IMAGING OF CELLULOSE

[Dar Do]

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Hello,
Does anybody know of microscopy techniques for 3D imaging of paper or other
cellulose based fabrics? We have been trying with confocal microscopy and
did imaging of the surface of the outer layer of cellulose fibers, but were
unable to go through the fibers and image the underlying layers.
Best regards,
Dario
--
Dario Donnarumma
PhD student
personal blog <http://dariodonnarumma.blogspot.it/>
tel: [phone]
fax: [phone]
*Chemical Engineering @ the Micro-Scale (ChemEMS)*
*Dipartimento di Ingegneria Chimica, *
*dei Materiali e della Produzione Industriale (DICMAPI)*
*Università degli Studi di Napoli Federico II*
*Piazzale V. Tecchio 80 Napoli*
*I-80125 ITALIA*

[Carol Heckman]

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Dario-
There is a long history of imaging paper by scanning electron microscopy.  Again one is limited to viewing the surface, but with stereo microscopy, one can see the surface in 3D.
Carol Heckman
Bowling Green State University
________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Dar Do <[hidden email]>
Sent: Tuesday, May 6, 2014 9:58 AM
To: [hidden email]
Subject: IMAGING OF CELLULOSE

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Hello,
Does anybody know of microscopy techniques for 3D imaging of paper or other
cellulose based fabrics? We have been trying with confocal microscopy and
did imaging of the surface of the outer layer of cellulose fibers, but were
unable to go through the fibers and image the underlying layers.
Best regards,
Dario
--
Dario Donnarumma
PhD student
personal blog <http://dariodonnarumma.blogspot.it/>
tel: [phone]
fax: [phone]
*Chemical Engineering @ the Micro-Scale (ChemEMS)*
*Dipartimento di Ingegneria Chimica, *
*dei Materiali e della Produzione Industriale (DICMAPI)*
*Università degli Studi di Napoli Federico II*
*Piazzale V. Tecchio 80 Napoli*
*I-80125 ITALIA*

[John Shields]

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*****

A good method might be to use a  Xray CT scanner.  Most manufacturers
include software for analysis and volume computations..


On Tue, May 6, 2014 at 9:58 AM, Dar Do <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello,
> Does anybody know of microscopy techniques for 3D imaging of paper or other
> cellulose based fabrics? We have been trying with confocal microscopy and
> did imaging of the surface of the outer layer of cellulose fibers, but were
> unable to go through the fibers and image the underlying layers.
> Best regards,
> Dario
> --
> Dario Donnarumma
> PhD student
> personal blog <http://dariodonnarumma.blogspot.it/>
> tel: [phone]
> fax: [phone]
> *Chemical Engineering @ the Micro-Scale (ChemEMS)*
> *Dipartimento di Ingegneria Chimica, *
> *dei Materiali e della Produzione Industriale (DICMAPI)*
> *Università degli Studi di Napoli Federico II*
> *Piazzale V. Tecchio 80 Napoli*
> *I-80125 ITALIA*
>

[Oshel, Philip Eugene]

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Scanning electron microscopy works very well for imaging papers and
other such cellulosic products and their coatings.
Also, the paper (etc.) can be "fractured" in liquid nitrogen to produce
high quality edges to image the inner layers of the paper (etc.), and
the coating layers. Cutting tends to smear the surface over the cut edge
- this is more of an issue with coated papers.

Phil

> Hello,
> Does anybody know of microscopy techniques for 3D imaging of paper or other
> cellulose based fabrics? We have been trying with confocal microscopy and
> did imaging of the surface of the outer layer of cellulose fibers, but were
> unable to go through the fibers and image the underlying layers.
> Best regards,
> Dario

--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
[phone]

[Smith, Benjamin E.]

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Heyo,
    On thing you could try would be to soak the paper in fluorescein, wash out the excess, and then mount the paper in water or glycerol (which is closer to the actual refractive index of cellulose - 1.469).  Multi-photon will allow you look even further into the sample, if available.

    As others suggested, a normally more expensive option, but one that would allow you to see through the full depth of the sample with equal resolution, would be to use a microCT.

