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Jeremy
Assuming pollen is similar to other cell wall material RI should be 1.53 to 1.55. We have successfully imaged pollen in both glycerol and immersion oil although immersion oil was better to see inside the grains. For very spiny pollen there may be some problems with air bubbles on the surface of the grains so warming the immersion oil on the slide may improve the mounting.
The material that is fluorescing is sporopollenin located in the exine or outer layer of the pollen wall. The exact composition of this material is not known but it is though to be a mixture of phenylpropanoids, phenolics, fatty acids and carotenoids.
Regards
Dr Lloyd Donaldson - Senior Scientist
Cellwall Biotechnology Centre
SCION - Next generation biomaterials
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Does anyone have a plausible RI for pollen grains, or the RI of a medium that produces the best images ?
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden
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From: Confocal Microscopy List on behalf of Guy Cox
Sent: Tue 6/17/2008 03:44
To: [hidden email]
Subject: Re: Pollen grain
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I think it's phenolics that are responsible for the fluorescence. It
is pretty broad but you can see different peaks (depending on the
pollen).
I had thought that spiky pollen grains would be a good TIRF test
sample but not so - the fluorescence is deep enough below the
surface to be out of TIRF range.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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From: Confocal Microscopy List [[hidden email]] On Behalf Of Shalin Mehta
Sent: Tuesday, 17 June 2008 10:08 AM
To: [hidden email]
Subject: Re: Pollen grain
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I also keep one of the pollen grain slides in the confocal room. When a user
says that there's something wrong with the scope, I ask them if they've checked
will the pollen slide. Since they inevitably haven't, I tell them to do that
first, and then come back if there's a problem with the microscope. Amazingly,
they rarely return.
This is interesting, do pollen's have nice excitation-emission properties? Do they have specific peaks or just broad excitation and emission? What would be the underlying biological organelle/molecule responsible for autofluorescence?
Cheers
Shalin
Kristi DeCourcy
--
~~~~~~~~~~~~~~~~~~~~~~~~~
Shalin Mehta
mobile: +65-90694182
blog: shalin.wordpress.com <http://shalin.wordpress.com/>
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Bioimaging Lab, Block-E3A, #7-10
Div of Bioengineering, NUS Singapore 117574
website: http://www.bioeng.nus.edu.sg/optbioimaging/colin/index.html
Liver Cancer Functional Genomics Lab, #6-05
National Cancer Centre, Singapore 169610
http://www.nccs.com.sg/researcher/02_04d.htm
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