Confocal Microscopy List

Commercial Reply: Mounting media for STED

classic Classic list List threaded Threaded
♦
♦
 2 messages Options
Christian Wurm Christian Wurm
Reply | Threaded
Open this post in threaded view
♦
♦
|

Commercial Reply: Mounting media for STED

*****
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, Dear all,

you are right, according to our observations every hardening mounting media will have an unwanted squashing effect on your sample. To overcome this, you should use a mounting medium that stays liquid in order to maintain the shape and structure of your sample. Abberior Mount Liquid Antifade is such a non-polymerizing mounting medium that preserves cell structure for imaging. It was designed exactly for the purpose of 3D STED and volume confocal imaging: https://www.abberior.com/jtl-shop/Abberior-Mount-Liquid-Antifade-2-x-15-ml

As Jakub has suggested, for very flat samples refractive index mismatches and aberrations are frequently not such a big deal.  However, for deep z-imaging you typically struggle with unwanted optical effects (z-distortion, signal reduction, loss of resolution).  These can be drastically reduced using adaptive optics based on a spatial light modulator and a deformable mirror: https://www.abberior-instruments.com/products/expert-line/adaptive-optics/

Concerning Alison’s question on TDE:
TDE is keeping its refractive index over time and will not harden.
So far, we have mixed it with buffers and reagents like antifade - but not yet with Prolong.

Best,
-Christian
___________________________
Christian Wurm
Head of Application
Abberior Instruments & Abberior

-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List <[hidden email]> Im Auftrag von Jakub Chojnacki
Gesendet: Dienstag, 14. April 2020 12:32
An: [hidden email]
Betreff: Mounting media for STED

*****
To join, leave or search the confocal microscopy listserv, go to:
Ahttp://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

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=JeTkUgVztGMmhKYjxsy2rfoW
> YibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rv
> qDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=WUlLvPIEiL3itAuXGle--4z0Hj
> CGcgEdNAY2apbQdng&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> IFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLN
> mFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ
> 2M&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=JeTkUgVztGMmhKYjxsy2rfoW
> YibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rv
> qDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=WUlLvPIEiL3itAuXGle--4z0Hj
> CGcgEdNAY2apbQdng&e=
> > Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> IFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLN
> mFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ
> 2M&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=JeTkUgVztGMmhKYjxsy2rfoW
> YibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0V
> emQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=UT_HGylOCutPMdVi0EcPvL1f1T
> K7bxfzTOSTBCZm8e0&e=
> > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> IFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLN
> mFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiom
> Gc&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-2Dmi
> crosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5F
> Preparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pd
> f&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5
> vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nr
> OCJiomGc&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>
Alison J. North Alison J. North
Reply | Threaded
Open this post in threaded view
♦
♦
|

Re: Commercial Reply: Mounting media for STED

*****
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 Christian,

Thanks for your reply!  But I am curious -  since people are recommending that one takes the sample through multiple steps of increasing TDE concentration, how does it work with your TDE mounting media?  Do you have to do the same then?  And if so, is there a protocol provided for doing so?

Again, thanks everybody for this useful discussion!  In full disclosure, we got our new Abberior Facility Line STED up and running fully at the beginning of February, and we were JUST getting going on it with real samples before the shutdown.   Now that we have a pause in operations, I am keen to make sure that we at least get all of our ducks in a row so that we are ready with the most optimal protocols for our users once we are fully open for business again.  After all, super-resolution systems must surely come into their own for studying the Coronavirus!!

All the best,
Alison

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Christian Wurm <[hidden email]>
Sent: Tuesday, April 14, 2020 3:50 PM
To: [hidden email] <[hidden email]>
Subject: Commercial Reply: 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=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=vJowLdGDdQKAqb_Z03B2v48nY5iSrqj82VG3LzfcBDk&s=zJKww-Bu9soxxKZNGq5s-wVR2epgdTLNbjVQ3W8ANgc&e=
Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=vJowLdGDdQKAqb_Z03B2v48nY5iSrqj82VG3LzfcBDk&s=0DI6v0RBV1AryzImm5VLJdf0bte82hPcESlVZ-rnaX0&e=  and include the link in your posting.
*****