Cheers,
   Ben Smith

Benjamin E. Smith, Ph.D.
Samuel Roberts Noble Microscopy Laboratory
Research Scientist II
University of Oklahoma
Norman, OK 73019
E-mail: [hidden email]
Voice   405-325-4391
FAX  [phone]
http://www.microscopy.ou.edu/
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Dar Do [[hidden email]]
Sent: Tuesday, May 06, 2014 8:58 AM
To: [hidden email]
Subject: IMAGING OF CELLULOSE

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello,
Does anybody know of microscopy techniques for 3D imaging of paper or other
cellulose based fabrics? We have been trying with confocal microscopy and
did imaging of the surface of the outer layer of cellulose fibers, but were
unable to go through the fibers and image the underlying layers.
Best regards,
Dario
--
Dario Donnarumma
PhD student
personal blog <http://dariodonnarumma.blogspot.it/>
tel: [phone]
fax: [phone]
*Chemical Engineering @ the Micro-Scale (ChemEMS)*
*Dipartimento di Ingegneria Chimica, *
*dei Materiali e della Produzione Industriale (DICMAPI)*
*Università degli Studi di Napoli Federico II*
*Piazzale V. Tecchio 80 Napoli*
*I-80125 ITALIA*

[Craig Brideau]

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*****

Second harmonic microscopy does an excellent job for this sort of thing if
you happen to have access to a 2-photon microscope:
http://www.opticsinfobase.org/ol/abstract.cfm?uri=ol-28-22-2207

Craig Brideau


On Tue, May 6, 2014 at 9:50 AM, Smith, Benjamin E. <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Heyo,
>     On thing you could try would be to soak the paper in fluorescein, wash
> out the excess, and then mount the paper in water or glycerol (which is
> closer to the actual refractive index of cellulose - 1.469).  Multi-photon
> will allow you look even further into the sample, if available.
>
>     As others suggested, a normally more expensive option, but one that
> would allow you to see through the full depth of the sample with equal
> resolution, would be to use a microCT.
>
> Cheers,
>    Ben Smith
>
> Benjamin E. Smith, Ph.D.
> Samuel Roberts Noble Microscopy Laboratory
> Research Scientist II
> University of Oklahoma
> Norman, OK 73019
> E-mail: [hidden email]
> Voice   405-325-4391
> FAX  [phone]
> http://www.microscopy.ou.edu/
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on
> behalf of Dar Do [[hidden email]]
> Sent: Tuesday, May 06, 2014 8:58 AM
> To: [hidden email]
> Subject: IMAGING OF CELLULOSE
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello,
> Does anybody know of microscopy techniques for 3D imaging of paper or other
> cellulose based fabrics? We have been trying with confocal microscopy and
> did imaging of the surface of the outer layer of cellulose fibers, but were
> unable to go through the fibers and image the underlying layers.
> Best regards,
> Dario
> --
> Dario Donnarumma
> PhD student
> personal blog <http://dariodonnarumma.blogspot.it/>
> tel: [phone]
> fax: [phone]
> *Chemical Engineering @ the Micro-Scale (ChemEMS)*
> *Dipartimento di Ingegneria Chimica, *
> *dei Materiali e della Produzione Industriale (DICMAPI)*
> *Università degli Studi di Napoli Federico II*
> *Piazzale V. Tecchio 80 Napoli*
> *I-80125 ITALIA*
>

[Ko, Alex]

Yes I think second harmonic generation is useful in imaging cellulose in paper. In fact I had some experience in imaging paper ( cigarette paper) using two-photon fluorescence, second harmonic generation and coherent anti-stokes Raman.  Based on the material you may be able to see strong 2-photon signal and second-harmonic signal.  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: May-06-14 11:16 AM
To: [hidden email]
Subject: Re: IMAGING OF CELLULOSE

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Second harmonic microscopy does an excellent job for this sort of thing if you happen to have access to a 2-photon microscope:
http://www.opticsinfobase.org/ol/abstract.cfm?uri=ol-28-22-2207

