Plant preparation for confocal microscopy

[Monique Vasseur]

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Dear all,

Do some of you could suggest how to prepare live sample of plant leaf for
confocal microscopy.  We don't have much idea how to "sandwich" a leaf
between slide and coverslip so that it is flat et not too thick for microscopy
(and without destroying it)? What should we use as mounting media? What
should we be aware of?  Our plant will have YFP in chloroplastides.

Thanks a lot in advance to all of you.

Monique

[Rosemary.White]

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Hi Monique,

You can image with little preparation, just mount in water and add a
coverslip. Some common problems:

Air bubbles within the tissue - bad if you want to look below the
epidermis - lots of reflection from the air/water (i.e. air/cell wall)
interface.
The laser illumination may stimulate photosynthesis so bubbles develop in
the leaf tissue - within cells as well as between them, and may accumulate
under the coverslip.
Both of these problems may be alleviated somewhat by gentle vacuum
infiltration with water or buffer before imaging.

Another option is to image the leaf dry without coverslip - you don't get
the bubble problems then, but still need to worry about reflection from
the leaf surface and about the leaf drying out during imaging.

Best is to just try it - since it's confocal, you don't have to worry
about leaf thickness. If you need to image deeper layers, just cut a thick
cross-section and mount in water; there will be enough undamaged cells
there.

good luck,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 25/01/12 8:31 AM, "Vasseur Monique" <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear all,
>
>Do some of you could suggest how to prepare live sample of plant leaf for
>confocal microscopy.  We don't have much idea how to "sandwich" a leaf
>between slide and coverslip so that it is flat et not too thick for
>microscopy
>(and without destroying it)? What should we use as mounting media? What
>should we be aware of?  Our plant will have YFP in chloroplastides.
>
>Thanks a lot in advance to all of you.
>
>Monique

[Littlejohn, George]

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Dear Monique,

I've been using a perfluorocarbon mounting medium for in vivo imaging of leaf tissue for a few years now (Littlejohn, G. R., Gouveia, J. D., Edner, C., Smirnoff, N. and Love, J. (2010), Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll. New Phytologist, 186: 1018–1025. doi: 10.1111/j.1469-8137.2010.03244.x). In addition we often use Carolina observation gel for mounting leaf samples.

We have an online protocol here: http://www.jove.com/video/3394/a-simple-method-for-imaging-arabidopsis-leaves-using-perfluorodecalin-as-an-infiltrative-imaging-medium
Hope it helps!

All the best,
George

******************************
Dr. George Littlejohn
School of Biosciences,
University of Exeter,
Mezzanine Laboratory,
Geoffrey Pope Building,
Stocker Road,
EX4 4QD, UK
******************************
Tel:  [phone] (Lab.)
        [phone] (Office)
Fax:  [phone]
E-mail:  [hidden email]
******************************
http://www.illuminatedcell.com/improved-imaging.html
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Vasseur Monique [[hidden email]]
Sent: Tuesday, January 24, 2012 9:31 PM
To: [hidden email]
Subject: Plant preparation for confocal microscopy

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Dear all,

Do some of you could suggest how to prepare live sample of plant leaf for
confocal microscopy.  We don't have much idea how to "sandwich" a leaf
between slide and coverslip so that it is flat et not too thick for microscopy
(and without destroying it)? What should we use as mounting media? What
should we be aware of?  Our plant will have YFP in chloroplastides.

Thanks a lot in advance to all of you.

Monique

[Lloyd Donaldson]

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It is often a problem with leaves to keep them flat. What we do is stick them to the slide with double sided tape and then mount in water or glycerol.


Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification
Scion - Forests . Products . Innovation
Private Bag 3020, Rotorua
49 Sala Street, Rotorua 3010
New Zealand

Ph: 64 7 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rosemary White
Sent: Wednesday, 25 January 2012 11:02 a.m.
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

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Hi Monique,

You can image with little preparation, just mount in water and add a
coverslip. Some common problems:

Air bubbles within the tissue - bad if you want to look below the
epidermis - lots of reflection from the air/water (i.e. air/cell wall)
interface.
The laser illumination may stimulate photosynthesis so bubbles develop in
the leaf tissue - within cells as well as between them, and may accumulate
under the coverslip.
Both of these problems may be alleviated somewhat by gentle vacuum
infiltration with water or buffer before imaging.

Another option is to image the leaf dry without coverslip - you don't get
the bubble problems then, but still need to worry about reflection from
the leaf surface and about the leaf drying out during imaging.

Best is to just try it - since it's confocal, you don't have to worry
about leaf thickness. If you need to image deeper layers, just cut a thick
cross-section and mount in water; there will be enough undamaged cells
there.

good luck,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 25/01/12 8:31 AM, "Vasseur Monique" <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear all,
>
>Do some of you could suggest how to prepare live sample of plant leaf for
>confocal microscopy.  We don't have much idea how to "sandwich" a leaf
>between slide and coverslip so that it is flat et not too thick for
>microscopy
>(and without destroying it)? What should we use as mounting media? What
>should we be aware of?  Our plant will have YFP in chloroplastides.
>
>Thanks a lot in advance to all of you.
>
>Monique

This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
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[Kitin, Peter]

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Also cutting small disks with a hole puncher may work, they’ll have lots of living cells. Small disks will stay flat in water or glycerol and normally (if there’s not too much trichomes or cuticle) you’ll be able to see chloroplasts beneath the epidermis (try each side of the leaf). Vacuoles may shrink in glycerol changing the look of the chloroplasts.
All the best,

Peter Kitin
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Lloyd Donaldson [[hidden email]]
Sent: Tuesday, January 24, 2012 3:07 PM
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

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It is often a problem with leaves to keep them flat. What we do is stick them to the slide with double sided tape and then mount in water or glycerol.


Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification
Scion - Forests . Products . Innovation
Private Bag 3020, Rotorua
49 Sala Street, Rotorua 3010
New Zealand

Ph: 64 7 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rosemary White
Sent: Wednesday, 25 January 2012 11:02 a.m.
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

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Hi Monique,

You can image with little preparation, just mount in water and add a
coverslip. Some common problems:

Air bubbles within the tissue - bad if you want to look below the
epidermis - lots of reflection from the air/water (i.e. air/cell wall)
interface.
The laser illumination may stimulate photosynthesis so bubbles develop in
the leaf tissue - within cells as well as between them, and may accumulate
under the coverslip.
Both of these problems may be alleviated somewhat by gentle vacuum
infiltration with water or buffer before imaging.

Another option is to image the leaf dry without coverslip - you don't get
the bubble problems then, but still need to worry about reflection from
the leaf surface and about the leaf drying out during imaging.

