fixing plant tissue from fluorescence

[Cromey, Douglas W - (dcromey)-2]

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I have a user who would like to use tetrazolium to label plant mitochondria in both slow and fast growing plants. He says that it is fluorescent. Since I know very little at all about preparing plant tissues for fluorescence microscopy, can they be fixed with aldehydes (formaldehyde or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra (structured illumination). Any "red flags"?

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
Life Sciences North, Room 463
1333 N. Martin Ave, Tucson, AZ  85721 USA

office:  LSN 463        email: [hidden email]<mailto:[hidden email]>
voice:  520-626-2824       fax:  [phone]

UA Microscopy Alliance - http://microscopy.arizona.edu/
A collaborative effort to bring information about shared microscopy
facilities to the University of Arizona and the community.

[Tobias Baskin]

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Doug,
         That is classic stuff. Literature I am aware of uses
tetrazolium on isolated mitochondria. This is a functional assay (the
tetrazolium gets reduced). In that standard sense, unlikely to work in a
fixed sample. Or even an intact plant organ. Having said that there
could be ways to use tetrazolium as a simple stain for mitochondria.
Yes, plant tissues are certainly well fixed with aldehydes. But both
formaldehyde and glutaraldehyde are capable of generating autofluoresent
compounds. The severity of the background depends on the organ, tissue,
and condition of the plants. What is more, many plant organs are
autofluorescent even without fixation: Chlorophyll in photosynthetic
tissue and many compounds in cell walls and vacuoles elsewhere. Clearly
some of this can be handled with spectral selection. As a rule, roots
tend to be easier to work with than leaves but not always. Depends what
your investigator wants to do.

     Good luck!
                     Tobias Baskin

On 3/8/21 5:47 PM, Cromey, Douglas W - (dcromey) wrote:

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> *****
>
> I have a user who would like to use tetrazolium to label plant mitochondria in both slow and fast growing plants. He says that it is fluorescent. Since I know very little at all about preparing plant tissues for fluorescence microscopy, can they be fixed with aldehydes (formaldehyde or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra (structured illumination). Any "red flags"?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
>
> office:  LSN 463        email: [hidden email]<mailto:[hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> UA Microscopy Alliance - http://microscopy.arizona.edu/
> A collaborative effort to bring information about shared microscopy
> facilities to the University of Arizona and the community.

.     __    __    ^    __   ___  Tobias I. Baskin                                 UMass counseling  |  413-545-2337
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[Timothy Chaya]

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Hey Doug,
Plant tissues can be fixed by vacuum or syringe infiltration with 2%
glutaraldehyde and 2% paraformaldehyde. Glutaraldehyde can autofluoresce,
but depending on the tissue and time before imaging, paraformaldehyde may
be sufficient.  If they are looking to fix these for an extended period of
time, then an additional step of 0.2M glycine for a few hours will quench
that autofluorescence.

What are they saying the spectra of the tetrazolium is? I am only aware of
nitroblue tetrazolium as a colorimetric stain for ROS stain. Plants are
full of things that autofluoresce, so depending on how sharp and isolated
the emission peak is, you may want to do a spectral image for confirmation.

That being said, live cell imaging of plant mitochondria can be performed
if you infiltrate the leaves with mitotracker. However, I do not know the
stability of this stain post fixation in plant tissue.

Hope this helps.

-Tim


On Mon, Mar 8, 2021 at 5:47 PM 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
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I have a user who would like to use tetrazolium to label plant
> mitochondria in both slow and fast growing plants. He says that it is
> fluorescent. Since I know very little at all about preparing plant tissues
> for fluorescence microscopy, can they be fixed with aldehydes (formaldehyde
> or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra
> (structured illumination). Any "red flags"?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
>
> office:  LSN 463        email: [hidden email]<mailto:
> [hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> UA Microscopy Alliance - http://microscopy.arizona.edu/
> A collaborative effort to bring information about shared microscopy
> facilities to the University of Arizona and the community.
>

[Rosemary White]

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You might get away with imaging fresh hand-sections of the tissue without fixation. It'd be worth imaging the fresh sections and making sure the fluorescence is still there after transferring a section through fixative - 2-4% formaldehyde in 25-50 mM phosphate buffer, pH 6.5-7.0.

cheers,
Rosemary

M: [phone]
E: [hidden email]
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Cromey, Douglas W - (dcromey) [[hidden email]]
Sent: Tuesday, 9 March 2021 9:47 AM
To: [hidden email]
Subject: fixing plant tissue from fluorescence

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I have a user who would like to use tetrazolium to label plant mitochondria in both slow and fast growing plants. He says that it is fluorescent. Since I know very little at all about preparing plant tissues for fluorescence microscopy, can they be fixed with aldehydes (formaldehyde or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra (structured illumination). Any "red flags"?

