YFP antifade for live plants

[Cromey, Douglas W - (dcromey)]

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I am helping with a short-term onsite demonstration of a SIM (Structured Illumination/superresolution) microscope. One of the users brought me a live plant sample (might have been part of a flower or where a branch comes off the stem, as you can perhaps tell, I have very little experience with botanical specimens) that is expressing YFP. The YFP seemed to fade pretty quickly. The sample is mounted in glycerine, with no additives. Is there a good antifade for YFP in live plant samples?

My other thought was that, since plants are mostly water, maybe we should be using the water-immersion objective, even at the sacrifice of some NA.

Any thoughts from people with far more experience with plants than me?

Thanks,
Doug


^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
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[Howard Berg]

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Hi Doug,

I would get the tissue out of that glycerol-- mount in water and use a water immersion lens.  For live plant cell imaging some have used ascorbate to scavenge oxygen.

Good luck and happy new year.

Howard Berg
Danforth Plant Science Center

> On Dec 30, 2013, at 11:30 AM, "Cromey, Douglas W - (dcromey)" <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I am helping with a short-term onsite demonstration of a SIM (Structured Illumination/superresolution) microscope. One of the users brought me a live plant sample (might have been part of a flower or where a branch comes off the stem, as you can perhaps tell, I have very little experience with botanical specimens) that is expressing YFP. The YFP seemed to fade pretty quickly. The sample is mounted in glycerine, with no additives. Is there a good antifade for YFP in live plant samples?
>
> My other thought was that, since plants are mostly water, maybe we should be using the water-immersion objective, even at the sacrifice of some NA.
>
> Any thoughts from people with far more experience with plants than me?
>
> Thanks,
> Doug
>
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"

[Lloyd Donaldson]

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Doug

Probably any of the commercial antifades will help but be aware that these can also greatly increase autofluorescence in plant tissues especially if you have lignified xylem in the sample (it looks like a bunch of springs). Glycerol solution will be quite acidic - pH about 4 so you could try making it in buffer to pH7 - phosphate buffer works fine.  Mounting in water or in sucrose solution are also options.
In my experience GFP in plant cells is often rather dim anyway.

Happy New Year

Lloyd Donaldson
Scion

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Berg, R. Howard [[hidden email]]
Sent: Tuesday, December 31, 2013 7:28 AM
To: [hidden email]
Subject: Re: YFP antifade for live plants

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Hi Doug,

I would get the tissue out of that glycerol-- mount in water and use a water immersion lens.  For live plant cell imaging some have used ascorbate to scavenge oxygen.

Good luck and happy new year.

Howard Berg
Danforth Plant Science Center

> On Dec 30, 2013, at 11:30 AM, "Cromey, Douglas W - (dcromey)" <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I am helping with a short-term onsite demonstration of a SIM (Structured Illumination/superresolution) microscope. One of the users brought me a live plant sample (might have been part of a flower or where a branch comes off the stem, as you can perhaps tell, I have very little experience with botanical specimens) that is expressing YFP. The YFP seemed to fade pretty quickly. The sample is mounted in glycerine, with no additives. Is there a good antifade for YFP in live plant samples?
>
> My other thought was that, since plants are mostly water, maybe we should be using the water-immersion objective, even at the sacrifice of some NA.
>
> Any thoughts from people with far more experience with plants than me?
>
> Thanks,
> Doug
>
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"

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[Stanislav Vitha-2]

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I had good luck with SIM imaging of plant tissue (YFP fused to a
cytoskeletal protein in Arabidopsis leaf chloroplasts) after vacuum
infiltrating the fixative -  3% fresh formaldehyde in phosphate buffer pH 7,
microwave-assisted fixation, followed by rinsing and gradual infiltration
with buffered glycerol (80% final concentration, pH 8.0).

With fresh tissue, I always vacuum infiltrate the tissue with water, then use
water immersion objective.  The silicon oil immersion objective (right now
only from Olympus, I believe) is even better. During a demo of the 60x SI  
I was getting about 25% deeper imaging compared to water immersion
(1-photon confocal, tobacco leaf).

In addition to the normally autofluorescent xylem elements, mechanical
damage to the tissue often causes strong green autofluorescence tissue.
Squeezing or pinching the tissue with tweezers can cause trouble.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University

On Mon, 30 Dec 2013 17:29:34 +0000, Cromey, Douglas W - (dcromey)
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>I am helping with a short-term onsite demonstration of a SIM (Structured
Illumination/superresolution) microscope. One of the users brought me a
live plant sample (might have been part of a flower or where a branch
comes off the stem, as you can perhaps tell, I have very little experience
with botanical specimens) that is expressing YFP. The YFP seemed to fade
pretty quickly. The sample is mounted in glycerine, with no additives. Is
there a good antifade for YFP in live plant samples?
>
>My other thought was that, since plants are mostly water, maybe we
should be using the water-immersion objective, even at the sacrifice of
some NA.
>
>Any thoughts from people with far more experience with plants than me?
>
>Thanks,
>Doug
>
>
>^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
^^^^^^^^^^^^^
>Douglas W. Cromey, M.S. - Associate Scientific Investigator
>Dept. of Cellular & Molecular Medicine, University of Arizona
>1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
>office:  AHSC 4212         email: [hidden email]
>voice:  520-626-2824       fax:  [phone]
>
>http://swehsc.pharmacy.arizona.edu/micro
>Home of: "Microscopy and Imaging Resources on the WWW"