2,2 Thiodiethanol as mounting medium - any experience?

[Steffen Dietzel]

Hi Everybody.

since the Staudt et al. paper (DOI:
10.1002/jemt.20396) is now out for over a year, I
assume that quite a number of people on this list
have played around with 2,2-Thiodiethanol (= TDE
= Thiodiethylene glycol = Thiodiglycol) as embedding medium.

In principle, its properties sound fantastic: no
more refraction index mismatch, protection
against bleaching. But how does it do in real life?

What is your experience with TDE? Does it really
make a difference for, let's say, cultured cells
on the coverslip, embedded tissues, other types
of preparations? How does it perform as
anti-bleaching reagent? Are there any dyes that
specifically do *not* work with it? Any dyes that
work better than usual? Any experience with two
photon microscopy? Maybe someone has tried TDE
even with living cells, just for the fun of it?
Does it get old fast? Any problems with residual
water on the specimen? Does it cause shrinkage? etcetc.

It would be be great if we could get a couple of
statements about what works and what maybe doesn't.

Steffen


--
---------------------------------------------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)

[Guy Cox]

OK, it works.  We used it for STED where index matching is pretty critical.  Mind you, the index matching is so good that getting a phase contrast image is tricky!  We were totally unable to find cells mounted on the coverslip (with the Leica STED system the fluorescence is in the near IR and hence invisible).  But with cells on a well slide we could focus on the blue background and then find our cells in phase.  And the index matching is so good we had no trouble getting 85nm resolution, which gets you into seriously uncharted territory.  Antifade properties seemed at least as good as Citifluor/glycerol, which doesn't give nearly as good an index match.  If you are chasing the ragged edge TDE is definitely the way to go.

                                              Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Thursday, 2 October 2008 7:57 PM
To: [hidden email]
Subject: 2,2 Thiodiethanol as mounting medium - any experience?

Hi Everybody.

since the Staudt et al. paper (DOI:
10.1002/jemt.20396) is now out for over a year, I assume that quite a number of people on this list have played around with 2,2-Thiodiethanol (= TDE = Thiodiethylene glycol = Thiodiglycol) as embedding medium.

In principle, its properties sound fantastic: no more refraction index mismatch, protection against bleaching. But how does it do in real life?

What is your experience with TDE? Does it really make a difference for, let's say, cultured cells on the coverslip, embedded tissues, other types of preparations? How does it perform as anti-bleaching reagent? Are there any dyes that specifically do *not* work with it? Any dyes that work better than usual? Any experience with two photon microscopy? Maybe someone has tried TDE even with living cells, just for the fun of it?
Does it get old fast? Any problems with residual water on the specimen? Does it cause shrinkage? etcetc.

It would be be great if we could get a couple of statements about what works and what maybe doesn't.

Steffen


--
---------------------------------------------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)

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[Paul Appleton]

I have been using TDE for over a  year now.  I think it's a great mounting medium - cheap,
easy to make up, non-toxic and has few disadvantages.  I use it for mounting fixed and
stained large gut specimens 2mm square x 0.5mm thick for confocal and 2-photon
microscopy.  One great benefit as mentioned in the original paper is it's clearing effect on
biological tissue.  We find no problem with bleaching in either confocal or 2-photon imaging.  
Specimens seem remarkably stable in TDE, I have some that have been in TDE for over a
year, travelled frequently and are still of the same quality as the day they were prepared!  
The only incompatibility I have found so far is with phalloidin (as stated in the original
paper).  For deep imaging the R.I matching makes a considerable difference.  I am just
making final revisions to a paper to be published in the journal of Microscopy that compares
TDE, BABB and glycerol as mountants for thick tissue imaging.  If I am not using phalloidin
then TDE would be my first choice for thick specimens.

[Phil Hertzler]

Paul,

Why would you not use TDE with phalloidin samples?  Not being familiar with this, how does one transition to TDE from aqueous washes?

Phil

At 07:22 AM 10/2/2008, you wrote:

I have been using TDE for over a  year now.  I think it's a great mounting medium - cheap,
easy to make up, non-toxic and has few disadvantages.  I use it for mounting fixed and
stained large gut specimens 2mm square x 0.5mm thick for confocal and 2-photon
microscopy.  One great benefit as mentioned in the original paper is it's clearing effect on
biological tissue.  We find no problem with bleaching in either confocal or 2-photon imaging. 
Specimens seem remarkably stable in TDE, I have some that have been in TDE for over a
year, travelled frequently and are still of the same quality as the day they were prepared! 
The only incompatibility I have found so far is with phalloidin (as stated in the original
paper).  For deep imaging the R.I matching makes a considerable difference.  I am just
making final revisions to a paper to be published in the journal of Microscopy that compares
TDE, BABB and glycerol as mountants for thick tissue imaging.  If I am not using phalloidin
then TDE would be my first choice for thick specimens.

---

Philip L. Hertzler
Associate Professor
Central Michigan University
Dept. of Biology, Brooks Hall 217
Mount Pleasant, MI 48859

Phone: [phone]
Fax: [phone]
Email: [hidden email]

"It is common sense to take a method and try it. If it fails, admit it frankly and try another. But above all, try something."
- Franklin D. Roosevelt

"Not everything that counts can be counted, and not everything that can be counted counts."
- attributed to Albert Einstein

[Paul Appleton]

TDE seems to unbind phalloidin, even if you fix again after staining.  If anyone has found a
solution to phalloidin staining and TDE mounting it would be good to hear.  Specimens are
mounted in TDE stepwise through a dilution series.  TDE is diluted in PBS in concentrations
of 10, 25, 50 and 97% (the final concentration determines the R.I you want).  I recommend
you check out the original paper that explains the simple procedure very clearly.

Paul