Experiment

[Sarah Kefayati]

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Hello all,

 

In order to do my experiment,I need to embed some fluorescent beads(.2 micron) inside my sample(which is Arabidopsis leaf).I was thinking of a simple way,just injecting the solution to the stem of the leaf(using a microlitre syringe and a 33 G needle).I am wondering if any one has any other suggestion to do that more efficiently?

 

Thanks

Sarah

[Russell Spear]

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Sarah

You might simply cut the petiole with razor blade and immerse the cut
end in the bead suspension contained in a microfuge tube.  I load
compounds into leaves using this technique all the time.

Russ



Russell N. Spear
Sr. Research Specialist
Dept. of Plant Pathology
Univ. of Wisconsin
1630 Linden Dr.
Madison WI 53706

voice 608.263.2093
fax     [phone]

>>> Sarah Kefayati <[hidden email]> 11/08/07 11:20 AM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 

Hello all,

In order to do my experiment,I need to embed some fluorescent beads(.2
micron) inside my sample(which is Arabidopsis leaf).I was thinking of
a
simple way,just injecting the solution to the stem of the leaf(using a
microlitre syringe and a 33 G needle).I am wondering if any one has
any
other suggestion to do that more efficiently?

Thanks
Sarah

[Rosemary.White]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal HI Sarah,

This depends on exactly what you mean by “embedding beads” in the leaf.  If you just want to see the beads in the xylem, you can let the cut petiole take them up from solution as Russell suggests.  However, they won’t get out of the xylem into other tissues – 200 nm is probably too big for that.  I say “probably”, because the xylem parenchyma can take up 3kD dextrans from xylem vessels under the right conditions, presumably by endocytosis.  However, cell wall porosity is usually limited to under 8 nm, so 200 nm probably won’t get through the xylem walls, even at the porous pits.

cheers,
Rosemary

Rosemary White                    [hidden email]
CSIRO Plant Industry            ph.     61 (0)2-6246 5475
GPO Box 1600                       fax.     [phone]
Canberra, ACT 2601            
Australia



On 9/11/07 4:20 AM, "Sarah Kefayati" <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello all,
 
In order to do my experiment,I need to embed some fluorescent beads(.2 micron) inside my sample(which is Arabidopsis leaf).I was thinking of a simple way,just injecting the solution to the stem of the leaf(using a microlitre syringe and a 33 G needle).I am wondering if any one has any other suggestion to do that more efficiently?
 
Thanks
Sarah

[Sarah Kefayati]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Rosemary:

 

Thanks for the great information,

actually I was thinking of this problem too,but I just tried the injection way once and I could tell it somehow worked,I mean I could see some of the beads when I imaged my sample by the two-photon microscopy,but the solution was too diluted,this time I was thinking of using more concentrated solution(5 microlitre bead solution and 5 microlitre water),I just have one more question:if we assume that xylem can take up the beads ,if I see them this means that I am looking at the beads in which part?

 

thanks

Sarah

On Nov 8, 2007 5:35 PM, Rosemary White <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

HI Sarah,

This depends on exactly what you mean by "embedding beads" in the leaf.  If you just want to see the beads in the xylem, you can let the cut petiole take them up from solution as Russell suggests.  However, they won't get out of the xylem into other tissues – 200 nm is probably too big for that.  I say "probably", because the xylem parenchyma can take up 3kD dextrans from xylem vessels under the right conditions, presumably by endocytosis.  However, cell wall porosity is usually limited to under 8 nm, so 200 nm probably won't get through the xylem walls, even at the porous pits.

cheers,
Rosemary

Rosemary White                    [hidden email]
CSIRO Plant Industry            ph.     61 (0)2-6246 5475
GPO Box 1600                       fax.     [phone]
Canberra, ACT 2601            
Australia

On 9/11/07 4:20 AM, "Sarah Kefayati" <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello all,
 
In order to do my experiment,I need to embed some fluorescent beads(.2 micron) inside my sample(which is Arabidopsis leaf).I was thinking of a simple way,just injecting the solution to the stem of the leaf(using a microlitre syringe and a 33 G needle).I am wondering if any one has any other suggestion to do that more efficiently?
 
Thanks
Sarah

[Rosemary.White]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Sarah,

What are you trying to label with these beads?  What’s the experiment?

Depending on how long you let the leaves take up the solution, you’ll see them throughout the xylem.  You really need to shine a bright light on the leaves while their petioles are in solution, then they’ll take it up faster and to a greater extent.  So, you’ll see the beads in the xylem.

cheers,
Rosemary


On 9/11/07 12:18 PM, "Sarah Kefayati" <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Rosemary:
 
Thanks for the great information,
actually I was thinking of this problem too,but I just tried the injection way once and I could tell it somehow worked,I mean I could see some of the beads when I imaged my sample by the two-photon microscopy,but the solution was too diluted,this time I was thinking of using more concentrated solution(5 microlitre bead solution and 5 microlitre water),I just have one more question:if we assume that xylem can take up the beads ,if I see them this means that I am looking at the beads in which part?
 
thanks
Sarah

On Nov 8, 2007 5:35 PM, Rosemary White <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
HI Sarah,

This depends on exactly what you mean by "embedding beads" in the leaf.  If you just want to see the beads in the xylem, you can let the cut petiole take them up from solution as Russell suggests.  However, they won't get out of the xylem into other tissues – 200 nm is probably too big for that.  I say "probably", because the xylem parenchyma can take up 3kD dextrans from xylem vessels under the right conditions, presumably by endocytosis.  However, cell wall porosity is usually limited to under 8 nm, so 200 nm probably won't get through the xylem walls, even at the porous pits.

cheers,
Rosemary

Rosemary White                    [hidden email]
CSIRO Plant Industry            ph.     61 (0)2-6246 5475
GPO Box 1600                       fax.     [phone]
Canberra, ACT 2601            
Australia




On 9/11/07 4:20 AM, "Sarah Kefayati" <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello all,
 
In order to do my experiment,I need to embed some fluorescent beads(.2 micron) inside my sample(which is Arabidopsis leaf).I was thinking of a simple way,just injecting the solution to the stem of the leaf(using a microlitre syringe and a 33 G needle).I am wondering if any one has any other suggestion to do that more efficiently?
 
Thanks
Sarah