GREAT CAREER OPPORTUNTIES

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Monica Wollman Monica Wollman
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GREAT CAREER OPPORTUNTIES

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

I have a client company that is looking for Confocal Microscopy Sales
Representatives in Virginia, Ohio/Pittsburg, Arizona, and Los Angeles.

They are also looking for a Field Applications Scientist for Southern CA.

This is with the leading manufacturer of confocal microscopy in the
industry. They offer a competitive base salary plus commissions and great
benefits.

Their ideal candidates will have strong confocal microscopy experience.
Someone from the lab who has strong hands on expereince in confocal would
be great.

If you are interested or have any questions please contact me ASAP!

Monica Wollman
Career Interchange, Inc.
818-597-0399
[hidden email]
Stephen Cody Stephen Cody
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MRC-1024 Pixel Dwell Time

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

Dear List,

We are just starting to play around with RICS (after Enrico Gratton). To
be able to calculate the diffusion co-efficients accurately we need to
know the pixel dwell time, and the time from one line to the next, when
using a Bio-Rad MRC-1024 at Zoom 10, on "SLOW" scan and a box size of
256 x 256.

Does anybody have this information?

I know at "Normal scan" at 756 x 512 each line is supposedly 6ms apart
(2ms scan, 2ms fly back, 2ms wasted turning around presumably). Does the
dwell time change at 256 x256 zoom 10? What happens to the line to line
timings?

I would appreciate any information people have on the slow scan dwell
time and on what happens to the normal scan dwell time when zoomed in
and using a smaller box size.

Regards

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria,      3050
Australia
Tel: 61 3 9341 3155    Fax: 61 3 9341 3104
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
http://www.ludwig.edu.au/confocal/Local/Booking_Hint.htm 



This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research.

Guy Cox Guy Cox
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Re: MRC-1024 Pixel Dwell Time

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

Stephen,

On the MRC 1024 the various cables are quite
accessible, so I'd suggest you just put a scope
on the scan line and/or the PMT output and
measure it.  Mind you Nuno Moreno has probably
already done that, so he may be able to post the
figure for you.

                                     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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody
Sent: Thursday, 16 August 2007 11:01 AM
To: [hidden email]
Subject: MRC-1024 Pixel Dwell Time

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

Dear List,

We are just starting to play around with RICS (after Enrico Gratton). To be able to calculate the diffusion co-efficients accurately we need to know the pixel dwell time, and the time from one line to the next, when using a Bio-Rad MRC-1024 at Zoom 10, on "SLOW" scan and a box size of
256 x 256.

Does anybody have this information?

I know at "Normal scan" at 756 x 512 each line is supposedly 6ms apart (2ms scan, 2ms fly back, 2ms wasted turning around presumably). Does the dwell time change at 256 x256 zoom 10? What happens to the line to line timings?

I would appreciate any information people have on the slow scan dwell time and on what happens to the normal scan dwell time when zoomed in and using a smaller box size.

Regards

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria,      3050
Australia
Tel: 61 3 9341 3155    Fax: 61 3 9341 3104
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
http://www.ludwig.edu.au/confocal/Local/Booking_Hint.htm 



This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research.


No virus found in this incoming message.
Checked by AVG Free Edition.
Version: 7.5.476 / Virus Database: 269.11.19/955 - Release Date: 15/08/2007 4:55 PM
 

No virus found in this outgoing message.
Checked by AVG Free Edition.
Version: 7.5.476 / Virus Database: 269.11.19/955 - Release Date: 15/08/2007 4:55 PM
 
Guy Cox Guy Cox
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Software flattening

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: MRC-1024 Pixel Dwell Time
One of our users is doing confocal / image analysis on
skink uterus - a cylinder of thin tissue which he dissects
out, opens and flattens on the slide.  However is is not
possible to get it completely flat, so it tends to get a bit
corrugated, which skews his quantification. It's east enough
to measure the curvature - does anyone know of software
which can computationally flatten the projection based on
this information?
 