Craig Brideau


On Tue, May 6, 2014 at 9:50 AM, Smith, Benjamin E. <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Heyo,
>     On thing you could try would be to soak the paper in fluorescein,
> wash out the excess, and then mount the paper in water or glycerol
> (which is closer to the actual refractive index of cellulose - 1.469).  
> Multi-photon will allow you look even further into the sample, if available.
>
>     As others suggested, a normally more expensive option, but one
> that would allow you to see through the full depth of the sample with
> equal resolution, would be to use a microCT.
>
> Cheers,
>    Ben Smith
>
> Benjamin E. Smith, Ph.D.
> Samuel Roberts Noble Microscopy Laboratory Research Scientist II
> University of Oklahoma Norman, OK 73019
> E-mail: [hidden email]
> Voice   405-325-4391
> FAX  [phone]
> http://www.microscopy.ou.edu/
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on
> behalf of Dar Do [[hidden email]]
> Sent: Tuesday, May 06, 2014 8:58 AM
> To: [hidden email]
> Subject: IMAGING OF CELLULOSE
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello,
> Does anybody know of microscopy techniques for 3D imaging of paper or
> other cellulose based fabrics? We have been trying with confocal
> microscopy and did imaging of the surface of the outer layer of
> cellulose fibers, but were unable to go through the fibers and image the underlying layers.
> Best regards,
> Dario
> --
> Dario Donnarumma
> PhD student
> personal blog <http://dariodonnarumma.blogspot.it/>
> tel: [phone]
> fax: [phone]
> *Chemical Engineering @ the Micro-Scale (ChemEMS)* *Dipartimento di
> Ingegneria Chimica, * *dei Materiali e della Produzione Industriale
> (DICMAPI)* *Università degli Studi di Napoli Federico II* *Piazzale V.
> Tecchio 80 Napoli*
> *I-80125 ITALIA*
>

[Lloyd Donaldson]

For confocal microscopy use congo red, pontamine fast scarlet or calcofluor to stain cellulose. Alternatively fluorescently labelled cellulose binding domains are available or you can just fluorescently label some cellulase enzyme. However visualising beyond the surface layer will be difficult. To see the cross-section you can embed the paper in resin and cut cross-sections and stain them with toluidine blue for brightfield microscopy. You can also polish the cross-section of embedded paper, stain with iodine vapour and image with SEM in backscattered mode (also useful for visualising clay fillers or coatings. There is an extensive literature on this topic mainly in specialist pulp & paper journals.


Dr Lloyd Donaldson
Project Leader – Microscopy & Wood Identification
Senior Scientist – Plant Cell Walls & Biomaterials
Scion – Forests, Products, Innovation
49 Sala Street, Rotorua 3010
New Zealand
Ph 07 343 5581
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Philip Oshel
Sent: Wednesday, 7 May 2014 2:48 a.m.
To: [hidden email]
Subject: Re: IMAGING OF CELLULOSE

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Scanning electron microscopy works very well for imaging papers and other such cellulosic products and their coatings.
Also, the paper (etc.) can be "fractured" in liquid nitrogen to produce high quality edges to image the inner layers of the paper (etc.), and the coating layers. Cutting tends to smear the surface over the cut edge
- this is more of an issue with coated papers.

Phil

> Hello,
> Does anybody know of microscopy techniques for 3D imaging of paper or
> other cellulose based fabrics? We have been trying with confocal
> microscopy and did imaging of the surface of the outer layer of
> cellulose fibers, but were unable to go through the fibers and image the underlying layers.
> Best regards,
> Dario

--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
[phone]



This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the  course of Scion’s usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.

[Craig Brideau]

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*****

I actually use paper as a crude test target for 2-p systems sometimes. The
white bleach in the paper fluoresces nicely, and I can create specific
emission wavelengths by tuning the paper to make sure that each channel is
detecting properly, at least for blue and green wavelengths.