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
Life Sciences North, Room 463
1333 N. Martin Ave, Tucson, AZ  85721 USA

office:  LSN 463        email: [hidden email]<mailto:[hidden email]>
voice:  520-626-2824       fax:  [phone]

UA Microscopy Alliance - http://microscopy.arizona.edu/
A collaborative effort to bring information about shared microscopy
facilities to the University of Arizona and the community.

[Rosemary White]

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What Tim said...

M: [phone]
E: [hidden email]
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Timothy Chaya [[hidden email]]
Sent: Tuesday, 9 March 2021 1:11 PM
To: [hidden email]
Subject: Re: fixing plant tissue from fluorescence

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Hey Doug,
Plant tissues can be fixed by vacuum or syringe infiltration with 2%
glutaraldehyde and 2% paraformaldehyde. Glutaraldehyde can autofluoresce,
but depending on the tissue and time before imaging, paraformaldehyde may
be sufficient.  If they are looking to fix these for an extended period of
time, then an additional step of 0.2M glycine for a few hours will quench
that autofluorescence.

What are they saying the spectra of the tetrazolium is? I am only aware of
nitroblue tetrazolium as a colorimetric stain for ROS stain. Plants are
full of things that autofluoresce, so depending on how sharp and isolated
the emission peak is, you may want to do a spectral image for confirmation.

That being said, live cell imaging of plant mitochondria can be performed
if you infiltrate the leaves with mitotracker. However, I do not know the
stability of this stain post fixation in plant tissue.

Hope this helps.

-Tim


On Mon, Mar 8, 2021 at 5:47 PM 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
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I have a user who would like to use tetrazolium to label plant
> mitochondria in both slow and fast growing plants. He says that it is
> fluorescent. Since I know very little at all about preparing plant tissues
> for fluorescence microscopy, can they be fixed with aldehydes (formaldehyde
> or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra
> (structured illumination). Any "red flags"?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
>
> office:  LSN 463        email: [hidden email]<mailto:
> [hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> UA Microscopy Alliance - http://microscopy.arizona.edu/
> A collaborative effort to bring information about shared microscopy
> facilities to the University of Arizona and the community.
>

[Craig Brideau]

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Not with plants, but we used Glutaraldehyde's autofluorescence as a label
when we imaged myelin:
https://www.sciencedirect.com/science/article/abs/pii/S1053811913010732
I could possibly be useful for your imaging, although I am uncertain what
it would label in plants.

Craig


On Mon, Mar 8, 2021 at 7:12 PM Timothy Chaya <[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.
> *****
>
> Hey Doug,
> Plant tissues can be fixed by vacuum or syringe infiltration with 2%
> glutaraldehyde and 2% paraformaldehyde. Glutaraldehyde can autofluoresce,
> but depending on the tissue and time before imaging, paraformaldehyde may
> be sufficient.  If they are looking to fix these for an extended period of
> time, then an additional step of 0.2M glycine for a few hours will quench
> that autofluorescence.
>
> What are they saying the spectra of the tetrazolium is? I am only aware of
> nitroblue tetrazolium as a colorimetric stain for ROS stain. Plants are
> full of things that autofluoresce, so depending on how sharp and isolated
> the emission peak is, you may want to do a spectral image for confirmation.
>
> That being said, live cell imaging of plant mitochondria can be performed
> if you infiltrate the leaves with mitotracker. However, I do not know the
> stability of this stain post fixation in plant tissue.
>
> Hope this helps.
>
> -Tim
>
>
> On Mon, Mar 8, 2021 at 5:47 PM 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
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > I have a user who would like to use tetrazolium to label plant
> > mitochondria in both slow and fast growing plants. He says that it is
> > fluorescent. Since I know very little at all about preparing plant
> tissues
> > for fluorescence microscopy, can they be fixed with aldehydes
> (formaldehyde
> > or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra
> > (structured illumination). Any "red flags"?
> >
> > Doug
> >
> > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> > Douglas W. Cromey, M.S. - Associate Scientific Investigator
> > Dept. of Cellular & Molecular Medicine, University of Arizona
> > Life Sciences North, Room 463
> > 1333 N. Martin Ave, Tucson, AZ  85721 USA
> >
> > office:  LSN 463        email: [hidden email]<mailto:
> > [hidden email]>
> > voice:  520-626-2824       fax:  [phone]
> >
> > UA Microscopy Alliance - http://microscopy.arizona.edu/
> > A collaborative effort to bring information about shared microscopy
> > facilities to the University of Arizona and the community.
> >
>

[Glen MacDonald]

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Hi Doug,
In dim memory, I recall obtaining a weak far red signal from NBT.  excites in the 633 nm range and emits > 700 nm.  

this paper may be useful.
BioTechniques 42:751-755 (June 2007)

Regards,
Glen

> On Mar 8, 2021, at 2:47 PM, 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
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I have a user who would like to use tetrazolium to label plant mitochondria in both slow and fast growing plants. He says that it is fluorescent. Since I know very little at all about preparing plant tissues for fluorescence microscopy, can they be fixed with aldehydes (formaldehyde or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra (structured illumination). Any "red flags"?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
>
> office:  LSN 463        email: [hidden email]<mailto:[hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> UA Microscopy Alliance - http://microscopy.arizona.edu/
> A collaborative effort to bring information about shared microscopy
> facilities to the University of Arizona and the community.