Best is to just try it - since it's confocal, you don't have to worry
about leaf thickness. If you need to image deeper layers, just cut a thick
cross-section and mount in water; there will be enough undamaged cells
there.

good luck,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 25/01/12 8:31 AM, "Vasseur Monique" <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear all,
>
>Do some of you could suggest how to prepare live sample of plant leaf for
>confocal microscopy.  We don't have much idea how to "sandwich" a leaf
>between slide and coverslip so that it is flat et not too thick for
>microscopy
>(and without destroying it)? What should we use as mounting media? What
>should we be aware of?  Our plant will have YFP in chloroplastides.
>
>Thanks a lot in advance to all of you.
>
>Monique

This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the  course of Scion's usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.

[Stanislav Vitha]

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Hi Monuique,
First you want to get rid of the air that is in the intercellular spaces in the
leaf. As George Littlejohn mentioned, perfluorodecalin is one option. One small
downside in my hands is that there are always some small air pockets left
every now and then (usually in the most interesting area of the leaf).  
Often, I just vacuum infiltrate a leaf segment with water, using a syringe
(works much better than a a vacuum dessicator: put the leaf in a 10 ml plastic
syringe, add about 1 to 1.5 ml water, insert the plunger and push all the air
out of the syringe. Then put your finger on the syringe outlet and pull the
plunger to creat vacuum. Shake the syringe vigorously to disodge air bubbles
sticking to the leaf. When the leaf is under water, release the plunger. The
water gets drawn into the leaf, the leaf should become translucent. If needed,
repeat few more times.    
Once infiltrated, I mount the leaf in water in a coverglass-bottom chamber (we
have an inverted microscope) - either a standard coverglass-bottom Petri
dish, or my home-made plexiglass chamber -
http://microscopy.tamu.edu/lab-protocols/light-microscopy-protocols.html

I then use a glass "brick" to keep the leaf flat - a piece of glass for making
glass knives for resin sectioning. It is a good idea to dull the edges of the
glass block with sandpaper, otherwise you will be cutting your fingers.
     
If you have the leaf infiltrated, the production of gas during illumination is not
noticeable (I do not see any new air bubbles in the infiltrated areas)

Sometimes, when in a hurry, I do not bother with vacuumm infiltration of
Arabidopsis leaves. i just put the leaf in the coerglass-bottom chamber in a
drop of water, put the glass brick on top and tap with the back of my
tweezers to knock out the air. Not perfect, but you can always find an area
without too many air bubbles. The downside is that it is fairly easy to break
the coverglass bottom by tapping.

Good luck!

Stan Vitha
Microscopy and Imaging Center
Texas A&M University

On Tue, 24 Jan 2012 15:31:42 -0600, Vasseur Monique
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear all,
>
>Do some of you could suggest how to prepare live sample of plant leaf for
>confocal microscopy.  We don't have much idea how to "sandwich" a leaf
>between slide and coverslip so that it is flat et not too thick for microscopy
>(and without destroying it)? What should we use as mounting media? What
>should we be aware of?  Our plant will have YFP in chloroplastides.
>
>Thanks a lot in advance to all of you.
>
>Monique

[Christian-103]

Localization to chloroplasts at low expression levels can be
challe
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Localization to chloroplasts at low expression levels can be
challenging depending on your system and familiarity with dealing with sample
with autofluorescence.  If you find you're having some difficulty, get
back to us, and as a group, I'm quite sure we can talk you through that part.



As for mounting samples, it depends on what plant you're dealing with. 
Zea mays, you would probably want to hand cut cross sections, mount in water
under a coverslip.  Arabidopsis, you will probably mount with the
"bottom" of the leaf towards the lens (are you upright or
inverted?)  This will help avoid not only most of the trichomes which
cause more air bubble issues and refraction, but the epidermal cells under them
require deep z-series to see.  Of course that is if you're working stable
lines, bombardment samples I usually view from the side which was
bombarded.  Lastly, tobacco, if transiently expressing from Agrobacterium,
is often infiltrated from the bottom of the leaf, so that’s where I look, on
the bottom.

As for air bubbles… I do not fret the air bubbles at all,
but this is mostly because I’m NOT trying to collect the transmitted light
image.  If you need it, then you might
worry about them, and I highly recommend checking out perfluorodecalin.  I personally have not had luck with pulling a
weak vacuum , actually, what I do is even more simplistic.

I place the leaf sample (disks are too small to be worth the
effort in my hands) with the side I’d like to image up on a slide.  Add water, and place a 22x60mm cover slip
over the sample, which is usually about 22x22mm.  I then tap out the air bubbles I can see, and
place the entire thing on a “steel slide” and then place two small magnets on
top of the cover slip to hold the works flat. 
If I avoid taking samples from area with large veins, I can do z-series
as long as I’d like without lateral shifting. 
The only major problem I will warn you about is that you’re making your “slide”
much thicker and if you use an automated stage, this could be a real
problem.  Secondly, and obviously, you
will not get a transmitted light image. 
Of course this only works on an upright, but it has worked for at least
four years.

Good luck.

Christian

 

 

>Dear all,

>

>Do some of you could suggest how to prepare live sample of plant leaf for

>confocal microscopy.  We don't have much idea how to
"sandwich" a leaf

>between slide and coverslip so that it is flat et not too thick for

>microscopy

>(and without destroying it)? What should we use as mounting media? What

>should we be aware of?  Our plant will have YFP in chloroplastides.

>

>Thanks a lot in advance to all of you.

>

[Monique Vasseur]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Yes it helps a lot.  Thank you very much!

Monique Vasseur
tél. [phone] poste 5148

-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Littlejohn, George
Envoyé : 24 janvier 2012 18:07
À : [hidden email]
Objet : Re: Plant preparation for confocal microscopy

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Dear Monique,

I've been using a perfluorocarbon mounting medium for in vivo imaging of leaf tissue for a few years now (Littlejohn, G. R., Gouveia, J. D., Edner, C., Smirnoff, N. and Love, J. (2010), Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll. New Phytologist, 186: 1018-1025. doi: 10.1111/j.1469-8137.2010.03244.x). In addition we often use Carolina observation gel for mounting leaf samples.

We have an online protocol here: http://www.jove.com/video/3394/a-simple-method-for-imaging-arabidopsis-leaves-using-perfluorodecalin-as-an-infiltrative-imaging-medium
Hope it helps!

