reflection contrast

[Ghafar Sarvestani]

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Dear All,
I am trying to collect some Z sections on a thick unstained sample from
spinal disc using reflective mode. I would appreciate any input from your
own experiences with reflection contrast. The disc material is mainly made
of very dense connective tissues and some cross-ties collagen fibres. I am
mainly interested to see the detail of collagen fibres between the
intervertebral disc lamellae. Obviously the sample is not
immunofluorescently labelled. Do you think normal H&E staining would help in
reflective mode of confocal imaging? What laser line would work better, Ar
488, or green HeNe 546?
With regards,
Ghafar

***********************************************
Ghafar T Sarvestani, PhD
Hanson Institute for Medical Research
Hanson Building level 2, Room 2-42-N
IMVS, Frome Road, Adelaide SA 5000

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[Urs Utzinger]

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Without having practical experience I would assume:

Scattering:
Smaller particles / longer wavelengths scatter more uniformly and since you
would want the opposite (more forward and backward scattering) you might
want to go with shorter wavelengths. However it's likely that the gain is
minimal as the ratio between fiber diameter and wavelength does not change
much from Ar to HeNe and you might not get more light back.

Absorption:
What is more critical is absorption and the more you have, the more
attenuation (less penetration) you will have. In that sense H&E staining
would not be useful and a red HeNe or a laser between 700 and 900 nms would
be best.

Perhaps a silver or gold stain could help for reflection.

I hope someone with practical experience will respond.

Urs


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Ghafar Sarvestani
Sent: Wednesday, January 09, 2008 7:59 PM
To: [hidden email]
Subject: reflection contrast

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear All,
I am trying to collect some Z sections on a thick unstained sample from
spinal disc using reflective mode. I would appreciate any input from your
own experiences with reflection contrast. The disc material is mainly made
of very dense connective tissues and some cross-ties collagen fibres. I am
mainly interested to see the detail of collagen fibres between the
intervertebral disc lamellae. Obviously the sample is not
immunofluorescently labelled. Do you think normal H&E staining would help in
reflective mode of confocal imaging? What laser line would work better, Ar
488, or green HeNe 546?
With regards,
Ghafar

***********************************************
Ghafar T Sarvestani, PhD
Hanson Institute for Medical Research
Hanson Building level 2, Room 2-42-N
IMVS, Frome Road, Adelaide SA 5000

Tel: [phone]
Fax: [phone]
Email:[hidden email]

This e-mail may contain confidential information, which also
may be legally privileged.  Only the intended recipient(s) may access, use,
distribute or copy this e-mail.  If this e-mail is received in error, please
inform the sender by return e-mail and delete the original.  It is the
recipient's responsibility to check the e-mail and any attached files for
viruses.

[Guy Cox]

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Ghafar,

         I'd try second harmonic if I were you.
It works best in transmission but lots of people
have used it in back-propagated mode.

                                        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)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ghafar Sarvestani
Sent: Thursday, 10 January 2008 1:59 PM
To: [hidden email]
Subject: reflection contrast

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear All,
I am trying to collect some Z sections on a thick unstained sample from spinal disc using reflective mode. I would appreciate any input from your own experiences with reflection contrast. The disc material is mainly made of very dense connective tissues and some cross-ties collagen fibres. I am mainly interested to see the detail of collagen fibres between the intervertebral disc lamellae. Obviously the sample is not immunofluorescently labelled. Do you think normal H&E staining would help in reflective mode of confocal imaging? What laser line would work better, Ar 488, or green HeNe 546?
With regards,
Ghafar

***********************************************
Ghafar T Sarvestani, PhD
Hanson Institute for Medical Research
Hanson Building level 2, Room 2-42-N
IMVS, Frome Road, Adelaide SA 5000

Tel: [phone]
Fax: [phone]
Email:[hidden email]

This e-mail may contain confidential information, which also may be legally privileged.  Only the intended recipient(s) may access, use, distribute or copy this e-mail.  If this e-mail is received in error, please inform the sender by return e-mail and delete the original.  It is the recipient's responsibility to check the e-mail and any attached files for viruses.

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[George McNamara]

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Hi Ghafar,

If you have an empty position on the emission side, try each laser
line in turn. Collagen fibrils can show up looking like needles in
live cells 3D matrix experiments (see also J. Paul Robinson's
papers). Different laser lines will have different artifacts -
easiest to see these by focusing on the coverglass - medium interface.

George


At 09:58 PM 1/9/2008, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear All,
>I am trying to collect some Z sections on a thick unstained sample from
>spinal disc using reflective mode. I would appreciate any input from your
>own experiences with reflection contrast. The disc material is mainly made
>of very dense connective tissues and some cross-ties collagen fibres. I am
>mainly interested to see the detail of collagen fibres between the
>intervertebral disc lamellae. Obviously the sample is not
>immunofluorescently labelled. Do you think normal H&E staining would help in
>reflective mode of confocal imaging? What laser line would work better, Ar
>488, or green HeNe 546?
>With regards,
>Ghafar
>
>***********************************************
>Ghafar T Sarvestani, PhD
>Hanson Institute for Medical Research
>Hanson Building level 2, Room 2-42-N
>IMVS, Frome Road, Adelaide SA 5000
>
>Tel: [phone]
>Fax: [phone]
>Email:[hidden email]
>
>This e-mail may contain confidential information, which also
>may be legally privileged.  Only the intended recipient(s) may access, use,
>distribute or copy this e-mail.  If this e-mail is received in error, please
>inform the sender by return e-mail and delete the original.  It is the
>recipient's responsibility to check the e-mail and any attached files for
>viruses.

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp (see
Analytical Imaging Core Facility)

[Gary Laevsky-2]

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Hi Ghafar,

We did quite a bit of reflected confocal on a homemade system at
Northeastern.  We put a quarter wave plate in the path to reduce back
reflections from optics/glass.

In terms of wavelength, a good deal depends on the coatings of your
optics.  We got our best images around 750nm.

Second harmomic also produced some pretty nice images of the collagen.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Ghafar Sarvestani
Sent: Wednesday, January 09, 2008 9:59 PM
To: [hidden email]
Subject: reflection contrast

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear All,
I am trying to collect some Z sections on a thick unstained sample from
spinal disc using reflective mode. I would appreciate any input from
your
own experiences with reflection contrast. The disc material is mainly
made
of very dense connective tissues and some cross-ties collagen fibres. I
am
mainly interested to see the detail of collagen fibres between the
intervertebral disc lamellae. Obviously the sample is not
immunofluorescently labelled. Do you think normal H&E staining would
help in
reflective mode of confocal imaging? What laser line would work better,
Ar
488, or green HeNe 546?
With regards,
Ghafar

***********************************************
Ghafar T Sarvestani, PhD
Hanson Institute for Medical Research
Hanson Building level 2, Room 2-42-N
IMVS, Frome Road, Adelaide SA 5000

Tel: [phone]
Fax: [phone]
Email:[hidden email]

This e-mail may contain confidential information, which also
may be legally privileged.  Only the intended recipient(s) may access,
use,
distribute or copy this e-mail.  If this e-mail is received in error,
please
inform the sender by return e-mail and delete the original.  It is the
recipient's responsibility to check the e-mail and any attached files
for
viruses.