Syto dyes for plant nuclei in living cells

[John Runions]

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

Do any of the plant biologists out there have experience using the Syto dyes from Molecular Probes to stain nuclei of living cells.  In particular, they list 5 different dyes in the blue emission range (Syto 40-45).  Syto 42 looks good to me on paper.  What do you think.  Thanks, John.

[Tobias Baskin]

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

John,

        One or two years ago, we got a group of syto dyes for this very purpose, imaging in arabidopsis roots. We were hoping to image DNA along with a GFP reporter and therefore we tested sytos that have long wavelength emission (we were working on confocal, without ability to do uv excitation). None of them worked for us. They tended to be either toxic or dim. The other thing we noticed was that because the cells are crammed with mitochondria that move all over the place, it was difficult to follow nuclear DNA against this hive of mitochondrial motion. But we never tested blue ones.

        Good luck,

                Tobias

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

Do any of the plant biologists out there have experience using the Syto dyes from Molecular Probes to stain nuclei of living cells.  In particular, they list 5 different dyes in the blue emission range (Syto 40-45).  Syto 42 looks good to me on paper.  What do you think.  Thanks, John.

--
Runions signature

*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]

Runions' lab web site

 

Visit The Illuminated Plant Cell dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology

-- 

      _      ____          __   ____   

     /  \   /          / \    /   \ \        Tobias I. Baskin

    /   /  /          /   \   \      \         Biology Department

   /_ /   __      /__ \   \       \__    611 N. Pleasant St.

  /      /          /       \   \       \        University of Massachusetts

 /      /          /         \   \       \          Amherst, MA, 01003

/      / ___   /           \   \__/  \ ____

http://www.bio.umass.edu/biology/baskin/

Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

[Alison J. North]

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

Hi John and Tobias,

This may be a completely unhelpful response because I was not working on
plants and I was looking for dyes in the red/far red range, not blue.
However, a few years ago I tested a whole range of the red Syto dyes to
try to use them to follow cell division using live cell imaging
(together with a GFP construct) and I found something very interesting
with Syto 61.  Although I had the same problem as Tobias when I imaged
through Texas Red filter sets - i.e. the mitochondria obscured the
nuclear signal - I found that if I acquired through the far red (Cy5)
filters the signal was now predominantly nuclear.  The longer I left it,
the more the nuclear signal seemed to intensify while the remaining
mitochondrial signal soon bleached away. I called Mol. Probes to ask
them about this and they said that the dye becomes red-shifted when it's
highly concentrated - which fits with the fact that the mitotic cells
were brightest.  Anyway, unlike most of the nuclear dyes it didn't
affect cell division so I could image away for hours/days.

One slight problem - the signal and localisation appeared to be highly
cell specific.  It was great in MDCK cells but I seem to remember I
couldn't get the same effect in HeLas.  Still - might be worth a shot if
a far red dye would do?

Best wishes,
Alison


Tobias Baskin wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> John,
>         One or two years ago, we got a group of syto dyes for this very
> purpose, imaging in arabidopsis roots. We were hoping to image DNA along
> with a GFP reporter and therefore we tested sytos that have long
> wavelength emission (we were working on confocal, without ability to do
> uv excitation). None of them worked for us. They tended to be either
> toxic or dim. The other thing we noticed was that because the cells are
> crammed with mitochondria that move all over the place, it was difficult
> to follow nuclear DNA against this hive of mitochondrial motion. But we
> never tested blue ones.
>
>         Good luck,
>                 Tobias
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Do any of the plant biologists out there have experience using the
>> Syto dyes from Molecular Probes to stain nuclei of living cells.  In
>> particular, they list 5 different dyes in the blue emission range
>> (Syto 40-45).  Syto 42 looks good to me on paper.  What do you think.  
>> Thanks, John.
>> --
>> Runions signature
>> *********************************
>> C. John Runions, Ph.D.
>> School of Life Sciences
>> Oxford Brookes University
>> Oxford, UK
>> OX3 0BP
>>
>> email:  [hidden email] <mailto:[hidden email]>
>> phone: [phone]
>> Runions' lab web site
>> <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>
>>  
>> Visit The Illuminated Plant Cell
>> <http://www.illuminatedcell.com/ER.html> dot com
>> Oxford Brookes Master's in Bioimaging with Molecular Technology
>> <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>
>
>
> --
>
>       _      ____          __   ____  
>      /  \   /          / \    /   \ \        Tobias I. Baskin
>     /   /  /          /   \   \      \         Biology Department
>    /_ /   __      /__ \   \       \__    611 N. Pleasant St.
>   /      /          /       \   \       \        University of Massachusetts
>  /      /          /         \   \       \          Amherst, MA, 01003
> /      / ___   /           \   \__/  \ ____
> http://www.bio.umass.edu/biology/baskin/
> Voice: 413 - 545 - 1533 Fax:/ 413 - 545 - 3243/

