Confocal Microscopy List

Need fluorescent stain for lignin

Classic

List

Threaded

7 messages Options

Tina Carvalho

Need fluorescent stain for lignin

I apologize for cross-posting...

A student here wants to find a fluorescent dye for lignin so that she can
see it on her (mostly red) autofluorescing grass. The grass will be
subjected to various harsh treatments to rid it of the lignin, but so far
it seems that all the treatments result in lignin being redopisited on the
material in droplets. We can see the droplets with SEM, but would like to
find a good way to quantify the amount of remaining cells walls
(cellulose) that is covered by lignin droplets. I think the ultimate goal
is to use image analysis to get some numbers.

Any ideas?

Aloha from sunny and warm Honolulu,
Tina

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

Dale Callaham

Re: Need fluorescent stain for lignin

Hi Tina,

What is the source of the red autofluorescence? Is it from chlorophyll?
If so, Chlorophyll can be extracted by acetone if that is a suitable
prep -you said harsh.....

The standard Schiff's Reagent (pararosanilin based) used in the PAS
reaction gives a strong specificity for untreated lignins (no periodic
acid step) - fluoresces red - a standard Rhodamine-type filter set is fine.

The animated helix thing on my Facility webpage is such staining - the
secondary thickenings of a common weed xylem (makes a great 3D and
durable confocal sample....).
http://www.bio.umass.edu/microscopy/

I have notes if this seems useful.

Dale

Tina Carvalho wrote:

> I apologize for cross-posting...
>
> A student here wants to find a fluorescent dye for lignin so that she can
> see it on her (mostly red) autofluorescing grass. The grass will be
> subjected to various harsh treatments to rid it of the lignin, but so far
> it seems that all the treatments result in lignin being redopisited on the
> material in droplets. We can see the droplets with SEM, but would like to
> find a good way to quantify the amount of remaining cells walls
> (cellulose) that is covered by lignin droplets. I think the ultimate goal
> is to use image analysis to get some numbers.
>
> Any ideas?
>
> Aloha from sunny and warm Honolulu,
> Tina
>
> ****************************************************************************
> * Tina (Weatherby) Carvalho * [hidden email] *
> * Biological Electron Microscope Facility * (808) 956-6251 *
> * University of Hawaii at Manoa * http://www.pbrc.hawaii.edu/bemf*
> ****************************************************************************

Lloyd Donaldson

Re: Need fluorescent stain for lignin

In reply to this post by Tina Carvalho

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.

Hope that helps. Let me know if you have further questions.

Regards

Dr Lloyd Donaldson
Senior Scientist
Scion - Next Generation Biomaterials
49 Sala St. Rotorua
Private Bag 3020, Rotorua 3046
NEW ZEALAND
Ph: 64 7 343 5581
Fx: 64 7 343 5507
www.scionresearch.com

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tina Carvalho
Sent: Thursday, 26 November 2009 7:08 a.m.
To: [hidden email]
Subject: Need fluorescent stain for lignin

I apologize for cross-posting...

A student here wants to find a fluorescent dye for lignin so that she can
see it on her (mostly red) autofluorescing grass. The grass will be
subjected to various harsh treatments to rid it of the lignin, but so far
it seems that all the treatments result in lignin being redopisited on the
material in droplets. We can see the droplets with SEM, but would like to
find a good way to quantify the amount of remaining cells walls
(cellulose) that is covered by lignin droplets. I think the ultimate goal
is to use image analysis to get some numbers.

Any ideas?

Aloha from sunny and warm Honolulu,
Tina

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

Tina Carvalho

Re: Need fluorescent stain for lignin

In reply to this post by Dale Callaham

We are getting red from chlorophyll, but also other cell wall material. We
were guessing cellulose...? Filter paper fluoresces because...? (I'm not a
plant person.) The goal here is to segregate lignin from cellulose with
imaging techniques. I think the ultimate goal is to get rid of all the
lignin so that cellulose can be extracted on an industrial scale, and they
tell me the lignin is a barrier to this.

We tried Acridine Orange, which is supposed to bind to lignin, but it
seemed to bind to everything else as well.

Aloha,
Tina

> What is the source of the red autofluorescence? Is it from chlorophyll?
> If so, Chlorophyll can be extracted by acetone if that is a suitable
> prep -you said harsh.....
>
> The standard Schiff's Reagent (pararosanilin based) used in the PAS
> reaction gives a strong specificity for untreated lignins (no periodic
> acid step) - fluoresces red - a standard Rhodamine-type filter set is fine.
>
> The animated helix thing on my Facility webpage is such staining - the
> secondary thickenings of a common weed xylem (makes a great 3D and
> durable confocal sample....).
> http://www.bio.umass.edu/microscopy/
>
> I have notes if this seems useful.

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

Tina Carvalho

Re: Need fluorescent stain for lignin

In reply to this post by Lloyd Donaldson

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.

Lloyd Donaldson

Re: Need fluorescent stain for lignin

In reply to this post by Tina Carvalho

Tina

Pure cellulose doesn't fluoresce. Filter paper fluoresces either from hemicellulose or because it contains fluorescent brightener (to make it look white). Filter paper might also have some lignin or degradation products. Hemicellulose fluorescence is very weak, I wouldn't expect this to be significant in a leaf.
If you are seeing red fluorescence in your leaf cell walls it is possible the chlorophyll has "stained" the cell walls if the samples have been in solvent. There could be all sorts of fluorescent components in your leaves.

There are several ways to stain cellulose. You can use calcofluor (UV excitation) but this will overlap completely with lignin autofluorescence, or you can use cellulose binding domains which come with HIS tags so you can label them. These are available commercially.

Regards - Lloyd

Dr Lloyd Donaldson
Senior Scientist
Scion - Next Generation Biomaterials
49 Sala St. Rotorua
Private Bag 3020, Rotorua 3046
NEW ZEALAND
Ph: 64 7 343 5581
Fx: 64 7 343 5507
www.scionresearch.com

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tina Carvalho
Sent: Thursday, 26 November 2009 9:30 a.m.
To: [hidden email]
Subject: Re: Need fluorescent stain for lignin

We are getting red from chlorophyll, but also other cell wall material. We
were guessing cellulose...? Filter paper fluoresces because...? (I'm not a
plant person.) The goal here is to segregate lignin from cellulose with
imaging techniques. I think the ultimate goal is to get rid of all the
lignin so that cellulose can be extracted on an industrial scale, and they
tell me the lignin is a barrier to this.

We tried Acridine Orange, which is supposed to bind to lignin, but it
seemed to bind to everything else as well.

Aloha,
Tina

G. Esteban Fernandez

Re: Need fluorescent stain for lignin

People in my lab use phloroglucinol reagent and Maule reagent to stain
lignin in root cross-sections. See Sibout et al., Plant Cell 17:2059
(2005).

--
G. Esteban Fernandez, Ph.D.

Associate Director
Molecular Cytology Core Facility
University of Missouri
120 Bond Life Sciences Center
Columbia, MO 65211

http://www.biotech.missouri.edu/mcc

(573)882-4895
(573)884-9676 fax