observation of GFP fusion by LSCM in root tips - observation of GFP fusion by LSCM in root tips (May/08/2008 )

Hi dear brothers & sisters:
Who has suggestions on improving the observation of GFP-target gene fusion by laser scanning confocal microscope in root tips of transgenic Arabidopsis? I found that, due to the thickness of root tips, cell iamges that from different layers overlap, which is a severe problem I think.
Any of your idea would be greatly appreciated .Thanks in advance!
Paul

In my experience, the observation of GFP in plant tissues is quite a pain.
First advice I can give you is to be careful about auto-fluorescent tissues. The most auto-fluorescent tissues are the light-coloured ones (white). In that case, even the best filter combination can lead you to mistakes. Dead cells are highly fluorescent too.

Moreover, long exposures to light can severly affect sensitive tissues such as root tips. In case you want to establish a conservative assay, you must optimize the scanning in order to keep your cells alive.

About the layers, probabily I dind't get the point. In what planes you get the overlap? Can't you elaborate your images once that they have been taken (for example eliminatig all the undesired/redundant information)?
Alternatively: can't you make sections of your samples???

Just curious: which GFP gene are you using?

Hope I've been of some help!
ILA

Thank you very much ILA!
Maybe I am not so experienced as you in LSCM observation that I even do not understand eaxctly the mean of your question "In what planes you get the overlap?". Could you please give me a more detailed explaination?
The GFP gene I used is the enhanced eGFP, which works well in my previous works using onion epidermal systems.
I also has tried sectioning of root tips before, but you know, it's difficult beacuse root tips of arabidopsis is so small! And also I doubt whether the GFP signals will "leak" from its native positions during the treatments such as fixation, dehydration etc when preparing sections.
Looking forward to hearing from you soon!

Ok, let's make a step behind: what do you mean with overlap?
If your sample is thick (and you can't make smaller sections) you can observe one plane at time proceeding along the axis. In this way you will obtain different images that together "compose" the whole specimen.
For a nice tutorial and more theoretical details, take a look here: http://micro.magnet.fsu.edu/primer/virtual...ocal/index.html

GFP signals will of course be lost if you try with fixed sections. You need to look at live cells, even if they are not going to survive the assay.
I'm using mgfp5, and it's quite complicated to cope with.

Hope this time my explanation was better!
Cheers!

Hi, ila and peixumol.
I have seen your discussion and am eager to request you. Now I use eGFP and mGFP5 to transfer into orchid. Our goal is to obtain green fluorescence orchids. And I want to request you this goal whether can be realized. And any experience or suggestion will be grateful. Thanks !