Clearing wood

[Jeremy Adler-4]

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We have a customer who wants to image a GFP tagged organism in wood.

Has anyone tried clearing wood in a way that would leave GFP functional ?


We suggested cutting the wood  and looking at the exposed surface as an effective way of imaging a gradient into the material, but they would like a more extensive method.


The problems seem to be lignin and RI mismatch.


so any ideas ?


Jeremy Adler

BioVis

Uppsala U

Sweden










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[Sacha Escamez]

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Dear Jeremy,

I work with the biology of wood cells for years and I have never encountered a method that allows for getting rid of the strong autofluorescence of lignin without strongly altering the wood samples (and destroying the fluorescent proteins). Wood cell biology always presents quite a challenge but what I found to work best is to either acquire spectral images and sort the lignin from the GFP by linear unmixing, or, even better, to use gating if the instrumentation allows for it. Indeed, lignin fluorescence has a shorter lifetime than GFP so lignin autofluorescence can be excluded by gating.

Another issue given the size of wood cells is the penetration so I would recommend using lenses with long working distance (even if this means sacrificing NA and resolution).

I hope this helps.

Sincerely

Dr. Sacha Escamez
Post-doctoral researcher
Department of Plant Physiology
Umeå Plant Science Centre
Umeå University
[hidden email]

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jeremy Adler
Sent: den 3 oktober 2018 10:21
To: [hidden email]
Subject: Clearing wood

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We have a customer who wants to image a GFP tagged organism in wood.

Has anyone tried clearing wood in a way that would leave GFP functional ?


We suggested cutting the wood  and looking at the exposed surface as an effective way of imaging a gradient into the material, but they would like a more extensive method.


The problems seem to be lignin and RI mismatch.


so any ideas ?


Jeremy Adler

BioVis

Uppsala U

Sweden










När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/

E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/om-uu/dataskydd-personuppgifter/

[Zdenek Svindrych-2]

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Hi Jeremy,
I'm not working with plants nor wood, but out of curiosity I saved these two
papers on clearing wood:

https://pubs.acs.org/doi/abs/10.1021/acs.biomac.6b00145

https://onlinelibrary.wiley.com/doi/full/10.1002/adma.201600427
The delignification was quite harsh (boiling with NaOH, Na2SO3, H2O2; or
NaClO2), I don't expect GFP would survive that. You may have some luck with
enzymatic approach ( https://www.sciencedirect.com/science/article/pii/S
0168165616314675 ), but it's quite an unexplored territory.

As to the RI matching, you can try to adapt the classical BA/BB (or more GFP
-friendly THF-DBE, see e.g. https://journals.plos.org/plosone/article?id=
10.1371/journal.pone.0033916 ) approaches, probably with some sort of vacuum
infiltration.

Or you can try and let your piece of wood sit in some water-based RI
matching solution (e.g. TDE) for a week and see how deep you can image...
You may also want to get rid of the cellulose (enzymatically), too.

Good luck!

Best, zdenek
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
email: [hidden email]

---------- Původní e-mail ----------
Od: Jeremy Adler <[hidden email]>
Komu: [hidden email]
Datum: 3. 10. 2018 4:24:12
Předmět: Clearing wood
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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.
*****


We have a customer who wants to image a GFP tagged organism in wood.

Has anyone tried clearing wood in a way that would leave GFP functional ?


We suggested cutting the wood and looking at the exposed surface as an
effective way of imaging a gradient into the material, but they would like a
more extensive method.


The problems seem to be lignin and RI mismatch.


so any ideas ?


Jeremy Adler

BioVis

Uppsala U

Sweden










När du har kontakt med oss på Uppsala universitet med e-post så innebär det
att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det
kan du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/

E-mailing Uppsala University means that we will process your personal data.
For more information on how this is performed, please read here: http://www.
uu.se/om-uu/dataskydd-personuppgifter/
"