                                                                  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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
 
Stephen Cody Stephen Cody
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Re: MRC-1024 Pixel Dwell Time

In reply to this post by Guy Cox
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks Guy,

This approach will give the line timing. However, I'm concerned that it
will not be possible to calculate pixel dwell time from this.

As I understand it, the signal measured on the oscilloscope will
determine when the mirrors start and finish each line. But I think the
image is a subset of this signal, from parameters that are set on each
individual confocal in the field during installation (there are various
offsets etc.). So if this is correct will the pixel dwell time vary from
instrument to instrument under a given imaging setup?

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria,      3050
Australia
Tel: 61 3 9341 3155    Fax: 61 3 9341 3104
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
http://www.ludwig.edu.au/confocal/Local/Booking_Hint.htm

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Guy Cox
Sent: Thursday, 16 August 2007 12:00 PM
To: [hidden email]
Subject: Re: MRC-1024 Pixel Dwell Time

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

Stephen,

On the MRC 1024 the various cables are quite
accessible, so I'd suggest you just put a scope
on the scan line and/or the PMT output and
measure it.  Mind you Nuno Moreno has probably
already done that, so he may be able to post the
figure for you.

                                     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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Stephen Cody
Sent: Thursday, 16 August 2007 11:01 AM
To: [hidden email]
Subject: MRC-1024 Pixel Dwell Time

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

Dear List,

We are just starting to play around with RICS (after Enrico Gratton). To
be able to calculate the diffusion co-efficients accurately we need to
know the pixel dwell time, and the time from one line to the next, when
using a Bio-Rad MRC-1024 at Zoom 10, on "SLOW" scan and a box size of
256 x 256.

Does anybody have this information?

I know at "Normal scan" at 756 x 512 each line is supposedly 6ms apart
(2ms scan, 2ms fly back, 2ms wasted turning around presumably). Does the
dwell time change at 256 x256 zoom 10? What happens to the line to line
timings?

I would appreciate any information people have on the slow scan dwell
time and on what happens to the normal scan dwell time when zoomed in
and using a smaller box size.

Regards

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy Ludwig Institute for Cancer
Research PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria,      3050
Australia
Tel: 61 3 9341 3155    Fax: 61 3 9341 3104
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
http://www.ludwig.edu.au/confocal/Local/Booking_Hint.htm 



This communication is intended only for the named recipient and may
contain information that is confidential, legally privileged or subject
to copyright; the Ludwig Institute for Cancer Research does not waiver
any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do
not necessarily reflect the views of the Ludwig Institute for Cancer
Research.


No virus found in this incoming message.
Checked by AVG Free Edition.
Version: 7.5.476 / Virus Database: 269.11.19/955 - Release Date:
15/08/2007 4:55 PM
 

No virus found in this outgoing message.
Checked by AVG Free Edition.
Version: 7.5.476 / Virus Database: 269.11.19/955 - Release Date:
15/08/2007 4:55 PM
 


This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research.

Jeremy Adler Jeremy Adler
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Re: Software flattening

In reply to this post by Guy Cox
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: MRC-1024 Pixel Dwell Time
a simple alternative is to transect the uterus and look at a cross section - no distortion whatsoever.
 
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden


From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One of our users is doing confocal / image analysis on
skink uterus - a cylinder of thin tissue which he dissects
out, opens and flattens on the slide.  However is is not
possible to get it completely flat, so it tends to get a bit
corrugated, which skews his quantification. It's east enough
to measure the curvature - does anyone know of software
which can computationally flatten the projection based on
this information?
 
                                                                  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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
 
Guy Cox Guy Cox
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Re: Software flattening

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: MRC-1024 Pixel Dwell Time
Well, sure, but it's hard to get the topology of vascular branching
from a cross-section.
 
                                                                            Guy


From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Thu 16/08/2007 5:51 PM
To: [hidden email]
Subject: Re: Software flattening

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
a simple alternative is to transect the uterus and look at a cross section - no distortion whatsoever.
 