Craig



On Tue, May 6, 2014 at 1:27 PM, Ko, Alex <[hidden email]> wrote:

> Yes I think second harmonic generation is useful in imaging cellulose in
> paper. In fact I had some experience in imaging paper ( cigarette paper)
> using two-photon fluorescence, second harmonic generation and coherent
> anti-stokes Raman.  Based on the material you may be able to see strong
> 2-photon signal and second-harmonic signal.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Craig Brideau
> Sent: May-06-14 11:16 AM
> To: [hidden email]
> Subject: Re: IMAGING OF CELLULOSE
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Second harmonic microscopy does an excellent job for this sort of thing if
> you happen to have access to a 2-photon microscope:
> http://www.opticsinfobase.org/ol/abstract.cfm?uri=ol-28-22-2207
>
> Craig Brideau
>
>
> On Tue, May 6, 2014 at 9:50 AM, Smith, Benjamin E. <[hidden email]
> >wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Heyo,
> >     On thing you could try would be to soak the paper in fluorescein,
> > wash out the excess, and then mount the paper in water or glycerol
> > (which is closer to the actual refractive index of cellulose - 1.469).
> > Multi-photon will allow you look even further into the sample, if
> available.
> >
> >     As others suggested, a normally more expensive option, but one
> > that would allow you to see through the full depth of the sample with
> > equal resolution, would be to use a microCT.
> >
> > Cheers,
> >    Ben Smith
> >
> > Benjamin E. Smith, Ph.D.
> > Samuel Roberts Noble Microscopy Laboratory Research Scientist II
> > University of Oklahoma Norman, OK 73019
> > E-mail: [hidden email]
> > Voice   405-325-4391
> > FAX  [phone]
> > http://www.microscopy.ou.edu/
> > ________________________________________
> > From: Confocal Microscopy List [[hidden email]] on
> > behalf of Dar Do [[hidden email]]
> > Sent: Tuesday, May 06, 2014 8:58 AM
> > To: [hidden email]
> > Subject: IMAGING OF CELLULOSE
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hello,
> > Does anybody know of microscopy techniques for 3D imaging of paper or
> > other cellulose based fabrics? We have been trying with confocal
> > microscopy and did imaging of the surface of the outer layer of
> > cellulose fibers, but were unable to go through the fibers and image the
> underlying layers.
> > Best regards,
> > Dario
> > --
> > Dario Donnarumma
> > PhD student
> > personal blog <http://dariodonnarumma.blogspot.it/>
> > tel: [phone]
> > fax: [phone]
> > *Chemical Engineering @ the Micro-Scale (ChemEMS)* *Dipartimento di
> > Ingegneria Chimica, * *dei Materiali e della Produzione Industriale
> > (DICMAPI)* *Università degli Studi di Napoli Federico II* *Piazzale V.
> > Tecchio 80 Napoli*
> > *I-80125 ITALIA*
> >
>

[Rosemary.White]

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*****

Yes, exactly as Lloyd says below.

There are two other things to overcome here - light scattering with depth
and light absorption with depth, and I think these are touched on in some
of the literature Lloyd refers to.

The inability to penetrate deeply - a depth/scattering problem - can be
ameliorated by matching the refractive index of the immersion medium with
that of the fabric. This might need to be determined empirically, but will
be around 1.5. Glycerol, at RI 1.47, is close.

However, there is also a tendency to overstain cellulose - it looks great
when it's really bright, but if it's very thoroughly stained, the fibres
will absorb more of the laser light, leaving less to penetrate into deeper
layers. You may find that with lighter staining, you can resolve some of
the deeper layers of the material. If your material has been dyed with
something very absorbant, this will be difficult....

cheers,
Rosemary White

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 7/05/14 5:55 AM, "Lloyd Donaldson" <[hidden email]>
wrote:

>For confocal microscopy use congo red, pontamine fast scarlet or
>calcofluor to stain cellulose. Alternatively fluorescently labelled
>cellulose binding domains are available or you can just fluorescently
>label some cellulase enzyme. However visualising beyond the surface layer
>will be difficult. To see the cross-section you can embed the paper in
>resin and cut cross-sections and stain them with toluidine blue for
>brightfield microscopy. You can also polish the cross-section of embedded
>paper, stain with iodine vapour and image with SEM in backscattered mode
>(also useful for visualising clay fillers or coatings. There is an
>extensive literature on this topic mainly in specialist pulp & paper
>journals.
>
>
>Dr Lloyd Donaldson
>Project Leader ­ Microscopy & Wood Identification
>Senior Scientist ­ Plant Cell Walls & Biomaterials
>Scion ­ Forests, Products, Innovation
>49 Sala Street, Rotorua 3010
>New Zealand
>Ph 07 343 5581
>www.scionresearch.com
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Philip Oshel
>Sent: Wednesday, 7 May 2014 2:48 a.m.
>To: [hidden email]
>Subject: Re: IMAGING OF CELLULOSE
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Scanning electron microscopy works very well for imaging papers and other
>such cellulosic products and their coatings.
>Also, the paper (etc.) can be "fractured" in liquid nitrogen to produce
>high quality edges to image the inner layers of the paper (etc.), and the
>coating layers. Cutting tends to smear the surface over the cut edge
>- this is more of an issue with coated papers.
>
>Phil
>
>> Hello,
>> Does anybody know of microscopy techniques for 3D imaging of paper or
>> other cellulose based fabrics? We have been trying with confocal
>> microscopy and did imaging of the surface of the outer layer of
>> cellulose fibers, but were unable to go through the fibers and image
>>the underlying layers.
>> Best regards,
>> Dario
>
>--
>Philip Oshel
>Microscopy Facility Supervisor
>Biology Department
>024C Brooks Hall
>Central Michigan University
>Mt. Pleasant, MI 48859
>[phone]
>
>
>
>This e-mail and any attachments may contain information which is
>confidential or subject to copyright. If you receive this e-mail in
>error, please delete it.
>Scion does not accept responsibility for anything in this e-mail which is
>not provided in the  course of Scion¹s usual business or for any computer
>virus, data corruption, interference or delay arising from this e-mail.

[Stanislav Vitha-2]

*****
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*****

I have recently done some confocal imaging of cotton fibers. For deeper
penetration, less scattering in single photon fluorescence imaging, rather
than Calcofluor White I suggest staining with orange or red-fluorescing
dyes - some have been already mentioned, like Congo Red. The fluorescent
version of Periodic Acid-Schiff staining works great (use propidium iodide
as pseudo-Schiff reagent; Truernit, E., H. Bauby, et al. (2008). "High-
resolution whole-mount imaging of three-dimensional tissue organization
and gene expression enables the study of Phloem development and
structure in Arabidopsis." Plant Cell 20(6): 1494-1503).
As an alternative to fluorescence staining, I have also used vapor fixation
with acrolein, which tends to induce very strong autofluorescence, at least
in cotton fibers.  This is useful if you do not want to expose the fibers to
water and induce swelling.
For imaging with oil immersion objectives I used 2,2-thioidiethanol as a
mounting medium with good success.
Staudt, T., M. C. Lang, et al. (2007). "2,2'-thiodiethanol: a new water
soluble mounting medium for high resolution optical microscopy."
Microscopy Research and Technique 70(1): 1-9.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University

[Guy Cox-2]

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Cellulose is a beta 1-4 linked glucan.   This gives it a degree of symmetry since each glucose molecule is reversed relative to its neighbour.  That in turn make it a relatively poor SH emitter.  Nevertheless it can be images, especially if nothing else is there.  Starch, an alpha 1-4 linked glucan has all the glucose molecules in the same orientation and is therefore non-symmetric and a much stronger emitter.  So imaging cellulose when starch is present will be difficult.  Also, in plant material, 3-photon fluorescence from lignin may be a confounding factor.  

