Yes, there will be starch in tea leaves, but it will be recognizably
confined to the chloroplasts so if caffeine is in crystalline granules
they could be recognizable. However, the cellulose of the cell walls is
also highly birefringent and this would create a pretty high background.
And the caffeine may well be in solution even if it is in discrete
vesicles. This is a tricky one - would caffeine have a recognizable
signature in Raman microscopy?
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Martin Wessendorf
Sent: Wednesday, 2 June 2010 1:32 AM
To: [log in to unmask]
Subject: Re: Immuno-labelling leaf material. Please help!
Dear Guy et al--
Never having done a bit of work in plants, I have no idea of the
considerations involved here. However, based on the chemistry of
caffeine--i.e., it's soluble in polar solvents and has no free primary
amines or carboxyl groups--it seems as if it'd be unlikely to stay in
place without fixation but hard to fix covalently. The only way I know
that might fix the stuff would be with McLean-Nakane fixation, where the
ketones would get oxidized to aldehydes, but that would probably render
it unrecognizable by the antibody.
Is there any way that polarized microscopy could be used, since caffeine
is birefringent? I realize that starch is also birefringent, but is
there likely to be much starch in tea leaves?
Martin
Guy Cox wrote:
> I have immuno-labelled leaf epidermal peels. I found that mild
> paraformaldehyde fixation and cellulase treatment were necessary to
get
> penetration. I can't answer for what that will do for caffeine, since
I
> don't know how easy it is to leach (though we do use boiling water to
> make coffee or tea) but short of microinjection I can't see what would
> do better. Anything that would allow the antibody to enter would
surely
> allow caffeine to dissolve, it's just a question of how fast it
> dissolves at room temperature. Another point - leaf tissue after this
> sort of treatment is not robust, so I found it was essential to fix
the
> epidermis to coverslips with poly-l-lysine. Otherwise, even if they
> looked OK, the intracellular structure was scrambled.
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
> http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
[mailto:[log in to unmask]]
> On Behalf Of Shane van Breda
> Sent: Monday, 31 May 2010 7:18 PM
> To: [log in to unmask]
> Subject: Immuno-labelling leaf material. Please help!
>
> I am currently trying to determine the tissue and intracellular
> localisation of
> caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine
> antibody Ig
> fraction that is directly labelled to FTC-ED.
>
> I am struggling to find literature of labelling leaves with
> flourescently labelled
> antibodies. Would it be possible to label fresh material without
fixing
> or
> dehydration (I am worried these steps will dissolve or remove caffeine
> from
> the leaf).
>
> Alternatively I will be doing high pressure freezing and freeze
> substitution (in a
> solvent that caffeine does not dissovle in) or maybe freeze drying in
3
> weeks
> to embedd my samples. But how would I ensure labelling with my
antibody?
>
> and will I still need to premeablise my cells?
>
> Another idea I have is to determine if caffeine has any type of
> autoflourescence and then use this to aid in visualising it.
>
> Any ideas, references or protocols that could give me a starting point
> will be
> much appreciated!
--
Martin Wessendorf, Ph.D. office: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [log in to unmask]
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