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden


From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One of our users is doing confocal / image analysis on
skink uterus - a cylinder of thin tissue which he dissects
out, opens and flattens on the slide.  However is is not
possible to get it completely flat, so it tends to get a bit
corrugated, which skews his quantification. It's east enough
to measure the curvature - does anyone know of software
which can computationally flatten the projection based on
this information?
 
                                                                  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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
 
Guy Cox Guy Cox
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Re: MRC-1024 Pixel Dwell Time

In reply to this post by Stephen Cody
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: MRC-1024 Pixel Dwell Time
It's a bit worrying if there would be a significant variation in
dwell time of different instruments at the same scan rate,
but I can see that it's possible.
 
You can see what part of the line scan is collecting
signal by setting another channel on the scope to
show the signal with a uniform bright sample (e.g.
Chroma fluorescent plastic slide) under the scope
(or just using the transmission detector with no
sample, which might be easier).
 
                                                               Guy
 


From: Confocal Microscopy List on behalf of Stephen Cody
Sent: Thu 16/08/2007 5:16 PM
To: [hidden email]
Subject: Re: MRC-1024 Pixel Dwell Time

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

Thanks Guy,

This approach will give the line timing. However, I'm concerned that it
will not be possible to calculate pixel dwell time from this.

As I understand it, the signal measured on the oscilloscope will
determine when the mirrors start and finish each line. But I think the
image is a subset of this signal, from parameters that are set on each
individual confocal in the field during installation (there are various
offsets etc.). So if this is correct will the pixel dwell time vary from
instrument to instrument under a given imaging setup?

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria,      3050
Australia
Tel: 61 3 9341 3155    Fax: 61 3 9341 3104
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
http://www.ludwig.edu.au/confocal/Local/Booking_Hint.htm

-----Original Message-----
From: Confocal Microscopy List [[hidden email]] On
Behalf Of Guy Cox
Sent: Thursday, 16 August 2007 12:00 PM
To: [hidden email]
Subject: Re: MRC-1024 Pixel Dwell Time

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

Stephen,

On the MRC 1024 the various cables are quite
accessible, so I'd suggest you just put a scope
on the scan line and/or the PMT output and
measure it.  Mind you Nuno Moreno has probably
already done that, so he may be able to post the
figure for you.

                                     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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

-----Original Message-----
From: Confocal Microscopy List [[hidden email]] On
Behalf Of Stephen Cody
Sent: Thursday, 16 August 2007 11:01 AM
To: [hidden email]
Subject: MRC-1024 Pixel Dwell Time

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

Dear List,

We are just starting to play around with RICS (after Enrico Gratton). To
be able to calculate the diffusion co-efficients accurately we need to
know the pixel dwell time, and the time from one line to the next, when
using a Bio-Rad MRC-1024 at Zoom 10, on "SLOW" scan and a box size of
256 x 256.

Does anybody have this information?

I know at "Normal scan" at 756 x 512 each line is supposedly 6ms apart
(2ms scan, 2ms fly back, 2ms wasted turning around presumably). Does the
dwell time change at 256 x256 zoom 10? What happens to the line to line
timings?

I would appreciate any information people have on the slow scan dwell
time and on what happens to the normal scan dwell time when zoomed in
and using a smaller box size.

Regards

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy Ludwig Institute for Cancer
Research PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria,      3050
Australia
Tel: 61 3 9341 3155    Fax: 61 3 9341 3104
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
http://www.ludwig.edu.au/confocal/Local/Booking_Hint.htm



This communication is intended only for the named recipient and may
contain information that is confidential, legally privileged or subject
to copyright; the Ludwig Institute for Cancer Research does not waiver
any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do
not necessarily reflect the views of the Ludwig Institute for Cancer
Research.


No virus found in this incoming message.
Checked by AVG Free Edition.
Version: 7.5.476 / Virus Database: 269.11.19/955 - Release Date:
15/08/2007 4:55 PM


No virus found in this outgoing message.
Checked by AVG Free Edition.
Version: 7.5.476 / Virus Database: 269.11.19/955 - Release Date:
15/08/2007 4:55 PM



This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research.