All the best,
George

******************************
Dr. George Littlejohn
School of Biosciences,
University of Exeter,
Mezzanine Laboratory,
Geoffrey Pope Building,
Stocker Road,
EX4 4QD, UK
******************************
Tel:  [phone] (Lab.)
        [phone] (Office)
Fax:  [phone]
E-mail:  [hidden email]
******************************
http://www.illuminatedcell.com/improved-imaging.html
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Vasseur Monique [[hidden email]]
Sent: Tuesday, January 24, 2012 9:31 PM
To: [hidden email]
Subject: Plant preparation for confocal microscopy

*****
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Dear all,

Do some of you could suggest how to prepare live sample of plant leaf for confocal microscopy.  We don't have much idea how to "sandwich" a leaf between slide and coverslip so that it is flat et not too thick for microscopy (and without destroying it)? What should we use as mounting media? What should we be aware of?  Our plant will have YFP in chloroplastides.

Thanks a lot in advance to all of you.

Monique

[Monique Vasseur]

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Hi Mr. Donaldson,

The tape doesn't interfer for the imaging?

Monique Vasseur
tél. [phone] poste 5148

-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Lloyd Donaldson
Envoyé : 24 janvier 2012 18:08
À : [hidden email]
Objet : Re: Plant preparation for confocal microscopy

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It is often a problem with leaves to keep them flat. What we do is stick them to the slide with double sided tape and then mount in water or glycerol.


Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification Scion - Forests . Products . Innovation Private Bag 3020, Rotorua
49 Sala Street, Rotorua 3010
New Zealand

Ph: 64 7 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rosemary White
Sent: Wednesday, 25 January 2012 11:02 a.m.
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

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Hi Monique,

You can image with little preparation, just mount in water and add a coverslip. Some common problems:

Air bubbles within the tissue - bad if you want to look below the epidermis - lots of reflection from the air/water (i.e. air/cell wall) interface.
The laser illumination may stimulate photosynthesis so bubbles develop in the leaf tissue - within cells as well as between them, and may accumulate under the coverslip.
Both of these problems may be alleviated somewhat by gentle vacuum infiltration with water or buffer before imaging.

Another option is to image the leaf dry without coverslip - you don't get the bubble problems then, but still need to worry about reflection from the leaf surface and about the leaf drying out during imaging.

Best is to just try it - since it's confocal, you don't have to worry about leaf thickness. If you need to image deeper layers, just cut a thick cross-section and mount in water; there will be enough undamaged cells there.

good luck,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 25/01/12 8:31 AM, "Vasseur Monique" <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear all,
>
>Do some of you could suggest how to prepare live sample of plant leaf
>for confocal microscopy.  We don't have much idea how to "sandwich" a
>leaf between slide and coverslip so that it is flat et not too thick
>for microscopy (and without destroying it)? What should we use as
>mounting media? What should we be aware of?  Our plant will have YFP in
>chloroplastides.
>
>Thanks a lot in advance to all of you.
>
>Monique

This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the  course of Scion's usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.

[Jeffrey Caplan]

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*****

Hi Monique,
On an inverted microscope we just use NUNC chambers and a glass block to
keep the leaf section down towards the cover glass and immerse the leaf
section in water. For an upright, we use Secure-Seal spacers that have a 13
mm diameter well that's 0.12mm deep.  It's the perfect depth for
sandwiching a leaf section and the spacers will stick to glass even when
wet with water or perfluorodecalin. We syringe vacuum infiltrate the leaves
with water for routine imaging of localization. But, if we are doing long
time courses, we use perfluorodecalin (as suggested by George) and then
mount them with a secure seal spacer in IBIDI 50mm dishes, which have gas
permeable cover glass. The combination of gas permeable plastic and
perflurodecalin helps keep the leaf section alive for many hours and keeps
it very stable during imaging.

Best Regards,

Jeff Caplan
Bioimaging Center
Delaware  Biotechnology Institute


On Tue, Jan 24, 2012 at 4:31 PM, Vasseur Monique <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Do some of you could suggest how to prepare live sample of plant leaf for
> confocal microscopy.  We don't have much idea how to "sandwich" a leaf
> between slide and coverslip so that it is flat et not too thick for
> microscopy
> (and without destroying it)? What should we use as mounting media? What
> should we be aware of?  Our plant will have YFP in chloroplastides.
>
> Thanks a lot in advance to all of you.
>
> Monique
>

[Lloyd Donaldson]

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Monique

The tape will be behind the sample for confocal imaging so it is not in the light path. We are able to image leaves still attached to the plant by this method but that is probably not required for your application. We are observing infiltration of the leaf by herbicide. You could also try Thiodiethanol as a mounting medium. It is water miscible but has a refractive index similar to immersion oil depending on the water content. I am not sure if it is suitable for live imaging though.


Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification
Scion - Forests . Products . Innovation
Private Bag 3020, Rotorua
49 Sala Street, Rotorua 3010
New Zealand

Ph: 64 7 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vasseur Monique
Sent: Thursday, 26 January 2012 7:56 a.m.
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

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Hi Mr. Donaldson,

The tape doesn't interfer for the imaging?

Monique Vasseur
tél. [phone] poste 5148

-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Lloyd Donaldson
Envoyé : 24 janvier 2012 18:08
À : [hidden email]
Objet : Re: Plant preparation for confocal microscopy

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

It is often a problem with leaves to keep them flat. What we do is stick them to the slide with double sided tape and then mount in water or glycerol.


Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification Scion - Forests . Products . Innovation Private Bag 3020, Rotorua
49 Sala Street, Rotorua 3010
New Zealand

Ph: 64 7 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rosemary White
Sent: Wednesday, 25 January 2012 11:02 a.m.
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Monique,

You can image with little preparation, just mount in water and add a coverslip. Some common problems:

Air bubbles within the tissue - bad if you want to look below the epidermis - lots of reflection from the air/water (i.e. air/cell wall) interface.
The laser illumination may stimulate photosynthesis so bubbles develop in the leaf tissue - within cells as well as between them, and may accumulate under the coverslip.
Both of these problems may be alleviated somewhat by gentle vacuum infiltration with water or buffer before imaging.

Another option is to image the leaf dry without coverslip - you don't get the bubble problems then, but still need to worry about reflection from the leaf surface and about the leaf drying out during imaging.

Best is to just try it - since it's confocal, you don't have to worry about leaf thickness. If you need to image deeper layers, just cut a thick cross-section and mount in water; there will be enough undamaged cells there.

good luck,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 25/01/12 8:31 AM, "Vasseur Monique" <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear all,
>
>Do some of you could suggest how to prepare live sample of plant leaf
>for confocal microscopy.  We don't have much idea how to "sandwich" a
>leaf between slide and coverslip so that it is flat et not too thick
>for microscopy (and without destroying it)? What should we use as
>mounting media? What should we be aware of?  Our plant will have YFP in
>chloroplastides.
>
>Thanks a lot in advance to all of you.
>
>Monique

This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the  course of Scion's usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.