--
Alison J. North, Ph.D.,
Research Assistant Professor and
Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab     ++ 212 327 7486
Fax:         ++ 212 327 7489

[Christian-103]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'll echo Tobias here.  It's very tough to get any dye into living plant cells and keep them that way.  We've tried nearly every nuclear stain available along with various other markers and trackers, such as mitotracker, all with no luck in the hands of the users.

Have you thought about making stables with RFP in the nuclei?  Three channel sequencing then allows you to visualize GFP, RFP and cholorplasts in the far red channel. 

Christian

[Glen MacDonald-2]

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

I only used the red Sytos once on live brain slices. The red Syto  
dyes seem to be lipophilic and wander through the intracellular  
membrane compartments on their way to the nucleus.  You have to wait  
for them to appear in the nuclei after staining.

Glen
On May 8, 2008, at 7:11 AM, Alison North wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi John and Tobias,
>
> This may be a completely unhelpful response because I was not  
> working on plants and I was looking for dyes in the red/far red  
> range, not blue. However, a few years ago I tested a whole range of  
> the red Syto dyes to try to use them to follow cell division using  
> live cell imaging (together with a GFP construct) and I found  
> something very interesting with Syto 61.  Although I had the same  
> problem as Tobias when I imaged through Texas Red filter sets -  
> i.e. the mitochondria obscured the nuclear signal - I found that if  
> I acquired through the far red (Cy5) filters the signal was now  
> predominantly nuclear.  The longer I left it, the more the nuclear  
> signal seemed to intensify while the remaining mitochondrial signal  
> soon bleached away. I called Mol. Probes to ask them about this and  
> they said that the dye becomes red-shifted when it's highly  
> concentrated - which fits with the fact that the mitotic cells were  
> brightest.  Anyway, unlike most of the nuclear dyes it didn't  
> affect cell division so I could image away for hours/days.
>
> One slight problem - the signal and localisation appeared to be  
> highly cell specific.  It was great in MDCK cells but I seem to  
> remember I couldn't get the same effect in HeLas.  Still - might be  
> worth a shot if a far red dye would do?
>
> Best wishes,
> Alison
>
>
> Tobias Baskin wrote:
>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/ 
>> cgi-bin/wa?S1=confocal
>> John,
>>         One or two years ago, we got a group of syto dyes for this  
>> very purpose, imaging in arabidopsis roots. We were hoping to  
>> image DNA along with a GFP reporter and therefore we tested sytos  
>> that have long wavelength emission (we were working on confocal,  
>> without ability to do uv excitation). None of them worked for us.  
>> They tended to be either toxic or dim. The other thing we noticed  
>> was that because the cells are crammed with mitochondria that move  
>> all over the place, it was difficult to follow nuclear DNA against  
>> this hive of mitochondrial motion. But we never tested blue ones.
>>         Good luck,
>>                 Tobias
>>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/ 
>>> cgi-bin/wa?S1=confocal
>>>
>>> Do any of the plant biologists out there have experience using  
>>> the Syto dyes from Molecular Probes to stain nuclei of living  
>>> cells.  In particular, they list 5 different dyes in the blue  
>>> emission range (Syto 40-45).  Syto 42 looks good to me on paper.  
>>> What do you think.  Thanks, John.
>>> --
>>> Runions signature
>>> *********************************
>>> C. John Runions, Ph.D.
>>> School of Life Sciences
>>> Oxford Brookes University
>>> Oxford, UK
>>> OX3 0BP
>>>
>>> email:  [hidden email] <mailto:[hidden email]>
>>> phone: [phone]
>>> Runions' lab web site <http://www.brookes.ac.uk/lifesci/runions/ 
>>> HTMLpages/index.html%21>
>>>  Visit The Illuminated Plant Cell <http://www.illuminatedcell.com/ 
>>> ER.html> dot com
>>> Oxford Brookes Master's in Bioimaging with Molecular Technology  
>>> <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>
>> --
>>       _      ____          __   ____        /  \   /          /  
>> \    /   \ \        Tobias I. Baskin
>>     /   /  /          /   \   \      \         Biology Department
>>    /_ /   __      /__ \   \       \__    611 N. Pleasant St.
>>   /      /          /       \   \       \        University of  
>> Massachusetts
>>  /      /          /         \   \       \          Amherst, MA,  
>> 01003
>> /      / ___   /           \   \__/  \ ____
>> http://www.bio.umass.edu/biology/baskin/
>> Voice: 413 - 545 - 1533 Fax:/ 413 - 545 - 3243/
>
> --
> Alison J. North, Ph.D.,
> Research Assistant Professor and
> Director of the Bio-Imaging Resource Center,
> The Rockefeller University,
> 1230 York Avenue,
> New York,
> NY 10065.
> Tel: office ++ 212 327 7488
> Tel: lab     ++ 212 327 7486
> Fax:         ++ 212 327 7489