Tony Collins-4 Tony Collins-4
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Re: Software flattening

In reply to this post by Guy Cox
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: MRC-1024 Pixel Dwell Time

Corrugated or curved? For a curved XZ image this ImageJ plugin may help:

http://rsb.info.nih.gov/ij/plugins/straighten.html

 

Best,

 

Tony

Tony J. Collins, Ph.D.
McMaster Biophotonics Facility
Dept. Biochemistry and Biomedical Sciences HSC 4H21A
McMaster University, Hamilton, ON, L8N 3Z5
(905) 525 9140 x28812(off.)/x26488(lab)
[hidden email]     www.macbiophotonics.ca


From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: August 16, 2007 3:11 AM
To: [hidden email]
Subject: [CONFOCAL] Software flattening

 

One of our users is doing confocal / image analysis on

skink uterus - a cylinder of thin tissue which he dissects

out, opens and flattens on the slide.  However is is not

possible to get it completely flat, so it tends to get a bit

corrugated, which skews his quantification. It's east enough

to measure the curvature - does anyone know of software

which can computationally flatten the projection based on

this information?

 

                                                                  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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

 

Sven Terclavers-2 Sven Terclavers-2
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Re: Software flattening

In reply to this post by Guy Cox
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: MRC-1024 Pixel Dwell Time

Not optimal either, but have you considered vibratome sections of +250µm thick? Or is it more important to look at vascular branching over a larger field than a small field, but in depth?

We’ve been using this approach a while ago for defining the branching topology of bloodvessels in the tail of a zebrafish embryo (which, by the way, we called the “sushi”-method).

Best,

 

Sven

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Saturday, August 18, 2007 10:49 AM
To: [hidden email]
Subject: Re: [CONFOCAL] Software flattening

 

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

Well, sure, but it's hard to get the topology of vascular branching

from a cross-section.

 

                                                                            Guy

 


From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Thu 16/08/2007 5:51 PM
To: [hidden email]
Subject: Re: Software flattening

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

a simple alternative is to transect the uterus and look at a cross section - no distortion whatsoever.

 

Jeremy Adler

Cell Biology

The Wenner-Gren Inst.

Arrhenius Laboratories E5

Stockholm University

Stockholm 106 91

Sweden

 


From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening

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

One of our users is doing confocal / image analysis on

skink uterus - a cylinder of thin tissue which he dissects

out, opens and flattens on the slide.  However is is not

possible to get it completely flat, so it tends to get a bit

corrugated, which skews his quantification. It's east enough

to measure the curvature - does anyone know of software

which can computationally flatten the projection based on

this information?

 

                                                                  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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

 



Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information.

Oshel, Philip Eugene Oshel, Philip Eugene
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Re: Software flattening

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

This isn't an optical method, but why not use
corrosion-casting and SEM? Perfuse the skink with
e.g. methylmethacrylate, then digest away the
tissue after polymerization. This leaves an
accurate cast of the blood vessels as they were
in life. The plastic captures details down to the
levels of endothethial cell boundaries in
capillaries.
This also creates an archival specimen for future studies.
Phil

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Not optimal either, but have you considered
vibratome sections of +250µm thick? Or is it more
important to look at vascular branching over a
larger field than a small field, but in depth?
We've been using this approach a while ago for
defining the branching topology of bloodvessels
in the tail of a zebrafish embryo (which, by the
way, we called the "sushi"-method).
Best,

Sven


From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf
Of Guy Cox
Sent: Saturday, August 18, 2007 10:49 AM
To: [hidden email]
Subject: Re: [CONFOCAL] Software flattening

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Well, sure, but it's hard to get the topology of vascular branching
from a cross-section.

                                                                             Guy


From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Thu 16/08/2007 5:51 PM
To: [hidden email]
Subject: Re: Software flattening
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
a simple alternative is to transect the uterus
and look at a cross section - no distortion
whatsoever.

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden


From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One of our users is doing confocal / image analysis on
skink uterus - a cylinder of thin tissue which he dissects
out, opens and flattens on the slide.  However is is not
possible to get it completely flat, so it tends to get a bit
corrugated, which skews his quantification. It's east enough
to measure the curvature - does anyone know of software
which can computationally flatten the projection based on
this information?