This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the  course of Scion's usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.

[Guy Cox-2]

What I do with leaves is to peel off one or other epidermis.  This enables you to flood the leaf and you can put both the peeled epidermis and the remaining leaf on a slide and image without air problems.  Chloroplast fluorescence is going to be a problem but if you can work with etiolated leaves that might help.

                                  Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian
Sent: Thursday, 26 January 2012 3:18 AM
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

Localization to chloroplasts at low expression levels can be
challe
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****



Localization to chloroplasts at low expression levels can be
challenging depending on your system and familiarity with dealing with sample
with autofluorescence.  If you find you're having some difficulty, get
back to us, and as a group, I'm quite sure we can talk you through that part.



As for mounting samples, it depends on what plant you're dealing with. 
Zea mays, you would probably want to hand cut cross sections, mount in water
under a coverslip.  Arabidopsis, you will probably mount with the
"bottom" of the leaf towards the lens (are you upright or
inverted?)  This will help avoid not only most of the trichomes which
cause more air bubble issues and refraction, but the epidermal cells under them
require deep z-series to see.  Of course that is if you're working stable
lines, bombardment samples I usually view from the side which was
bombarded.  Lastly, tobacco, if transiently expressing from Agrobacterium,
is often infiltrated from the bottom of the leaf, so that’s where I look, on
the bottom.

As for air bubbles… I do not fret the air bubbles at all,
but this is mostly because I’m NOT trying to collect the transmitted light
image.  If you need it, then you might
worry about them, and I highly recommend checking out perfluorodecalin.  I personally have not had luck with pulling a
weak vacuum , actually, what I do is even more simplistic.

I place the leaf sample (disks are too small to be worth the
effort in my hands) with the side I’d like to image up on a slide.  Add water, and place a 22x60mm cover slip
over the sample, which is usually about 22x22mm.  I then tap out the air bubbles I can see, and
place the entire thing on a “steel slide” and then place two small magnets on
top of the cover slip to hold the works flat. 
If I avoid taking samples from area with large veins, I can do z-series
as long as I’d like without lateral shifting. 
The only major problem I will warn you about is that you’re making your “slide”
much thicker and if you use an automated stage, this could be a real
problem.  Secondly, and obviously, you
will not get a transmitted light image. 
Of course this only works on an upright, but it has worked for at least
four years.

Good luck.

Christian

 

 

>Dear all,

>

>Do some of you could suggest how to prepare live sample of plant leaf for

>confocal microscopy.  We don't have much idea how to
"sandwich" a leaf

>between slide and coverslip so that it is flat et not too thick for

>microscopy

>(and without destroying it)? What should we use as mounting media? What

>should we be aware of?  Our plant will have YFP in chloroplastides.

>

>Thanks a lot in advance to all of you.

>

[Stanislav Vitha]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

2,2-Thiodiethanol is nice for dead samples but is not compatible with live
imaging. In the optimal concentration (97% TDE, 3% buffer) it also quenches
fluorescent protein signals.

Stan Vitha
 
On Thu, 26 Jan 2012 08:56:24 +1300, Lloyd Donaldson
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Monique
>
>The tape will be behind the sample for confocal imaging so it is not in the
light path. We are able to image leaves still attached to the plant by this
method but that is probably not required for your application. We are
observing infiltration of the leaf by herbicide. You could also try Thiodiethanol
as a mounting medium. It is water miscible but has a refractive index similar to
immersion oil depending on the water content. I am not sure if it is suitable for
live imaging though.

>
>
>Dr Lloyd Donaldson
>
>Senior Scientist, Project Leader - Microscopy/Wood Identification
>Scion - Forests . Products . Innovation
>Private Bag 3020, Rotorua
>49 Sala Street, Rotorua 3010
>New Zealand
>
>Ph: 64 7 343 5581
>www.scionresearch.com
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List

[mailto:[hidden email]] On Behalf Of Vasseur
Monique

>Sent: Thursday, 26 January 2012 7:56 a.m.
>To: [hidden email]
>Subject: Re: Plant preparation for confocal microscopy
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Mr. Donaldson,
>
>The tape doesn't interfer for the imaging?
>
>Monique Vasseur
>tél. [phone] poste 5148
>
>-----Message d'origine-----
>De : Confocal Microscopy List [mailto:[hidden email]]

De la part de Lloyd Donaldson

>Envoyé : 24 janvier 2012 18:08
>À : [hidden email]
>Objet : Re: Plant preparation for confocal microscopy
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>It is often a problem with leaves to keep them flat. What we do is stick them

to the slide with double sided tape and then mount in water or glycerol.
>
>
>Dr Lloyd Donaldson
>
>Senior Scientist, Project Leader - Microscopy/Wood Identification Scion -
Forests . Products . Innovation Private Bag 3020, Rotorua

>49 Sala Street, Rotorua 3010
>New Zealand
>
>Ph: 64 7 343 5581
>www.scionresearch.com
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List

[mailto:[hidden email]] On Behalf Of Rosemary White

>Sent: Wednesday, 25 January 2012 11:02 a.m.
>To: [hidden email]
>Subject: Re: Plant preparation for confocal microscopy
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Monique,
>
>You can image with little preparation, just mount in water and add a

coverslip. Some common problems:
>
>Air bubbles within the tissue - bad if you want to look below the epidermis -
lots of reflection from the air/water (i.e. air/cell wall) interface.
>The laser illumination may stimulate photosynthesis so bubbles develop in the
leaf tissue - within cells as well as between them, and may accumulate under
the coverslip.
>Both of these problems may be alleviated somewhat by gentle vacuum
infiltration with water or buffer before imaging.
>
>Another option is to image the leaf dry without coverslip - you don't get the
bubble problems then, but still need to worry about reflection from the leaf
surface and about the leaf drying out during imaging.
>
>Best is to just try it - since it's confocal, you don't have to worry about leaf
thickness. If you need to image deeper layers, just cut a thick cross-section
and mount in water; there will be enough undamaged cells there.

