Photobleach (FRAP) problem in Zeiss 510Meta

[Rosemary.White]

All good suggestions.  I used to mount plant tissues in a home-made well
(hole drilled through glass slide, coverslip glued over hole with valap,
tissue placed onto coverslip in well).  Then I'd drop one third of a small
circular coverslip onto each end of the tissue, and glue in place with more
valap - taking care not to touch the tissue with the warm valap.  Then the
tissue was tissue anchored down and I could change solutions if needed, and
also microinject from above - on an inverted scope.

As John says, cyanoacrylate glue will work as a fixative and kill the
tissue.

You could also stabilise the actin cytoskeleton with a protein crosslinker,
like MBS, but as John says, if you're trying to image dynamics of a
relatively unperturbed system, modifying the cytoskeleton will probably
affect recovery into the bleached area.  Nuclei do move around, especially
if the cells have been perturbed in any way.

cheers,
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 15/05/10 1:56 AM, "John Runions" <[hidden email]> wrote:

> Also, I meant to say -
>
> There are much better ways to immobilize whole tissues and leaf pieces
> than with glue.  Try mounting the leaf piece in low-melting temperature
> agarose and then sealing the slide with VALAP (which is vaseline / lanolin
> / paraffin wax mixed 1:1:1 and heated to homogenize.  You use a hot metal
> spatula to 'draw' the warm liquid VALAP around the edges of the coverslip
> and then it sets and seals the coverslip without being toxic to living
> cells).   Tissue will stay happy and immobilized for hours in this setup.
>
>
> !!!! Don't use Krazy Glue or any solvent based glue on leaves. Even at
> some distance from the cells, this will kill them!!!
>
> I would suggest removing the actin cytoskeleton using, e.g. latrunculinB,
> if nuclear movement is an issue.  This, of course depends on what your
> experiment is.  If you are trying to observe FRAP of a protein or
> mechanism that transports on microfilaments, then depolymerising the actin
> won't work.
>
> John.
>
>
>
> Dear All,
>
> Thanks you very much for all of your input (comments, suggestions,
> protocols
> etc ) in regard to the photobleaching in our Zeiss510. The good news is
> that
> we got some progress on this but bad news is that a new question needs to
> be
> addressed: the movement of the bleaching subject/organelle.
>
> We are using transiently GFP-expressed tobacco leaves and want to bleach
> whole or partial nucleus. However the nucleus seems to move at lease about
> 1-2um in a 5min period. we tried to use larger pinhole and larger ROI but
> the plane of focus is still an issue (so only saw partial bleach). Zeiss
> specialist suggest to stick the leaves on the slide with krazy glue and
> surrounded by something to avoid the leave floating/vibrating (we are
> using
> inverted microscope). We tried but the movement was still noticeable. Have
> some of you meet a similar problem and how did it solved?
>
> Meantime I am want to install a macro that could track the cell movement
> (developed by Ellenberg). Any experience on it?
>
> Thank you in advance.
> --
> View this message in context:
> http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-
> in-Zeiss-510Meta-tp5038048p5051254.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions¹ lab web site
> (http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)
>
> Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)
>
> Oxford Brookes Master's in Bioimaging with Molecular Technology
> (http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions¹ lab web site
> (http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)
>
> Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)
>
> Oxford Brookes Master's in Bioimaging with Molecular Technology
> (http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)

[Stanislav Vitha]

Just like some others, I use a home-made coverslip-bottom chamber for FRAP
in leaf tissue. My chamber is a piece of 3 x 2 " plexiglass 3/8" or 10 mm
thick, with the short edges slightly beveled so that the microscope slide
holder can grab the plexiglass and hold it down. The piece has a rectangular
hole about 2 mm  narrower than the coverslip used (such as 22x40mm
coverslips). The coverslip is glued to the bottom side of the chamber with
silicone caulk (clear GE Silicone II from a hardware store) - this way if
the coverslip breaks, it can be scraped off with a razor blade and replaced
with a new one. The coverslips are individually measured with a micrometer
gauge and only those close to 0.170 mm in thickness are used (0.165 to 0.175
mm range).
The specimen, such as plant leaf tissue, is held in place with a small glass
"brick" made on a glass knife maker from a leftover glass strip used to make
knives for EM. One or two of those glass bricks are enough to flatten and
press the leaf down against the coverslip. I also like to vacuum infiltrate
the leaf with water to get rid of the air.
The picture of the chamber is on our web site:
http://microscopy.tamu.edu/lab-protocols/light-microscopy-protocols.html