[Marc Green]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal (another Red-not-blue and not in plant comment)

I tried several concentrations and incubation times for the syto dyes in the red sampler kit on live yeast,
But never got any specific nuclear staining. Sometimes the opposite.
I was watching on both “Texas-Red” and “Cy5” channels.

Please let me know if you get a working blue past the cell wall at a non-toxic concentration.

Marc

On 8/5/08 03:13, "John Runions" <[hidden email]> wrote:

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

Do any of the plant biologists out there have experience using the Syto dyes from Molecular Probes to stain nuclei of living cells.  In particular, they list 5 different dyes in the blue emission range (Syto 40-45).  Syto 42 looks good to me on paper.  What do you think. Thanks, John.

------------------
Marc D Green
Pombe.net
[hidden email]

[John Runions]

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

Thanks Christian,  good suggestion about using FPs.  We already have almost every organelle marked in almost every color and that clearly is the way to go.  We were only interested in nuclear stains that emit in the blue as a 'quick and dirty' way of looking at nuclei in lines without nuclear marking by FPs.  Best, John.

Christian wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'll echo Tobias here.  It's very tough to get any dye into living plant cells and keep them that way.  We've tried nearly every nuclear stain available along with various other markers and trackers, such as mitotracker, all with no luck in the hands of the users.

Have you thought about making stables with RFP in the nuclei?  Three channel sequencing then allows you to visualize GFP, RFP and cholorplasts in the far red channel. 

Christian

[Ignatius, Mike]

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

For those interested I can send a recently organized listing of dyes that have seen utility for plant cell work (respond off-line).  It is fairly long and includes an extensive bibliography.  We have never made it available before, but in the queue for future publication.

 

For example for Nuclei: 

·         Commonly used stains for nucleic acids in plants are DAPI, Hoechst 33258, YOYO®-1 iodide, YO-PRO®-1 iodide , acridine orange, ethidium bromide, and propidium iodide. 

But as Christian points out, these may not work.  However a new reagent has emerged that might be of interest.   It isn't a simple stain, but it readily labels dividing plant cell nuclei (alfalfa) in a workflow similar to BrDu but much improved with our new Click-iT technology.