>
>good luck,
>Rosemary
>
>Dr Rosemary White
>CSIRO Plant Industry
>GPO Box 1600
>Canberra, ACT 2601
>Australia
>
>T 61 2 6246 5475
>F 61 2 6246 5334
>E [hidden email]
>
>
>On 25/01/12 8:31 AM, "Vasseur Monique" <[hidden email]>
>wrote:
>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>Dear all,
>>
>>Do some of you could suggest how to prepare live sample of plant leaf
>>for confocal microscopy.  We don't have much idea how to "sandwich" a
>>leaf between slide and coverslip so that it is flat et not too thick
>>for microscopy (and without destroying it)? What should we use as
>>mounting media? What should we be aware of?  Our plant will have YFP in
>>chloroplastides.
>>
>>Thanks a lot in advance to all of you.
>>
>>Monique
>
>
>
>This e-mail and any attachments may contain information which is

confidential or subject to copyright. If you receive this e-mail in error, please
delete it.
>Scion does not accept responsibility for anything in this e-mail which is not
provided in the  course of Scion's usual business or for any computer virus,
data corruption, interference or delay arising from this e-mail.
>
>
>
>This e-mail and any attachments may contain information which is
confidential or subject to copyright. If you receive this e-mail in error, please
delete it.
>Scion does not accept responsibility for anything in this e-mail which is not
provided in the  course of Scion's usual business or for any computer virus,
data corruption, interference or delay arising from this e-mail.

[Christian-103]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Guy,

I will have to disagree with you on the etiolated plant material.  Large, healthy chloroplasts have very strong autofluorescence in the "far red" channel, and much smaller peak in the "GFP" range.  When plants are stressed, not only do they not produce normal chlorophylls, but they can also produce an entire gambit of other pigments.  These pigments may throw the autofluorescence curves all over the place making identification of GFP or YFP much more difficult.  The same thing occurs in leaves which are stressed or experiencing hypersensitive responses to transformation.

The best bet is to keep the plants healthy as possible.

Christian


--- On Wed, 1/25/12, Guy Cox <[hidden email]> wrote:

From: Guy Cox <[hidden email]>
Subject: Re: Plant preparation for confocal microscopy
To: [hidden email]
Date: Wednesday, January 25, 2012, 10:50 PM

What I do with leaves is to peel off one or other epidermis.  This enables you to flood the leaf and you can put both the peeled epidermis and the remaining leaf on a slide and image without air problems.  Chloroplast fluorescence is going to be a problem but if you can work with etiolated leaves that might help.

                                  Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm

______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net

 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian
Sent: Thursday, 26 January 2012 3:18 AM
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

Localization to chloroplasts at low expression levels can be
challe
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

*****



Localization to chloroplasts at low expression levels can be
challenging depending on your system and familiarity with dealing with sample
with autofluorescence.  If you find you're having some difficulty, get
back to us, and as a group, I'm quite sure we can talk you through that part.



As for mounting samples, it depends on what plant you're dealing with. 
Zea mays, you would probably want to hand cut cross sections, mount in water
under a coverslip.  Arabidopsis, you will probably mount with the
"bottom" of the leaf towards the lens (are you upright or
inverted?)  This will help avoid not only most of the trichomes which
cause more air bubble issues and refraction, but the epidermal cells under them
require deep z-series to see.  Of course that is if you're working stable
lines, bombardment samples I usually view from the side which was
bombarded.  Lastly, tobacco, if transiently expressing from Agrobacterium,
is often infiltrated from the bottom of the leaf, so that’s where I look, on
the bottom.

As for air bubbles… I do not fret the air bubbles at all,
but this is mostly because I’m NOT trying to collect the transmitted light
image.  If you need it, then you might
worry about them, and I highly recommend checking out perfluorodecalin.  I personally have not had luck with pulling a
weak vacuum , actually, what I do is even more simplistic.

I place the leaf sample (disks are too small to be worth the
effort in my hands) with the side I’d like to image up on a slide.  Add water, and place a 22x60mm cover slip
over the sample, which is usually about 22x22mm.  I then tap out the air bubbles I can see, and
place the entire thing on a “steel slide” and then place two small magnets on
top of the cover slip to hold the works flat. 
If I avoid taking samples from area with large veins, I can do z-series
as long as I’d like without lateral shifting. 
The only major problem I will warn you about is that you’re making your “slide”
much thicker and if you use an automated stage, this could be a real
problem.  Secondly, and obviously, you
will not get a transmitted light image. 
Of course this only works on an upright, but it has worked for at least
four years.

Good luck.

Christian

 

 

>Dear all,

>

>Do some of you could suggest how to prepare live sample of plant leaf for

>confocal microscopy.  We don't have much idea how to
"sandwich" a leaf

>between slide and coverslip so that it is flat et not too thick for

>microscopy

>(and without destroying it)? What should we use as mounting media? What

>should we be aware of?  Our plant will have YFP in chloroplastides.

>

>Thanks a lot in advance to all of you.

>

[Monique Vasseur]

Christian,

Do you have experience with variegated leaves?  
Also, as control of the chlorophylls, some people use 458 nm as excitation, others 633 nm. What would you suggest the best?  Thanks again.

Monique Vasseur
tél. [phone] poste 5148

-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Christian
Envoyé : 26 janvier 2012 11:06
À : [hidden email]
Objet : Re: Plant preparation for confocal microscopy

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Guy,

I will have to disagree with you on the etiolated plant material.  Large, healthy chloroplasts have very strong autofluorescence in the "far red" channel, and much smaller peak in the "GFP" range.  When plants are stressed, not only do they not produce normal chlorophylls, but they can also produce an entire gambit of other pigments.  These pigments may throw the autofluorescence curves all over the place making identification of GFP or YFP much more difficult.  The same thing occurs in leaves which are stressed or experiencing hypersensitive responses to transformation.

The best bet is to keep the plants healthy as possible.

Christian


--- On Wed, 1/25/12, Guy Cox <[hidden email]> wrote:

From: Guy Cox <[hidden email]>
Subject: Re: Plant preparation for confocal microscopy
To: [hidden email]
Date: Wednesday, January 25, 2012, 10:50 PM

What I do with leaves is to peel off one or other epidermis.  This enables you to flood the leaf and you can put both the peeled epidermis and the remaining leaf on a slide and image without air problems.  Chloroplast fluorescence is going to be a problem but if you can work with etiolated leaves that might help.

                                  Guy

Optical Imaging Techniques in Cell Biology by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm

______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net

 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian
Sent: Thursday, 26 January 2012 3:18 AM
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

Localization to chloroplasts at low expression levels can be challe
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

*****



Localization to chloroplasts at low expression levels can be challenging depending on your system and familiarity with dealing with sample with autofluorescence.  If you find you're having some difficulty, get back to us, and as a group, I'm quite sure we can talk you through that part.



As for mounting samples, it depends on what plant you're dealing with. Zea mays, you would probably want to hand cut cross sections, mount in water under a coverslip.  Arabidopsis, you will probably mount with the "bottom" of the leaf towards the lens (are you upright or
inverted?)  This will help avoid not only most of the trichomes which cause more air bubble issues and refraction, but the epidermal cells under them require deep z-series to see.  Of course that is if you're working stable lines, bombardment samples I usually view from the side which was bombarded.  Lastly, tobacco, if transiently expressing from Agrobacterium, is often infiltrated from the bottom of the leaf, so that’s where I look, on the bottom.