                                                                   Guy
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Ko, Alex [[hidden email]]
Sent: 07 May 2014 05:27
To: [hidden email]
Subject: Re: IMAGING OF CELLULOSE

Yes I think second harmonic generation is useful in imaging cellulose in paper. In fact I had some experience in imaging paper ( cigarette paper) using two-photon fluorescence, second harmonic generation and coherent anti-stokes Raman.  Based on the material you may be able to see strong 2-photon signal and second-harmonic signal.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: May-06-14 11:16 AM
To: [hidden email]
Subject: Re: IMAGING OF CELLULOSE

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Second harmonic microscopy does an excellent job for this sort of thing if you happen to have access to a 2-photon microscope:
http://www.opticsinfobase.org/ol/abstract.cfm?uri=ol-28-22-2207

Craig Brideau


On Tue, May 6, 2014 at 9:50 AM, Smith, Benjamin E. <[hidden email]>wrote:

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> *****
>
> Heyo,
>     On thing you could try would be to soak the paper in fluorescein,
> wash out the excess, and then mount the paper in water or glycerol
> (which is closer to the actual refractive index of cellulose - 1.469).
> Multi-photon will allow you look even further into the sample, if available.
>
>     As others suggested, a normally more expensive option, but one
> that would allow you to see through the full depth of the sample with
> equal resolution, would be to use a microCT.
>
> Cheers,
>    Ben Smith
>
> Benjamin E. Smith, Ph.D.
> Samuel Roberts Noble Microscopy Laboratory Research Scientist II
> University of Oklahoma Norman, OK 73019
> E-mail: [hidden email]
> Voice   405-325-4391
> FAX  [phone]
> http://www.microscopy.ou.edu/
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on
> behalf of Dar Do [[hidden email]]
> Sent: Tuesday, May 06, 2014 8:58 AM
> To: [hidden email]
> Subject: IMAGING OF CELLULOSE
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello,
> Does anybody know of microscopy techniques for 3D imaging of paper or
> other cellulose based fabrics? We have been trying with confocal
> microscopy and did imaging of the surface of the outer layer of
> cellulose fibers, but were unable to go through the fibers and image the underlying layers.
> Best regards,
> Dario
> --
> Dario Donnarumma
> PhD student
> personal blog <http://dariodonnarumma.blogspot.it/>
> tel: [phone]
> fax: [phone]
> *Chemical Engineering @ the Micro-Scale (ChemEMS)* *Dipartimento di
> Ingegneria Chimica, * *dei Materiali e della Produzione Industriale
> (DICMAPI)* *Università degli Studi di Napoli Federico II* *Piazzale V.
> Tecchio 80 Napoli*
> *I-80125 ITALIA*
>

[Guy Cox-2]

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I totally endorse TDE as an embedding medium but I am puzzled by acrolein inducing fluorescence in cellulose.  Can someone explain the chemistry?  I thought acroleon reacted with proteins.

                                                            Guy
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Stanislav Vitha [[hidden email]]
Sent: 08 May 2014 00:04
To: [hidden email]
Subject: Re: IMAGING OF CELLULOSE

*****
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I have recently done some confocal imaging of cotton fibers. For deeper
penetration, less scattering in single photon fluorescence imaging, rather
than Calcofluor White I suggest staining with orange or red-fluorescing
dyes - some have been already mentioned, like Congo Red. The fluorescent
version of Periodic Acid-Schiff staining works great (use propidium iodide
as pseudo-Schiff reagent; Truernit, E., H. Bauby, et al. (2008). "High-
resolution whole-mount imaging of three-dimensional tissue organization
and gene expression enables the study of Phloem development and
structure in Arabidopsis." Plant Cell 20(6): 1494-1503).
As an alternative to fluorescence staining, I have also used vapor fixation
with acrolein, which tends to induce very strong autofluorescence, at least
in cotton fibers.  This is useful if you do not want to expose the fibers to
water and induce swelling.
For imaging with oil immersion objectives I used 2,2-thioidiethanol as a
mounting medium with good success.
Staudt, T., M. C. Lang, et al. (2007). "2,2'-thiodiethanol: a new water
soluble mounting medium for high resolution optical microscopy."
Microscopy Research and Technique 70(1): 1-9.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University

[Lloyd Donaldson]

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Cotton does contain small amounts of protein. It is unlikely that the cellulose would fluoresce.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Thursday, 8 May 2014 3:54 a.m.
To: [hidden email]
Subject: Re: IMAGING OF CELLULOSE

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I totally endorse TDE as an embedding medium but I am puzzled by acrolein inducing fluorescence in cellulose.  Can someone explain the chemistry?  I thought acroleon reacted with proteins.