 

Full text available at:  http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/bioprobes/bioprobes55.Par.79287.File.dat/bp55_1_EdU.pdf 

From that article:  Figure 5—Cell wall digestion is not required with Click-iT™ EdU. Medicago sativa (alfalfa) suspension cultures were incubated with 10 μM EdU for 3 hours. Cells were then fixed and permeabilized. EdU that had been incorporated into newly synthesized DNA was detected with the Click-iT™ EdU Alexa Fluor® 488 Imaging Kit (green fluorescence, Cat. no. C10083). Nuclei were stained with blue-fluorescent DAPI. Six confocal sections were overlaid onto a differential interference contrast image. Image contributed by Ferhan Ayaydin, Cellular Imaging Laboratory, Biological Research Center, Szeged, Hungary.

Kind Regards,

Mike Ignatius, Molecular Probes/Invitrogen

Eugene, Oregon, Site of the 2008 US Track and Field Trials.

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'll echo Tobias here.  It's very tough to get any dye into living plant cells and keep them that way.  We've tried nearly every nuclear stain available along with various other markers and trackers, such as mitotracker, all with no luck in the hands of the users.

Have you thought about making stables with RFP in the nuclei?  Three channel sequencing then allows you to visualize GFP, RFP and cholorplasts in the far red channel. 

Christian

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Runions
Sent: Thursday, May 08, 2008 9:25 AM
To: [hidden email]
Subject: Re: Syto dyes for plant nuclei in living cells

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

Thanks Christian,  good suggestion about using FPs.  We already have almost every organelle marked in almost every color and that clearly is the way to go.  We were only interested in nuclear stains that emit in the blue as a 'quick and dirty' way of looking at nuclei in lines without nuclear marking by FPs.  Best, John.

Christian wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'll echo Tobias here.  It's very tough to get any dye into living plant cells and keep them that way.  We've tried nearly every nuclear stain available along with various other markers and trackers, such as mitotracker, all with no luck in the hands of the users.

Have you thought about making stables with RFP in the nuclei?  Three channel sequencing then allows you to visualize GFP, RFP and cholorplasts in the far red channel. 

Christian

[Christian-103]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Quick and dirty... hmm, might I suggest animal cells then? 

Yes, I'm joking.  I've done primarily plant confocal since 2001, and I don't know anything quick and dirty yet, but I'm always hopeful.

Maybe a few of the other responses here will show some promise.  I will say what works in one species may not in another, no matter how much time you throw at it.

If you have success, please share it here.

Christian


John Runions <[hidden email]> wrote:

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

Thanks Christian,  good suggestion about using FPs.  We already have almost every organelle marked in almost every color and that clearly is the way to go.  We were only interested in nuclear stains that emit in the blue as a 'quick and dirty' way of looking at nuclei in lines without nuclear marking by FPs.  Best, John.

Christian wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'll echo Tobias here.  It's very tough to get any dye into living plant cells and keep them that way.  We've tried nearly every nuclear stain available along with various other markers and trackers, such as mitotracker, all with no luck in the hands of the users.

Have you thought about making stables with RFP in the nuclei?  Three channel sequencing then allows you to visualize GFP, RFP and cholorplasts in the far red channel. 

Christian

[Rosemary.White]

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

I tried a whole range of them at one stage, but in my hands, they all labelled quite a bit more than the nucleus, some just went in and lit up the whole cytoplasm.  Didn’t pursue it further than that, it may be that with different buffers, etc. they would be less likely to rush into the cell.
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 8/5/08 8:13 PM, "John Runions" <[hidden email]> wrote:

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

Do any of the plant biologists out there have experience using the Syto dyes from Molecular Probes to stain nuclei of living cells.  In particular, they list 5 different dyes in the blue emission range (Syto 40-45).  Syto 42 looks good to me on paper.  What do you think. Thanks, John.

[Rosemary.White]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Oh yes, I forgot to mention that we also found a few to be toxic (had forgotten....).  We tried the green and orange ones.
cheers,
Rosemary


On 8/5/08 11:03 PM, "Tobias Baskin" <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
John,
        One or two years ago, we got a group of syto dyes for this very purpose, imaging in arabidopsis roots. We were hoping to image DNA along with a GFP reporter and therefore we tested sytos that have long wavelength emission (we were working on confocal, without ability to do uv excitation). None of them worked for us. They tended to be either toxic or dim. The other thing we noticed was that because the cells are crammed with mitochondria that move all over the place, it was difficult to follow nuclear DNA against this hive of mitochondrial motion. But we never tested blue ones.

