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Guy,
I will have to disagree with you on the etiolated plant material. Large, healthy chloroplasts have very strong autofluorescence in the "far red" channel, and much smaller peak in the "GFP" range. When plants are stressed, not only do they not produce normal chlorophylls, but they can also produce an entire gambit of other pigments. These pigments may throw the autofluorescence curves all over the place making identification of GFP or YFP much more difficult. The same thing occurs in leaves which are stressed or experiencing hypersensitive responses to transformation.
The best bet is to keep the plants healthy as possible.
Christian
--- On Wed, 1/25/12, Guy Cox <[log in to unmask]> wrote:
From: Guy Cox <[log in to unmask]>
Subject: Re: Plant preparation for confocal microscopy
To: [log in to unmask]
Date: Wednesday, January 25, 2012, 10:50 PM
What I do with leaves is to peel off one or other epidermis. This enables you to flood the leaf and you can put both the peeled epidermis and the remaining leaf on a slide and image without air problems. Chloroplast fluorescence is going to be a problem but if you can work with etiolated leaves that might help.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
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Phone +61 2 9351 3176 Fax +61 2 9351 7682
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Christian
Sent: Thursday, 26 January 2012 3:18 AM
To: [log in to unmask]
Subject: Re: Plant preparation for confocal microscopy
Localization to chloroplasts at low expression levels can be
challe
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Localization to chloroplasts at low expression levels can be
challenging depending on your system and familiarity with dealing with sample
with autofluorescence. If you find you're having some difficulty, get
back to us, and as a group, I'm quite sure we can talk you through that part.
As for mounting samples, it depends on what plant you're dealing with.
Zea mays, you would probably want to hand cut cross sections, mount in water
under a coverslip. Arabidopsis, you will probably mount with the
"bottom" of the leaf towards the lens (are you upright or
inverted?) This will help avoid not only most of the trichomes which
cause more air bubble issues and refraction, but the epidermal cells under them
require deep z-series to see. Of course that is if you're working stable
lines, bombardment samples I usually view from the side which was
bombarded. Lastly, tobacco, if transiently expressing from Agrobacterium,
is often infiltrated from the bottom of the leaf, so that’s where I look, on
the bottom.
As for air bubbles… I do not fret the air bubbles at all,
but this is mostly because I’m NOT trying to collect the transmitted light
image. If you need it, then you might
worry about them, and I highly recommend checking out perfluorodecalin. I personally have not had luck with pulling a
weak vacuum , actually, what I do is even more simplistic.
I place the leaf sample (disks are too small to be worth the
effort in my hands) with the side I’d like to image up on a slide. Add water, and place a 22x60mm cover slip
over the sample, which is usually about 22x22mm. I then tap out the air bubbles I can see, and
place the entire thing on a “steel slide” and then place two small magnets on
top of the cover slip to hold the works flat.
If I avoid taking samples from area with large veins, I can do z-series
as long as I’d like without lateral shifting.
The only major problem I will warn you about is that you’re making your “slide”
much thicker and if you use an automated stage, this could be a real
problem. Secondly, and obviously, you
will not get a transmitted light image.
Of course this only works on an upright, but it has worked for at least
four years.
Good luck.
Christian
>Dear all,
>
>Do some of you could suggest how to prepare live sample of plant leaf for
>confocal microscopy. We don't have much idea how to
"sandwich" a leaf
>between slide and coverslip so that it is flat et not too thick for
>microscopy
>(and without destroying it)? What should we use as mounting media? What
>should we be aware of? Our plant will have YFP in chloroplastides.
>
>Thanks a lot in advance to all of you.
>
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