                                                            Guy ________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Stanislav Vitha [[hidden email]]
Sent: 08 May 2014 00:04
To: [hidden email]
Subject: Re: IMAGING OF CELLULOSE

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I have recently done some confocal imaging of cotton fibers. For deeper penetration, less scattering in single photon fluorescence imaging, rather than Calcofluor White I suggest staining with orange or red-fluorescing dyes - some have been already mentioned, like Congo Red. The fluorescent version of Periodic Acid-Schiff staining works great (use propidium iodide as pseudo-Schiff reagent; Truernit, E., H. Bauby, et al. (2008). "High- resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of Phloem development and structure in Arabidopsis." Plant Cell 20(6): 1494-1503).
As an alternative to fluorescence staining, I have also used vapor fixation with acrolein, which tends to induce very strong autofluorescence, at least in cotton fibers.  This is useful if you do not want to expose the fibers to water and induce swelling.
For imaging with oil immersion objectives I used 2,2-thioidiethanol as a mounting medium with good success.
Staudt, T., M. C. Lang, et al. (2007). "2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy."
Microscopy Research and Technique 70(1): 1-9.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University



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[Rosemary.White]

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*****

Acridine orange - the "RNA stain" also stains cell walls very nicely and
uniformly, especially cotton cell walls. It overcomes the irritating
effect of laser polarisation seen when using calcofluor white.

Propidium iodide will stain the pectic components of cotton fibres, but
you also see the effect of laser polarisation there too.

On 8/05/14 6:00 AM, "Lloyd Donaldson" <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Cotton does contain small amounts of protein. It is unlikely that the
>cellulose would fluoresce.
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Guy Cox
>Sent: Thursday, 8 May 2014 3:54 a.m.
>To: [hidden email]
>Subject: Re: IMAGING OF CELLULOSE
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>I totally endorse TDE as an embedding medium but I am puzzled by acrolein
>inducing fluorescence in cellulose.  Can someone explain the chemistry?
>I thought acroleon reacted with proteins.
>
>                                                            Guy
>________________________________________
>From: Confocal Microscopy List [[hidden email]] on
>behalf of Stanislav Vitha [[hidden email]]
>Sent: 08 May 2014 00:04
>To: [hidden email]
>Subject: Re: IMAGING OF CELLULOSE
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>I have recently done some confocal imaging of cotton fibers. For deeper
>penetration, less scattering in single photon fluorescence imaging,
>rather than Calcofluor White I suggest staining with orange or
>red-fluorescing dyes - some have been already mentioned, like Congo Red.
>The fluorescent version of Periodic Acid-Schiff staining works great (use
>propidium iodide as pseudo-Schiff reagent; Truernit, E., H. Bauby, et al.
>(2008). "High- resolution whole-mount imaging of three-dimensional tissue
>organization and gene expression enables the study of Phloem development
>and structure in Arabidopsis." Plant Cell 20(6): 1494-1503).
>As an alternative to fluorescence staining, I have also used vapor
>fixation with acrolein, which tends to induce very strong
>autofluorescence, at least in cotton fibers.  This is useful if you do
>not want to expose the fibers to water and induce swelling.
>For imaging with oil immersion objectives I used 2,2-thioidiethanol as a
>mounting medium with good success.
>Staudt, T., M. C. Lang, et al. (2007). "2,2'-thiodiethanol: a new water
>soluble mounting medium for high resolution optical microscopy."
>Microscopy Research and Technique 70(1): 1-9.
>
>Stan Vitha
>Microscopy and Imaging Center
>Texas A&M University
>
>
>
>This e-mail and any attachments may contain information which is
>confidential or subject to copyright. If you receive this e-mail in
>error, please delete it.
>Scion does not accept responsibility for anything in this e-mail which is
>not provided in the  course of Scion's usual business or for any computer
>virus, data corruption, interference or delay arising from this e-mail.