concerning a commercial mailing were a bit fast.
Best wishes,
Christoph


BIOIMAGING CENTER
University of Geneva - Science II
Room 245
30, Quai Ernest Ansermet
CH - 1211 Genève 4

Dr. Christoph R. Bauer
Managing Director
Tel.: + 41 22 3796632
Fax: + 41 22 3796868
email: [hidden email]
website: http://bioimaging.unige.ch/



-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Ralf Palmisano
Sent: 14 April 2020 15:41
To: [hidden email]
Subject: Re: New Leica Stellaris vs SP8

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Hi Director Arvydas of LEICA Microscopy Core,

This mail rang so many bells while reading. It is this sort of masked commercial mailings that I don't like to see here.
I was wondering why this initial mail was subjected a re-post. In fact it was not are-post. It is a sort of undercover commercial post.
Why are you hiding that this is the initial post?
Why are you hiding that you are director of Leica microscopy core.
Why do make me search online who "Director of Microscopy Core" is?

I don't think that Leica supports this type of misguiding this community, by pretending to be sort of a fair balanced user and starting a promotional tour.

Don't get me wrong. This is not about Leica pro or con. This is about your rather shabby behaviour here.
Don't get me wrong. If you would have made yourself known to be a Leica sponsored facility manager and asked for the communities opion, well fair enough, then everys would be aware what one is stepping into.

Leica Microsystems deserve much better than people like you coming in such questionable disguise (yes, that's a Shakespeare quote). Apart from it, Leica would certainly have phrased this commercial way better than you, Sir, ever will be able, too.

I sincerely apologise if you think this was personal, it is not and I believe this sort of posts always prove to be of disservice for the respective company.

Have a nice day,

Ralph Palmisano
Head Optical Imaging Centre Erlangen
Central Institute
Friedrich-Alexander University of Erlangen-Nuremberg

Am 14/04/2020 um 14:40 schrieb Arvydas Matiukas:

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> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello Microscopists,
>
>
> What do you think of the last week presented Leica Stellaris new generation of confocal/STED microscopes? I was planning to buy SP8 but now I think it is worth to wait till Stellaris is available. Second generation WLL with 350 nm range and most sensitiive new HyD detectors are worth it.
>
>
> Thanks,
> Arvydas
>
>
> Director of Microscopy Core

--
Ralph Palmisano
Head - Optical Imaging Centre Erlangen

Fellow Royal Microscopical Society
Member Royal Society of Medicine

Speaker Scientific Advisory Board "German BioImaging"
Board of Directors Core Technologies for Life Science

Cauerstr. 3
91058 Erlangen, Germany

[phone] (Office)
[phone] (Secretary)

www.oice.uni-erlangen.de