Re: CONFOCALMICROSCOPY Digest - 12 Apr 2020 to 13 Apr 2020 (#2020-79)

[Dr. Franziska Curdt]

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Dear Xavi,

to minimize shrinking when mounting in TDE, you need to increase the
TDE  concentration step-wise as explained in:

Staudt, T (2007), 2,2′‐Thiodiethanol: A new water soluble mounting
medium for high resolution optical microscopy. Microsc. Res. Tech., 70:
1-9. doi:10.1002/jemt.20396.

Here are the important steps:

"After fixation of the cells and depending on the sample, various
dilutions with increasing TDE content were used to exchange the water
with TDE in a continuous or step-wise manner. Good results have been
obtained with the following steps: 10% TDE , 25% TDE (250 lL TDE, 50 lL
PBS53, 700 lL water), 50% TDE , and then three times 97% TDE. The
samples were successively immersed in the above solutions with
increasing TDE concentrations for about 5–10 min each."

Hope that is helpful,

Franziska

Am 14/04/2020 um 07:00 schrieb CONFOCALMICROSCOPY automatic digest system:

> There are 7 messages totaling 1440 lines in this issue.
>
> Topics of the day:
>
>    1. Mounting media for STED (7)
>
> ----------------------------------------------------------------------
>
> Date:    Mon, 13 Apr 2020 14:20:24 +0000
> From:    "Alison J. North" <[hidden email]>
> Subject: Re: 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,
>
> 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=
>
> ------------------------------
>
> Date:    Mon, 13 Apr 2020 16:57:06 +0200
> From:    Jakub Chojnacki <[hidden email]>
> Subject: 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 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=
>>
>