Dear Xavi, Dear all,

you are right, according to our observations every hardening mounting media will have an unwanted squashing effect on your sample. To overcome this, you should use a mounting medium that stays liquid in order to maintain the shape and structure of your sample. Abberior Mount Liquid Antifade is such a non-polymerizing mounting medium that preserves cell structure for imaging. It was designed exactly for the purpose of 3D STED and volume confocal imaging: https://urldefense.proofpoint.com/v2/url?u=https-3A__www.abberior.com_jtl-2Dshop_Abberior-2DMount-2DLiquid-2DAntifade-2D2-2Dx-2D15-2Dml&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=vJowLdGDdQKAqb_Z03B2v48nY5iSrqj82VG3LzfcBDk&s=nuFNY4-AGKLWPch-eMleJhPXLHD24poe8Eo-N9JH9pg&e=

As Jakub has suggested, for very flat samples refractive index mismatches and aberrations are frequently not such a big deal.  However, for deep z-imaging you typically struggle with unwanted optical effects (z-distortion, signal reduction, loss of resolution).  These can be drastically reduced using adaptive optics based on a spatial light modulator and a deformable mirror: https://urldefense.proofpoint.com/v2/url?u=https-3A__www.abberior-2Dinstruments.com_products_expert-2Dline_adaptive-2Doptics_&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=vJowLdGDdQKAqb_Z03B2v48nY5iSrqj82VG3LzfcBDk&s=Uo3r6FU8EMRUnubZGhtZzC5B0skeZKyo_cD8GX3dpDQ&e=

Concerning Alison’s question on TDE:
TDE is keeping its refractive index over time and will not harden.
So far, we have mixed it with buffers and reagents like antifade - but not yet with Prolong.

Best,
-Christian
___________________________
Christian Wurm
Head of Application
Abberior Instruments & Abberior

-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List <[hidden email]> Im Auftrag von Jakub Chojnacki
Gesendet: Dienstag, 14. April 2020 12:32
An: [hidden email]
Betreff: Mounting media for STED

*****
To join, leave or search the confocal microscopy listserv, go to:
Ahttp://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=vJowLdGDdQKAqb_Z03B2v48nY5iSrqj82VG3LzfcBDk&s=0DI6v0RBV1AryzImm5VLJdf0bte82hPcESlVZ-rnaX0&e=  and include the link in your posting.
*****

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:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=vJowLdGDdQKAqb_Z03B2v48nY5iSrqj82VG3LzfcBDk&s=zJKww-Bu9soxxKZNGq5s-wVR2epgdTLNbjVQ3W8ANgc&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=vJowLdGDdQKAqb_Z03B2v48nY5iSrqj82VG3LzfcBDk&s=0DI6v0RBV1AryzImm5VLJdf0bte82hPcESlVZ-rnaX0&e=  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=JeTkUgVztGMmhKYjxsy2rfoW
> YibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rv
> qDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=WUlLvPIEiL3itAuXGle--4z0Hj
> CGcgEdNAY2apbQdng&e=
> Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> IFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLN
> mFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ
> 2M&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=JeTkUgVztGMmhKYjxsy2rfoW
> YibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=rv
> qDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ2M&s=WUlLvPIEiL3itAuXGle--4z0Hj
> CGcgEdNAY2apbQdng&e=
> > Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> IFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLN
> mFbqYvikvIZS5ns3-USwvMOuLo&m=rvqDSJTsgzJnEdr0hu2NokkGnsDIXKJjscILNckYZ
> 2M&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=JeTkUgVztGMmhKYjxsy2rfoW
> YibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0V
> emQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiomGc&s=UT_HGylOCutPMdVi0EcPvL1f1T
> K7bxfzTOSTBCZm8e0&e=
> > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> IFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLN
> mFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nrOCJiom
> Gc&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-2Dmi
> crosystems.com_fileadmin_academy_2019_Quick-5FGuide-5FSTED-5FSample-5F
> Preparation_STED-5FSample-5FPreparation-5FGuide-5FOnline-5F20190705.pd
> f&d=DwIFaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5
> vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=0VemQzJmtronf8nlxMjnSQIf5Vh0elEu5nr
> OCJiomGc&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://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_IrsiCaixa&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=vJowLdGDdQKAqb_Z03B2v48nY5iSrqj82VG3LzfcBDk&s=b-Oio0rbsL1t2bKf_u9gZTOv_QtD5TjF5xuvXdRpu58&e= >
Free forum by Nabble Edit this page