In my case (FRAP of chloroplast localized cytoskeletal proteins) the
light-avoidance movement of chloroplasts is really a problem - laterally and
also going out of focus.  For the focus problem I just have to sit there and
try to re-focus - this means that I may have to discard some datapoints
during analysis because of the focus "hunting". Lateral movement is
corrected in acquired dataset by stack alignment using ImageJ and the
"stackreg" plugin; generaly the "Translation" or "Rigid Body" method works
the best for my samples, as long as you have a single moving object in the
image (crop the stack).
My FRAP experiments last only about 10 min. The leaf tissue seems reasonably
happy for about 1 hour.
I have the pinhole open to ~ 3 Airy, but that is to get more signal rather
than to increase the optical section thickness.

Stan

Dr. Stanislav Vitha      
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257
http://microscopy.tamu.edu


On Sun, 16 May 2010 16:12:18 +1000, Rosemary White <[hidden email]>
wrote:

>All good suggestions.  I used to mount plant tissues in a home-made well
>(hole drilled through glass slide, coverslip glued over hole with valap,
>tissue placed onto coverslip in well).  Then I'd drop one third of a small
>circular coverslip onto each end of the tissue, and glue in place with more
>valap - taking care not to touch the tissue with the warm valap.  Then the
>tissue was tissue anchored down and I could change solutions if needed, and
>also microinject from above - on an inverted scope.
>
>As John says, cyanoacrylate glue will work as a fixative and kill the
>tissue.
>
>You could also stabilise the actin cytoskeleton with a protein crosslinker,
>like MBS, but as John says, if you're trying to image dynamics of a
>relatively unperturbed system, modifying the cytoskeleton will probably
>affect recovery into the bleached area.  Nuclei do move around, especially
>if the cells have been perturbed in any way.
>
>cheers,
>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 15/05/10 1:56 AM, "John Runions" <[hidden email]> wrote:
>
>> Also, I meant to say -
>>
>> There are much better ways to immobilize whole tissues and leaf pieces
>> than with glue.  Try mounting the leaf piece in low-melting temperature
>> agarose and then sealing the slide with VALAP (which is vaseline / lanolin
>> / paraffin wax mixed 1:1:1 and heated to homogenize.  You use a hot metal
>> spatula to 'draw' the warm liquid VALAP around the edges of the coverslip
>> and then it sets and seals the coverslip without being toxic to living
>> cells).   Tissue will stay happy and immobilized for hours in this setup.
>>
>>
>> !!!! Don't use Krazy Glue or any solvent based glue on leaves. Even at
>> some distance from the cells, this will kill them!!!
>>
>> I would suggest removing the actin cytoskeleton using, e.g. latrunculinB,
>> if nuclear movement is an issue.  This, of course depends on what your
>> experiment is.  If you are trying to observe FRAP of a protein or
>> mechanism that transports on microfilaments, then depolymerising the actin
>> won't work.
>>
>> John.
>>
>>
>>
>> Dear All,
>>
>> Thanks you very much for all of your input (comments, suggestions,
>> protocols
>> etc ) in regard to the photobleaching in our Zeiss510. The good news is
>> that
>> we got some progress on this but bad news is that a new question needs to
>> be
>> addressed: the movement of the bleaching subject/organelle.
>>
>> We are using transiently GFP-expressed tobacco leaves and want to bleach
>> whole or partial nucleus. However the nucleus seems to move at lease about
>> 1-2um in a 5min period. we tried to use larger pinhole and larger ROI but
>> the plane of focus is still an issue (so only saw partial bleach). Zeiss
>> specialist suggest to stick the leaves on the slide with krazy glue and
>> surrounded by something to avoid the leave floating/vibrating (we are
>> using
>> inverted microscope). We tried but the movement was still noticeable. Have
>> some of you meet a similar problem and how did it solved?
>>
>> Meantime I am want to install a macro that could track the cell movement
>> (developed by Ellenberg). Any experience on it?
>>
>> Thank you in advance.
>> --
>> View this message in context:
>>