As for air bubbles… I do not fret the air bubbles at all, but this is mostly because I’m NOT trying to collect the transmitted light image.  If you need it, then you might worry about them, and I highly recommend checking out perfluorodecalin.  I personally have not had luck with pulling a weak vacuum , actually, what I do is even more simplistic.

I place the leaf sample (disks are too small to be worth the effort in my hands) with the side I’d like to image up on a slide.  Add water, and place a 22x60mm cover slip over the sample, which is usually about 22x22mm.  I then tap out the air bubbles I can see, and place the entire thing on a “steel slide” and then place two small magnets on top of the cover slip to hold the works flat. If I avoid taking samples from area with large veins, I can do z-series as long as I’d like without lateral shifting. The only major problem I will warn you about is that you’re making your “slide”
much thicker and if you use an automated stage, this could be a real problem.  Secondly, and obviously, you will not get a transmitted light image. Of course this only works on an upright, but it has worked for at least four years.

Good luck.

Christian

 

 

>Dear all,

>

>Do some of you could suggest how to prepare live sample of plant leaf
>for

>confocal microscopy.  We don't have much idea how to
"sandwich" a leaf

>between slide and coverslip so that it is flat et not too thick for

>microscopy

>(and without destroying it)? What should we use as mounting media? What

>should we be aware of?  Our plant will have YFP in chloroplastides.

>

>Thanks a lot in advance to all of you.

>

[Christian-103]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Monique,

Yes, I have worked in variegated leaves. 

As for imaging techniques, nearly all of my imaging is done with only a 488 laser and a "GFP" and "CY5" emission.  The 488 excites the pigments in the chloroplasts just fine, and allows for simultaneous acquisition, which matters greatly in living cells which have cytoplasmic streaming.  Using sequential imaging with the two laser lines maybe too slow.

In the variegated section of the leaves, the proplastids, plastids, and chloroplasts can be maddening to image.  The reason again comes down to what pigments are there, and where in development those organelles are.  Furthermore, how successful you are is often tied to how strong a promoter is being used.  Identifying YFP or GFP which is highly expressed is quite easy, but at low levels, autofluorescence becomes a real problem.  I have actually been urging labs to switch to constructs using RFP or mCherry when possible.  It is a very "clean" channel to image in for plants.

Please contact me off-list if you'd like more details, or would like to share images.

Christian


 
--- On Thu, 1/26/12, Vasseur Monique <[hidden email]> wrote:

From: Vasseur Monique <[hidden email]>
Subject: Re: Plant preparation for confocal microscopy
To: [hidden email]
Date: Thursday, January 26, 2012, 10:28 AM

Christian,

Do you have experience with variegated leaves? 
Also, as control of the chlorophylls, some people use 458 nm as excitation, others 633 nm. What would you suggest the best?  Thanks again.

Monique Vasseur
tél. [phone] poste 5148

-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Christian
Envoyé : 26 janvier 2012 11:06
À : [hidden email]
Objet : Re: Plant preparation for confocal microscopy

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

*****

Guy,

I will have to disagree with you on the etiolated plant material.  Large, healthy chloroplasts have very strong autofluorescence in the "far red" channel, and much smaller peak in the "GFP" range.  When plants are stressed, not only do they not produce normal chlorophylls, but they can also produce an entire gambit of other pigments.  These pigments may throw the autofluorescence curves all over the place making identification of GFP or YFP much more difficult.  The same thing occurs in leaves which are stressed or experiencing hypersensitive responses to transformation.

The best bet is to keep the plants healthy as possible.

Christian


--- On Wed, 1/25/12, Guy Cox <[hidden email]> wrote:

From: Guy Cox <[hidden email]>
Subject: Re: Plant preparation for confocal microscopy
To: [hidden email]
Date: Wednesday, January 25, 2012, 10:50 PM

What I do with leaves is to peel off one or other epidermis.  This enables you to flood the leaf and you can put both the peeled epidermis and the remaining leaf on a slide and image without air problems.  Chloroplast fluorescence is going to be a problem but if you can work with etiolated leaves that might help.

                                  Guy

Optical Imaging Techniques in Cell Biology by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm


______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net


 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian
Sent: Thursday, 26 January 2012 3:18 AM
To: [hidden email]
Subject: Re: Plant preparation for confocal microscopy

Localization to chloroplasts at low expression levels can be challe
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy


*****



Localization to chloroplasts at low expression levels can be challenging depending on your system and familiarity with dealing with sample with autofluorescence.  If you find you're having some difficulty, get back to us, and as a group, I'm quite sure we can talk you through that part.



As for mounting samples, it depends on what plant you're dealing with. Zea mays, you would probably want to hand cut cross sections, mount in water under a coverslip.  Arabidopsis, you will probably mount with the "bottom" of the leaf towards the lens (are you upright or
inverted?)  This will help avoid not only most of the trichomes which cause more air bubble issues and refraction, but the epidermal cells under them require deep z-series to see.  Of course that is if you're working stable lines, bombardment samples I usually view from the side which was bombarded.  Lastly, tobacco, if transiently expressing from Agrobacterium, is often infiltrated from the bottom of the leaf, so that’s where I look, on the bottom.

As for air bubbles… I do not fret the air bubbles at all, but this is mostly because I’m NOT trying to collect the transmitted light image.  If you need it, then you might worry about them, and I highly recommend checking out perfluorodecalin.  I personally have not had luck with pulling a weak vacuum , actually, what I do is even more simplistic.

I place the leaf sample (disks are too small to be worth the effort in my hands) with the side I’d like to image up on a slide.  Add water, and place a 22x60mm cover slip over the sample, which is usually about 22x22mm.  I then tap out the air bubbles I can see, and place the entire thing on a “steel slide” and then place two small magnets on top of the cover slip to hold the works flat. If I avoid taking samples from area with large veins, I can do z-series as long as I’d like without lateral shifting. The only major problem I will warn you about is that you’re making your “slide”
much thicker and if you use an automated stage, this could be a real problem.  Secondly, and obviously, you will not get a transmitted light image. Of course this only works on an upright, but it has worked for at least four years.

Good luck.

Christian

 

 

>Dear all,

>

>Do some of you could suggest how to prepare live sample of plant leaf
>for

>confocal microscopy.  We don't have much idea how to
"sandwich" a leaf

>between slide and coverslip so that it is flat et not too thick for

>microscopy

>(and without destroying it)? What should we use as mounting media? What

>should we be aware of?  Our plant will have YFP in chloroplastides.

