GFP-clatrin imaging in Arabidopsis

[Michał Majkowski-2]

*****
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 List Members,
One of our facility users wants to image clathrin-GFP in Arabidopsis (in
living parenchyma cells of wood of Arabidopsis hypocotyl/stem ) in living
vibratome/hand made sections. We experienced two problems:

   1. Immediately after sectioning (both vibratome and hand) vesicles
   stopped moving

      2. We are not sure but the impression was that the signal from GFP
disappeared (or/and fluorescence lifetime contrast disappeared; I wanted to
use FALCON Leica mode to discriminate between autofluorescence and GFP
signal).

Roots from the same plant are excellent for imaging - clear movement of
vesicles was seen for at least 5-7 minutes.  Any hints how to overcome the
problem? Mayby mounting media?
Thank you in advance for your help.
Michal Majkowski
Imaging Specialist

University of Wroclaw
Biotechnology Faculty
Joliot-Curie 14a
50-383 Wroclaw POLAND

[Sacha Escamez]

*****
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 Michal,

Working with the cell biology of xylem/wood development (including in Arabidopsis), I can tell that this is generally a very difficult tissue to work with. It is difficult to image in whole mount, living samples due to how deep it is within the stem. If your imaging equipment would anyway allow for imaging several hundreds of microns deep, then whole mount might be best over making sections.


Another thing to take into account is that the cells in xylem/wood tend to be very elongated. Hence, if you make cross-sections you are very likely to cut most/all of them through. This will often lead to breaking their vacuoles, which may acidify the cell content and quench the GFP, and/or release of the many hydrolytic enzymes contained in the wood cell vacuoles, which tend to degrade all other proteins. If you have to make cross sections, maybe try to make them rather thick (e.g. 200-300 microns) so that some cells in the middle (in the z-direction) remain intact. Then, try to image in the middle (~100 microns deep) of the cross section to find the still intact, physiologically still functional (for a little while) cells. Hopefully, in these cells, your GFP should still be visible.

Finally, it might be important noting that the physiological, extra-cellular pH for xylem/wood cells is low, around 5.5-5.8 in the cell walls. Hence, to keep the few intact cells in the section in as close to normal conditions as possible (so that they do not interrupt vesicle trafficking for example), you might need to use a mounting medium at pH5.5-5.8.


Sincerely


Sacha


Sacha Escamez, PhD
Independent post-doctoral researcher
Umeå Plant Science Centre (UPSC)
Department of Forest Genetics and Plant Physiology
The Swedish University of Agricultural Sciences (SLU)
901 83 Umeå, Sweden
[hidden email]
[hidden email]


________________________________
De : Confocal Microscopy List <[hidden email]> de la part de Michał Majkowski <[hidden email]>
Envoyé : lundi 29 mars 2021 10:05
À : [hidden email]
Objet : GFP-clatrin imaging in Arabidopsis

*****
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 List Members,
One of our facility users wants to image clathrin-GFP in Arabidopsis (in
living parenchyma cells of wood of Arabidopsis hypocotyl/stem ) in living
vibratome/hand made sections. We experienced two problems:

   1. Immediately after sectioning (both vibratome and hand) vesicles
   stopped moving

      2. We are not sure but the impression was that the signal from GFP
disappeared (or/and fluorescence lifetime contrast disappeared; I wanted to
use FALCON Leica mode to discriminate between autofluorescence and GFP
signal).

Roots from the same plant are excellent for imaging - clear movement of
vesicles was seen for at least 5-7 minutes.  Any hints how to overcome the
problem? Mayby mounting media?
Thank you in advance for your help.
Michal Majkowski
Imaging Specialist

University of Wroclaw
Biotechnology Faculty
Joliot-Curie 14a
50-383 Wroclaw POLAND

[Michał Majkowski-2]

*****
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.
*****

Thank you Sacha for you e-mail. It helps a lot.
With kind regards,
Michal Majkowski

pon., 29 mar 2021 o 12:13 Sacha Escamez <[hidden email]> napisał(a):

> *****
> 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 Michal,
>
> Working with the cell biology of xylem/wood development (including in
> Arabidopsis), I can tell that this is generally a very difficult tissue to
> work with. It is difficult to image in whole mount, living samples due to
> how deep it is within the stem. If your imaging equipment would anyway
> allow for imaging several hundreds of microns deep, then whole mount might
> be best over making sections.
>
>
> Another thing to take into account is that the cells in xylem/wood tend to
> be very elongated. Hence, if you make cross-sections you are very likely to
> cut most/all of them through. This will often lead to breaking their
> vacuoles, which may acidify the cell content and quench the GFP, and/or
> release of the many hydrolytic enzymes contained in the wood cell vacuoles,
> which tend to degrade all other proteins. If you have to make cross
> sections, maybe try to make them rather thick (e.g. 200-300 microns) so
> that some cells in the middle (in the z-direction) remain intact. Then, try
> to image in the middle (~100 microns deep) of the cross section to find the
> still intact, physiologically still functional (for a little while) cells.
> Hopefully, in these cells, your GFP should still be visible.
>
> Finally, it might be important noting that the physiological,
> extra-cellular pH for xylem/wood cells is low, around 5.5-5.8 in the cell
> walls. Hence, to keep the few intact cells in the section in as close to
> normal conditions as possible (so that they do not interrupt vesicle
> trafficking for example), you might need to use a mounting medium at
> pH5.5-5.8.
>
>
> Sincerely
>
>
> Sacha
>
>
> Sacha Escamez, PhD
> Independent post-doctoral researcher
> Umeå Plant Science Centre (UPSC)
> Department of Forest Genetics and Plant Physiology
> The Swedish University of Agricultural Sciences (SLU)
> 901 83 Umeå, Sweden
> [hidden email]
> [hidden email]
>
>
> ________________________________
> De : Confocal Microscopy List <[hidden email]> de la
> part de Michał Majkowski <[hidden email]>
> Envoyé : lundi 29 mars 2021 10:05
> À : [hidden email]
> Objet : GFP-clatrin imaging in Arabidopsis
>
> *****
> 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 List Members,
> One of our facility users wants to image clathrin-GFP in Arabidopsis (in
> living parenchyma cells of wood of Arabidopsis hypocotyl/stem ) in living
> vibratome/hand made sections. We experienced two problems:
>
>    1. Immediately after sectioning (both vibratome and hand) vesicles
>    stopped moving
>
>       2. We are not sure but the impression was that the signal from GFP
> disappeared (or/and fluorescence lifetime contrast disappeared; I wanted to
> use FALCON Leica mode to discriminate between autofluorescence and GFP
> signal).
>
> Roots from the same plant are excellent for imaging - clear movement of
> vesicles was seen for at least 5-7 minutes.  Any hints how to overcome the
> problem? Mayby mounting media?
> Thank you in advance for your help.
> Michal Majkowski
> Imaging Specialist
>
> University of Wroclaw
> Biotechnology Faculty
> Joliot-Curie 14a
> 50-383 Wroclaw POLAND
>