http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-

>> in-Zeiss-510Meta-tp5038048p5051254.html
>> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>>
>>
>>
>> *********************************
>> C. John Runions, Ph.D.
>> School of Life Sciences
>> Oxford Brookes University
>> Oxford, UK
>> OX3 0BP
>>
>> email:  [hidden email]
>> phone: [phone]
>>
>> Runions¹ lab web site
>> (http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)
>>
>> Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)
>>
>> Oxford Brookes Master's in Bioimaging with Molecular Technology
>> (http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)
>>
>>
>>
>> *********************************
>> C. John Runions, Ph.D.
>> School of Life Sciences
>> Oxford Brookes University
>> Oxford, UK
>> OX3 0BP
>>
>> email:  [hidden email]
>> phone: [phone]
>>
>> Runions¹ lab web site
>> (http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)
>>
>> Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)
>>
>> Oxford Brookes Master's in Bioimaging with Molecular Technology
>> (http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)

[Littlejohn, George]

Dear John and List,

For mounting whole live plant leaves, I use a perfluorocarbon as a mounting medium. I use perfluorodecalin (PFD, F2 Chemicals, no commercial interest), which improves imaging depth penetration by filling up the airspaces with a non-fluorescent medium of refractive index close to that of cytosol, thereby making the leaf more optically homogenous. Perfluorocarbons also have an unparalleled capacity for dissolving O2 and CO2, which allows gas exchange to happen, with minimal physiological impact on the mounted leaf / seedling. We can also get away with using minimally-bleaching laser intensity as the signal is not scattered as badly as with other mounting media which don’t fill the airspaces of the leaf.

In addition I use polydimethylsiloxane (Carolina Observation Gel, no commercial interest) to make a little gas-permeable gasket/chamber between slide and coverslip or two coverslips. PFD-incubated leaves/seedlings can be mounted well in these chambers. Observation Gel is a viscoelastic polymer, and as such allows for a good deal of customization for individual experimental requirements.

We've just published this use of PFD as a new method. The paper can be found at:
http://www3.interscience.wiley.com/journal/123335070/abstract.
I'm happy to provide a reprint or further details to any interested parties!

Hope this helps,
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]
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of John Runions [[hidden email]]
Sent: Friday, May 14, 2010 4:56 PM
To: [hidden email]
Subject: Re: New question - Photobleach (FRAP) problem in Zeiss 510Meta

Also, I meant to say -

There are much better ways to immobilize whole tissues and leaf pieces
than with glue.  Try mounting the leaf piece in low-melting temperature
agarose and then sealing the slide with VALAP (which is vaseline / lanolin
/ paraffin wax mixed 1:1:1 and heated to homogenize.  You use a hot metal
spatula to 'draw' the warm liquid VALAP around the edges of the coverslip
and then it sets and seals the coverslip without being toxic to living
cells).   Tissue will stay happy and immobilized for hours in this setup.


!!!! Don't use Krazy Glue or any solvent based glue on leaves. Even at
some distance from the cells, this will kill them!!!

I would suggest removing the actin cytoskeleton using, e.g. latrunculinB,
if nuclear movement is an issue.  This, of course depends on what your
experiment is.  If you are trying to observe FRAP of a protein or
mechanism that transports on microfilaments, then depolymerising the actin
won't work.

John.



Dear All,

Thanks you very much for all of your input (comments, suggestions,
protocols
etc ) in regard to the photobleaching in our Zeiss510. The good news is
that
we got some progress on this but bad news is that a new question needs to
be
addressed: the movement of the bleaching subject/organelle.

We are using transiently GFP-expressed tobacco leaves and want to bleach
whole or partial nucleus. However the nucleus seems to move at lease about
1-2um in a 5min period. we tried to use larger pinhole and larger ROI but
the plane of focus is still an issue (so only saw partial bleach). Zeiss
specialist suggest to stick the leaves on the slide with krazy glue and
surrounded by something to avoid the leave floating/vibrating (we are
using
inverted microscope). We tried but the movement was still noticeable. Have
some of you meet a similar problem and how did it solved?

Meantime I am want to install a macro that could track the cell movement
(developed by Ellenberg). Any experience on it?

Thank you in advance.
--
View this message in context:
http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5051254.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.