[Timothy Chaya]

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Craig, interesting use of glutes autofluorescence, but most plant
microscopy is working around the abundance of autofluorescent molecules.

This review article might be useful to others in the plant microscopy
community.
Autofluorescence in Plants - https://www.mdpi.com/1420-3049/25/10/2393/htm


On Mon, Mar 8, 2021 at 9:41 PM Craig Brideau <[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.
> *****
>
> Not with plants, but we used Glutaraldehyde's autofluorescence as a label
> when we imaged myelin:
> https://www.sciencedirect.com/science/article/abs/pii/S1053811913010732
> I could possibly be useful for your imaging, although I am uncertain what
> it would label in plants.
>
> Craig
>
>
> On Mon, Mar 8, 2021 at 7:12 PM Timothy Chaya <[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.
> > *****
> >
> > Hey Doug,
> > Plant tissues can be fixed by vacuum or syringe infiltration with 2%
> > glutaraldehyde and 2% paraformaldehyde. Glutaraldehyde can autofluoresce,
> > but depending on the tissue and time before imaging, paraformaldehyde may
> > be sufficient.  If they are looking to fix these for an extended period
> of
> > time, then an additional step of 0.2M glycine for a few hours will quench
> > that autofluorescence.
> >
> > What are they saying the spectra of the tetrazolium is? I am only aware
> of
> > nitroblue tetrazolium as a colorimetric stain for ROS stain. Plants are
> > full of things that autofluoresce, so depending on how sharp and isolated
> > the emission peak is, you may want to do a spectral image for
> confirmation.
> >
> > That being said, live cell imaging of plant mitochondria can be performed
> > if you infiltrate the leaves with mitotracker. However, I do not know the
> > stability of this stain post fixation in plant tissue.
> >
> > Hope this helps.
> >
> > -Tim
> >
> >
> > On Mon, Mar 8, 2021 at 5:47 PM 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
> > > Post images on http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > I have a user who would like to use tetrazolium to label plant
> > > mitochondria in both slow and fast growing plants. He says that it is
> > > fluorescent. Since I know very little at all about preparing plant
> > tissues
> > > for fluorescence microscopy, can they be fixed with aldehydes
> > (formaldehyde
> > > or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra
> > > (structured illumination). Any "red flags"?
> > >
> > > Doug
> > >
> > > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> > > Douglas W. Cromey, M.S. - Associate Scientific Investigator
> > > Dept. of Cellular & Molecular Medicine, University of Arizona
> > > Life Sciences North, Room 463
> > > 1333 N. Martin Ave, Tucson, AZ  85721 USA
> > >
> > > office:  LSN 463        email: [hidden email]<mailto:
> > > [hidden email]>
> > > voice:  520-626-2824       fax:  [phone]
> > >
> > > UA Microscopy Alliance - http://microscopy.arizona.edu/
> > > A collaborative effort to bring information about shared microscopy
> > > facilities to the University of Arizona and the community.
> > >
> >
>

[Jeffrey Caplan]

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Hi Doug,
Not sure which Elyra you have, but the red flag on the Elyra PS.1 is that
most filter sets have a long pass 750.  If you are working with any green
tissue, the chlorophyll autofluorescence will blow out any other
fluorescence.  The solution is to put a SP740 filter in the slider
underneath the filter turret.

Best,

Jeff Caplan

Director of bioimaging
University of Delaware
302-831-3403



On Mon, Mar 8, 2021 at 5:47 PM 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
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I have a user who would like to use tetrazolium to label plant
> mitochondria in both slow and fast growing plants. He says that it is
> fluorescent. Since I know very little at all about preparing plant tissues
> for fluorescence microscopy, can they be fixed with aldehydes (formaldehyde
> or glutaraldehyde)? Our hope is to image them using our Zeiss Elyra
> (structured illumination). Any "red flags"?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> Life Sciences North, Room 463
> 1333 N. Martin Ave, Tucson, AZ  85721 USA
>
> office:  LSN 463        email: [hidden email]<mailto:
> [hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> UA Microscopy Alliance - http://microscopy.arizona.edu/
> A collaborative effort to bring information about shared microscopy
> facilities to the University of Arizona and the community.
>