Lloyd, thank you for your reply. I forwarded this to the student to see if
she wants to order the book. It sounds like a resource she needs!

This is a student's Masters project. She is trying to find a way to
quantify what they tell me is the removal and redeposition of lignin on
the grass during processing. Based on a paper she showed up at my door
with, we used ultrarapid cryofixation and freeze drying for SEM. The
lignin is (supposedly) in small to very small droplets on the mashed-up
material. I really don't want her to have to take a zillion high-mag SEMs
and manually try to figure out how many lignin droplets there are for each
treatment. I am hoping (for her sake) that she can use fluorescence to get
a better idea of the distribution and coverage of the lignin. 

It looks like we will have a nice list of dyes to try!

Aloha,
Tina

> The most reliable way to image lignin is by autofluorescence. With 488
> nm excitation the lignin will fluoresce green but will be very dim
> compared to the chlorophyll autofluorescence (red). If you have a
> spectral confocal you should be able to distinguish the lignin by its
> autofluorescence spectrum. I can send you a spectrum for pine lignin
> but grass lignin will be different. There are some dyes you can try.
> Acriflavine, Berberine sulphate and basic fuchsin can be used to stain
> lignified tissues but the specificity for lignin depends on exactly
> how you prepare the tissue. Quenching can be a problem as well so you
> need to try different dilutions. Protocols for these dyes for use with
> wood can be found in "Fluoresce microscopy of wood" by Donaldson and
> Bond. Let me know if you would like to order a copy. Applications to
> wheat straw can be found in Donaldson L.A., Hague J.R.B., Snell R.
> 2001: Lignin distribution in coppice poplar, linseed and wheat straw.
> Holzforschung 55: 379-385.
> 
> There are also brightfield reagents that can be used to detect lignin
> but I suspect they may not work well for what you are doing. Try 1%
> phloroglucinol in 95% ethanol and look for a red colour.
> 
> If you want to remove the lignin try peracetic acid. I have used this
> sucessfully on wheat straw. Make a 50:50 mixture of glacial acetic
> acid and hydrogen peroxide. Cook the grass tissue in this reagent at
> about 90 deg C in a water bath for a few hours until the tissue is
> bleached. The tissue may disintegrate so wash carefully, you should be
> able to keep the tissue intact. Not sure how well this will work for
> SEM ?
> 
> If you have a TEM you can stain with 1% potassium permanganate in 1%
> sodium citrate for 5-6 mins. Embedding and sectioning grass will be a
> challenge.

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* Tina (Weatherby) Carvalho               * [log in to unmask]           * 
* Biological Electron Microscope Facility * (808) 956-6251                 *
* University of Hawaii at Manoa           * http://www.pbrc.hawaii.edu/bemf* 
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