*********************************
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!)

Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)

Oxford Brookes Master's in Bioimaging with Molecular Technology
(http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)



*********************************
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!)

Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)

Oxford Brookes Master's in Bioimaging with Molecular Technology
(http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)

[Rosemary.White]

I've wondered about using these compounds as mounting agents to maintain
cell viability ever since the possibly apocryphal story of a mouse breathing
it (and in The Abyss, of course....), glad to see someone actually tried it
out!
cheers,
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 18/05/10 5:39 AM, "Littlejohn, George" <[hidden email]>
wrote:

> Dear John and List,
>
> For mounting whole live plant leaves, I use a perfluorocarbon as a mounting
> medium. I use perfluorodecalin (PFD, F2 Chemicals, no commercial interest),
> which improves imaging depth penetration by filling up the airspaces with a
> non-fluorescent medium of refractive index close to that of cytosol, thereby
> making the leaf more optically homogenous. Perfluorocarbons also have an
> unparalleled capacity for dissolving O2 and CO2, which allows gas exchange to
> happen, with minimal physiological impact on the mounted leaf / seedling. We
> can also get away with using minimally-bleaching laser intensity as the signal
> is not scattered as badly as with other mounting media which don¹t fill the
> airspaces of the leaf.
>
> In addition I use polydimethylsiloxane (Carolina Observation Gel, no
> commercial interest) to make a little gas-permeable gasket/chamber between
> slide and coverslip or two coverslips. PFD-incubated leaves/seedlings can be
> mounted well in these chambers. Observation Gel is a viscoelastic polymer, and
> as such allows for a good deal of customization for individual experimental
> requirements.
>
> We've just published this use of PFD as a new method. The paper can be found
> at:
> http://www3.interscience.wiley.com/journal/123335070/abstract.
> I'm happy to provide a reprint or further details to any interested parties!
>
> Hope this helps,
> 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]
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On Behalf Of
> John Runions [[hidden email]]
> Sent: Friday, May 14, 2010 4:56 PM
> To: [hidden email]
> Subject: Re: New question - Photobleach (FRAP) problem in Zeiss 510Meta
>
> Also, I meant to say -
>
> There are much better ways to immobilize whole tissues and leaf pieces
> than with glue.  Try mounting the leaf piece in low-melting temperature
> agarose and then sealing the slide with VALAP (which is vaseline / lanolin
> / paraffin wax mixed 1:1:1 and heated to homogenize.  You use a hot metal
> spatula to 'draw' the warm liquid VALAP around the edges of the coverslip
> and then it sets and seals the coverslip without being toxic to living
> cells).   Tissue will stay happy and immobilized for hours in this setup.
>
>
> !!!! Don't use Krazy Glue or any solvent based glue on leaves. Even at
> some distance from the cells, this will kill them!!!
>
> I would suggest removing the actin cytoskeleton using, e.g. latrunculinB,
> if nuclear movement is an issue.  This, of course depends on what your
> experiment is.  If you are trying to observe FRAP of a protein or
> mechanism that transports on microfilaments, then depolymerising the actin
> won't work.
>
> John.
>
>
>
> Dear All,
>
> Thanks you very much for all of your input (comments, suggestions,
> protocols
> etc ) in regard to the photobleaching in our Zeiss510. The good news is
> that
> we got some progress on this but bad news is that a new question needs to
> be
> addressed: the movement of the bleaching subject/organelle.
>
> We are using transiently GFP-expressed tobacco leaves and want to bleach
> whole or partial nucleus. However the nucleus seems to move at lease about
> 1-2um in a 5min period. we tried to use larger pinhole and larger ROI but
> the plane of focus is still an issue (so only saw partial bleach). Zeiss
> specialist suggest to stick the leaves on the slide with krazy glue and
> surrounded by something to avoid the leave floating/vibrating (we are
> using
> inverted microscope). We tried but the movement was still noticeable. Have
> some of you meet a similar problem and how did it solved?
>
> Meantime I am want to install a macro that could track the cell movement
> (developed by Ellenberg). Any experience on it?
>
> Thank you in advance.
> --
> View this message in context:
> http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-
> in-Zeiss-510Meta-tp5038048p5051254.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions¹ lab web site
> (http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)
>
> Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)
>
> Oxford Brookes Master's in Bioimaging with Molecular Technology
> (http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email:  [hidden email]
> phone: [phone]
>
> Runions¹ lab web site
> (http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)
>
> Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)
>
> Oxford Brookes Master's in Bioimaging with Molecular Technology
> (http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)