>

>Thanks a lot in advance to all of you.

>

[Christian-103]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

As we're on plants...  has anyone imaged GFP or RFP in the vacuole of plants?  Tonoplast is quite routine, but what about the lumen of the vacuole?  I have a user who believes there is a cleavage event which should lead to the build up of GFP inside the vacuole, my concern was degradation of the signal due to pH issues.

Does anyone have any experience with imaging inside the vacuole of plants?  This will be in tobacco until the stable Arabidopsis lines are available.

Thanks

Christian
 

[Jeffrey Caplan]

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Hi Monique,
You can do a fairly good job problem separating YFP from chloroplast
autofluorescence on a confocal using fast line switching between the 514 nm
and 458/633 for YFP and chloroplasts respectively. I find that 458 is
slightly better for chloroplasts, but either one is fine. For very, very
weak YFP signals, I have seen a significant amount of bleed through from
the chloroplasts using a 520-550 BP. In that case, spectral unmixing was
able to easily separate the two. I also agree with Christian that RFP,
although somewhat counter intuitive, is the easiest to separate from
chloroplast autofluorescence.

Jeff Caplan
Bioimaging Center
Delaware  Biotechnology Institute

On Thu, Jan 26, 2012 at 12:13 PM, Christian <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Monique,
>
> Yes, I have worked in variegated leaves.
>
> As for imaging techniques, nearly all of my imaging is done with only a
> 488 laser and a "GFP" and "CY5" emission.  The 488 excites the pigments in
> the chloroplasts just fine, and allows for simultaneous acquisition, which
> matters greatly in living cells which have cytoplasmic streaming.  Using
> sequential imaging with the two laser lines maybe too slow.
>
> In the variegated section of the leaves, the proplastids, plastids, and
> chloroplasts can be maddening to image.  The reason again comes down to
> what pigments are there, and where in development those organelles are.
> Furthermore, how successful you are is often tied to how strong a promoter
> is being used.  Identifying YFP or GFP which is highly expressed is quite
> easy, but at low levels, autofluorescence becomes a real problem.  I have
> actually been urging labs to switch to constructs using RFP or mCherry when
> possible.  It is a very "clean" channel to image in for plants.
>
> Please contact me off-list if you'd like more details, or would like to
> share images.
>
> Christian
>
>
>
> --- On Thu, 1/26/12, Vasseur Monique <[hidden email]> wrote:
>
> From: Vasseur Monique <[hidden email]>
> Subject: Re: Plant preparation for confocal microscopy
> To: [hidden email]
> Date: Thursday, January 26, 2012, 10:28 AM
>
> Christian,
>
> Do you have experience with variegated leaves?
> Also, as control of the chlorophylls, some people use 458 nm as
> excitation, others 633 nm. What would you suggest the best?  Thanks again.
>
> Monique Vasseur
> tél. [phone] poste 5148
>
> -----Message d'origine-----
> De : Confocal Microscopy List [mailto:[hidden email]]
> De la part de Christian
> Envoyé : 26 janvier 2012 11:06
> À : [hidden email]
> Objet : Re: Plant preparation for confocal microscopy
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
> Guy,
>
> I will have to disagree with you on the etiolated plant material.  Large,
> healthy chloroplasts have very strong autofluorescence in the "far red"
> channel, and much smaller peak in the "GFP" range.  When plants are
> stressed, not only do they not produce normal chlorophylls, but they can
> also produce an entire gambit of other pigments.  These pigments may throw
> the autofluorescence curves all over the place making identification of GFP
> or YFP much more difficult.  The same thing occurs in leaves which are
> stressed or experiencing hypersensitive responses to transformation.
>
> The best bet is to keep the plants healthy as possible.
>
> Christian
>
>
> --- On Wed, 1/25/12, Guy Cox <[hidden email]> wrote:
>
> From: Guy Cox <[hidden email]>
> Subject: Re: Plant preparation for confocal microscopy
> To: [hidden email]
> Date: Wednesday, January 25, 2012, 10:50 PM
>
> What I do with leaves is to peel off one or other epidermis.  This enables
> you to flood the leaf and you can put both the peeled epidermis and the
> remaining leaf on a slide and image without air problems.  Chloroplast
> fluorescence is going to be a problem but if you can work with etiolated
> leaves that might help.
>
>                                   Guy
>
> Optical Imaging Techniques in Cell Biology by Guy Cox    CRC Press /
> Taylor & Francis
>      http://www.guycox.com/optical.htm
>
>
> ______________________________________________
> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
> Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW
> 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Christian
> Sent: Thursday, 26 January 2012 3:18 AM
> To: [hidden email]
> Subject: Re: Plant preparation for confocal microscopy
>
> Localization to chloroplasts at low expression levels can be challe
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>
> *****
>
>
>
> Localization to chloroplasts at low expression levels can be challenging
> depending on your system and familiarity with dealing with sample with
> autofluorescence.  If you find you're having some difficulty, get back to
> us, and as a group, I'm quite sure we can talk you through that part.
>
>
>
> As for mounting samples, it depends on what plant you're dealing with. Zea
> mays, you would probably want to hand cut cross sections, mount in water
> under a coverslip.  Arabidopsis, you will probably mount with the "bottom"
> of the leaf towards the lens (are you upright or
> inverted?)  This will help avoid not only most of the trichomes which
> cause more air bubble issues and refraction, but the epidermal cells under
> them require deep z-series to see.  Of course that is if you're working
> stable lines, bombardment samples I usually view from the side which was
> bombarded.  Lastly, tobacco, if transiently expressing from Agrobacterium,
> is often infiltrated from the bottom of the leaf, so that’s where I look,
> on the bottom.
>
> As for air bubbles… I do not fret the air bubbles at all, but this is
> mostly because I’m NOT trying to collect the transmitted light image.  If
> you need it, then you might worry about them, and I highly recommend
> checking out perfluorodecalin.  I personally have not had luck with pulling
> a weak vacuum , actually, what I do is even more simplistic.
>
> I place the leaf sample (disks are too small to be worth the effort in my
> hands) with the side I’d like to image up on a slide.  Add water, and place
> a 22x60mm cover slip over the sample, which is usually about 22x22mm.  I
> then tap out the air bubbles I can see, and place the entire thing on a
> “steel slide” and then place two small magnets on top of the cover slip to
> hold the works flat. If I avoid taking samples from area with large veins,
> I can do z-series as long as I’d like without lateral shifting. The only
> major problem I will warn you about is that you’re making your “slide”
> much thicker and if you use an automated stage, this could be a real
> problem.  Secondly, and obviously, you will not get a transmitted light
> image. Of course this only works on an upright, but it has worked for at
> least four years.
>
> Good luck.
>
> Christian
>
>
>
>
>
> >Dear all,
>
> >
>
> >Do some of you could suggest how to prepare live sample of plant leaf
> >for
>
> >confocal microscopy.  We don't have much idea how to
> "sandwich" a leaf
>
> >between slide and coverslip so that it is flat et not too thick for
>
> >microscopy
>
> >(and without destroying it)? What should we use as mounting media? What
>
> >should we be aware of?  Our plant will have YFP in chloroplastides.
>
> >
>
> >Thanks a lot in advance to all of you.
>
> >
>