        Good luck,
              Tobias

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

Do any of the plant biologists out there have experience using the Syto dyes from Molecular Probes to stain nuclei of living cells. In particular, they list 5 different dyes in the blue emission range (Syto 40-45).  Syto 42 looks good to me on paper.  What do you think.  Thanks, John.
--
Runions signature
*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

email:  [hidden email]
phone: [phone]
Runions' lab web site <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>
 
Visit The Illuminated Plant Cell <http://www.illuminatedcell.com/ER.html>  dot com
Oxford Brookes Master's in Bioimaging with Molecular Technology <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>

[Christian-103]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'd like to point out that three researchers on three continents have now stated the difficulty getting only the nuclei labeled.  I know we had some input from a commercial source, but the literature is lacking.

Nearly every day I'm asked "What's that spotting in the cytoplasm?"  And I answer "I don't know without a counter stain."

The point I'm trying to make is that plant confocal is challenging, and none of us are likely to get bored any time soon.  Now if we could just have 1/10th the dyes the animal systems people do.

I'd also like to make a public plea for any protocols which may lead to nuclei only labeling in living plants.

Christian

 

[Rosemary.White]

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

Yes, those common stains do work - sometimes.  I used to think DAPI was good for all living plant cells, but have met many plant tissues, cell cultures and protoplasts that won’t take it up.  Ditto for all the rest (including DRAQ5).  My feeling is that these dyes can enter certain types of plant plasma membranes but not others.  My guess is that the charge on these dyes keeps them out, and maybe if they were modified to appear less polar to the membrane, they’d get in more readily.

And we use propidium iodide in our live/dead cell test – it is either excluded from or takes a long time to get into living plant cells.  The plant cells it gets into quickly are dying or undergoing programmed cell death – e.g. sloughing root cap cells.

regards,
Rosemary

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



On 9/5/08 3:33 AM, "Ignatius, Mike" <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
For those interested I can send a recently organized listing of dyes that have seen utility for plant cell work (respond off-line).  It is fairly long and includes an extensive bibliography.  We have never made it available before, but in the queue for future publication.

For example for Nuclei:  

·         Commonly used stains for nucleic acids in plants are DAPI, Hoechst 33258, YOYO®-1 iodide, YO-PRO®-1 iodide , acridine orange, ethidium bromide, and propidium iodide.  

But as Christian points out, these may not work.  However a new reagent has emerged that might be of interest.   It isn't a simple stain, but it readily labels dividing plant cell nuclei (alfalfa) in a workflow similar to BrDu but much improved with our new Click-iT technology.

Full text available at:  http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/bioprobes/bioprobes55.Par.79287.File.dat/bp55_1_EdU.pdf <http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/bioprobes/bioprobes55.Par.79287.File.dat/bp55_1_EdU.pdf>  
From that article:  Figure 5—Cell wall digestion is not required with Click-iT™ EdU. Medicago sativa (alfalfa) suspension cultures were incubated with 10 μM EdU for 3 hours. Cells were then fixed and permeabilized. EdU that had been incorporated into newly synthesized DNA was detected with the Click-iT™ EdU Alexa Fluor® 488 Imaging Kit (green fluorescence, Cat. no. C10083). Nuclei were stained with blue-fluorescent DAPI. Six confocal sections were overlaid onto a differential interference contrast image. Image contributed by Ferhan Ayaydin, Cellular Imaging Laboratory, Biological Research Center, Szeged, Hungary.

Kind Regards,

Mike Ignatius, Molecular Probes/Invitrogen

Eugene, Oregon, Site of the 2008 US Track and Field Trials.

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal <http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>  I'll echo Tobias here.  It's very tough to get any dye into living plant cells and keep them that way.  We've tried nearly every nuclear stain available along with various other markers and trackers, such as mitotracker, all with no luck in the hands of the users.

Have you thought about making stables with RFP in the nuclei?  Three channel sequencing then allows you to visualize GFP, RFP and cholorplasts in the far red channel.  

Christian