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 <[log in to unmask]>
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 [log in to unmask]
>
>
>
>On 15/05/10 1:56 AM, "John Runions" <[log in to unmask]> 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:  [log in to unmask]
>> phone: +44 (0) 1865 483 964
>> 
>> 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:  [log in to unmask]
>> phone: +44 (0) 1865 483 964
>> 
>> 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)