[Jeffrey Caplan]

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Hi Christian,
Many years ago, a colleague of mine brought in Cerulean targeted to the
large vacuole and we were able to see it filling the vacuoles in Nicotiana.
I don't remember how she targeted it there, but I can try to find out for
you. I believe Cerulean is fairly insensitive to low pH, unlike GFP.  The
pH in the vacuole is about 5 and the pKa of eGFP is 6ish. My guess is over
80% of the fluorescence will be lost, but that will be somewhat dependent
on the GFP variant.

Best Regards,

Jeff Caplan
Bioimaging Center
Delaware  Biotechnology Institute

On Thu, Jan 26, 2012 at 12:32 PM, Christian <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> As we're on plants...  has anyone imaged GFP or RFP in the vacuole of
> plants?  Tonoplast is quite routine, but what about the lumen of the
> vacuole?  I have a user who believes there is a cleavage event which should
> lead to the build up of GFP inside the vacuole, my concern was degradation
> of the signal due to pH issues.
>
> Does anyone have any experience with imaging inside the vacuole of
> plants?  This will be in tobacco until the stable Arabidopsis lines are
> available.
>
> Thanks
>
> Christian
>
>

[Littlejohn, George]

I agree. A really nice tuneable mounting medium from the Hell lab, but we also found it to be unsuitable for use with live tissue when we tried it.

All the best,
George


Dr. George Littlejohn

University of Exeter
College of Life and Environmental Sciences
Biosciences
Geoffrey Pope Building
Mezzanine Lab
Stocker Road
Exeter
United Kingdom
EX4 4QD

******************************
Tel:  [phone] (Lab.)
       [phone] (Office)
Fax:  [phone]
E-mail:  [hidden email]
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http://www.illuminatedcell.com/improved-imaging.html


On 26 Jan 2012, at 16:56, "Stanislav Vitha" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 2,2-Thiodiethanol is nice for dead samples but is not compatible with live
> imaging. In the optimal concentration (97% TDE, 3% buffer) it also quenches
> fluorescent protein signals.
>
> Stan Vitha
>
> On Thu, 26 Jan 2012 08:56:24 +1300, Lloyd Donaldson
> <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Monique
>>
>> The tape will be behind the sample for confocal imaging so it is not in the
> light path. We are able to image leaves still attached to the plant by this
> method but that is probably not required for your application. We are
> observing infiltration of the leaf by herbicide. You could also try Thiodiethanol
> as a mounting medium. It is water miscible but has a refractive index similar to
> immersion oil depending on the water content. I am not sure if it is suitable for
> live imaging though.
>>
>>
>> Dr Lloyd Donaldson
>>
>> Senior Scientist, Project Leader - Microscopy/Wood Identification
>> Scion - Forests . Products . Innovation
>> Private Bag 3020, Rotorua
>> 49 Sala Street, Rotorua 3010
>> New Zealand
>>
>> Ph: 64 7 343 5581
>> www.scionresearch.com
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Vasseur
> Monique
>> Sent: Thursday, 26 January 2012 7:56 a.m.
>> To: [hidden email]
>> Subject: Re: Plant preparation for confocal microscopy
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Mr. Donaldson,
>>
>> The tape doesn't interfer for the imaging?
>>
>> Monique Vasseur
>> tél. [phone] poste 5148
>>
>> -----Message d'origine-----
>> De : Confocal Microscopy List [mailto:[hidden email]]
> De la part de Lloyd Donaldson
>> Envoyé : 24 janvier 2012 18:08
>> À : [hidden email]
>> Objet : Re: Plant preparation for confocal microscopy
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> It is often a problem with leaves to keep them flat. What we do is stick them
> to the slide with double sided tape and then mount in water or glycerol.
>>
>>
>> Dr Lloyd Donaldson
>>
>> Senior Scientist, Project Leader - Microscopy/Wood Identification Scion -
> Forests . Products . Innovation Private Bag 3020, Rotorua
>> 49 Sala Street, Rotorua 3010
>> New Zealand
>>
>> Ph: 64 7 343 5581
>> www.scionresearch.com
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Rosemary White
>> Sent: Wednesday, 25 January 2012 11:02 a.m.
>> To: [hidden email]
>> Subject: Re: Plant preparation for confocal microscopy
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Monique,
>>
>> You can image with little preparation, just mount in water and add a
> coverslip. Some common problems:
>>
>> Air bubbles within the tissue - bad if you want to look below the epidermis -
> lots of reflection from the air/water (i.e. air/cell wall) interface.
>> The laser illumination may stimulate photosynthesis so bubbles develop in the
> leaf tissue - within cells as well as between them, and may accumulate under
> the coverslip.
>> Both of these problems may be alleviated somewhat by gentle vacuum
> infiltration with water or buffer before imaging.
>>
>> Another option is to image the leaf dry without coverslip - you don't get the
> bubble problems then, but still need to worry about reflection from the leaf
> surface and about the leaf drying out during imaging.
>>
>> Best is to just try it - since it's confocal, you don't have to worry about leaf
> thickness. If you need to image deeper layers, just cut a thick cross-section
> and mount in water; there will be enough undamaged cells there.
>>
>> good luck,
>> Rosemary
>>
>> Dr Rosemary White
>> CSIRO Plant Industry
>> GPO Box 1600
>> Canberra, ACT 2601
>> Australia
>>
>> T 61 2 6246 5475
>> F 61 2 6246 5334
>> E [hidden email]
>>
>>
>> On 25/01/12 8:31 AM, "Vasseur Monique" <[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear all,
>>>
>>> Do some of you could suggest how to prepare live sample of plant leaf
>>> for confocal microscopy.  We don't have much idea how to "sandwich" a
>>> leaf between slide and coverslip so that it is flat et not too thick
>>> for microscopy (and without destroying it)? What should we use as
>>> mounting media? What should we be aware of?  Our plant will have YFP in
>>> chloroplastides.
>>>
>>> Thanks a lot in advance to all of you.
>>>
>>> Monique
>>
>>
>>
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