SIM or other super high resolution techniques in leaves?

[Christian-103]

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Listers,
I am looking for anyone with direct experience with better than confocal resolution techniques in live plants.  We are having quite a time of colocalizing a few proteins in chloroplasts or more to the point suborganelle localization.  The difficulty of fixation is also a barrier.  If anyone may have some suggestions or even relevant information regarding true leaves over hypocotyls, I'd be much appreciative.  It seems once again working plants makes life just a little more challenging.  Thank you.
Christian

[Stanislav Vitha-2]

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Hi Christian,
we have done SIM of GFP-tagged cytoskeletal protein in Arabidopsis
chloroplasts, but not in live tissue. I vacuum infiltrated the tissue with 3%
formaldehyde and then fixed using a microwave processor. Afterwards  
washed, infiltrated gradually with glycerol-based mountant (80% glycerol
buffered with p-penylenediamine, if I remember correctly) again assisted
with microwave irradiation to speed it up.

It worked quite well; The fluorescence held up well enough to survive
having the slide put in a mailer and shipped off for imaging (no
refrigeration).
If I was doing it again I would use one of those high-index mounting media
(not thiodiethanol, but there are some glycerol-based commercial products
with R.I. 1.52)  to reduce spherical aberration.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University