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It is often a problem with leaves to keep them flat. What we do is stick them to the slide with double sided tape and then mount in water or glycerol.


Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification
Scion - Forests . Products . Innovation
Private Bag 3020, Rotorua
49 Sala Street, Rotorua 3010
New Zealand

Ph: 64 7 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Rosemary White
Sent: Wednesday, 25 January 2012 11:02 a.m.
To: [log in to unmask]
Subject: Re: Plant preparation for confocal microscopy

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Hi Monique,

You can image with little preparation, just mount in water and add a
coverslip. Some common problems:

Air bubbles within the tissue - bad if you want to look below the
epidermis - lots of reflection from the air/water (i.e. air/cell wall)
interface.
The laser illumination may stimulate photosynthesis so bubbles develop in
the leaf tissue - within cells as well as between them, and may accumulate
under the coverslip.
Both of these problems may be alleviated somewhat by gentle vacuum
infiltration with water or buffer before imaging.

Another option is to image the leaf dry without coverslip - you don't get
the bubble problems then, but still need to worry about reflection from
the leaf surface and about the leaf drying out during imaging.

Best is to just try it - since it's confocal, you don't have to worry
about leaf thickness. If you need to image deeper layers, just cut a thick
cross-section and mount in water; there will be enough undamaged cells
there.

good luck,
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 25/01/12 8:31 AM, "Vasseur Monique" <[log in to unmask]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>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.
>
>Monique



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