                                                                   Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    <http://www.guycox.com/optical.htm>http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
<http://www.guycox.net/>http://www.guycox.net



Disclaimer:
http://www.kuleuven.be/cwis/email_disclaimer.htm 
for more information.


--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
Guy Cox Guy Cox
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Re: Software flattening

In reply to this post by Guy Cox
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Sure, that works but it doesn't easily
solve the quantification problem.  You've
just got a different type of image to
untangle.  And the image quality from the
confocal approach is fine

We'll have a look at the  Image J plugin
that Tony suggested but it doesn't quite
seem to do the right thing either.  It's
a tricky issue.  Maybe geographical information
systems could help - finding the true length
and distance between forks in a road network in
undulating country would have the same problem.

                                   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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Philip Oshel
Sent: Wednesday, 22 August 2007 1:35 AM
To: [hidden email]
Subject: Re: Software flattening

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

This isn't an optical method, but why not use corrosion-casting and SEM? Perfuse the skink with e.g. methylmethacrylate, then digest away the tissue after polymerization. This leaves an accurate cast of the blood vessels as they were in life. The plastic captures details down to the levels of endothethial cell boundaries in capillaries.
This also creates an archival specimen for future studies.
Phil

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Not optimal either, but have you considered vibratome sections of +250µm thick? Or is it more important to look at vascular branching over a larger field than a small field, but in depth?
We've been using this approach a while ago for defining the branching topology of bloodvessels in the tail of a zebrafish embryo (which, by the way, we called the "sushi"-method).
Best,

Sven


From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Saturday, August 18, 2007 10:49 AM
To: [hidden email]
Subject: Re: [CONFOCAL] Software flattening

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Well, sure, but it's hard to get the topology of vascular branching from a cross-section.

                                                                             Guy


From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Thu 16/08/2007 5:51 PM
To: [hidden email]
Subject: Re: Software flattening
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
a simple alternative is to transect the uterus and look at a cross section - no distortion whatsoever.

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden


From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One of our users is doing confocal / image analysis on skink uterus - a cylinder of thin tissue which he dissects out, opens and flattens on the slide.  However is is not possible to get it completely flat, so it tends to get a bit corrugated, which skews his quantification. It's east enough to measure the curvature - does anyone know of software which can computationally flatten the projection based on this information?

                                                                   Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    <http://www.guycox.com/optical.htm>http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
<http://www.guycox.net/>http://www.guycox.net



Disclaimer:
http://www.kuleuven.be/cwis/email_disclaimer.htm
for more information.


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

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Jeremy Adler Jeremy Adler
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Re: Software flattening

In reply to this post by Guy Cox
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

your basic ploy of opening up the specimen effectively reduces its thickness by 50%, at the expense of heterogenous distortion.
you could deal with the distortion if you can relate the position of a large number of points in the final images to their location in the original specimen ~ except that this really requires images of the original, you can of course estimate/guess the original positions but there goes the accuracy

However, assuming that you can satifactorily image through half the speciemen depth, simply make one Z series of the unopened specimen, turn it over and make a second Z series, then align the two Z series. Distortion free, no assumptions. clearly you need to be able to turn the tissue over without distorting it.


1) transecting the tissue. having made a transection you can obviously make a Z series at the cut surface to produce an undistorted 3D dataset to whatever depth you can image.

2) if you want high res undistorted images of the whole organ, the whole specimen can be mounted on a microscope equipped with microtome and run through a section, image, section cycle / imaging the fresh surface of the tissue block / which ensures alignment. A few years ago a US based company offered a commercial version of this idea (I forget who) but similar approches have been implemented by others.

3) casting the vessels is a very good idea, you then mount them in an RI matched media. The advantage is that most the causes of image degradation with depth have been eliminated.

4) if you only want to make a topological measurement of blood vessels ~ topology, at least with the normal mathematical meaning,  is not altered by distortion.

5) stereology / 3D answers from 2D sections.

in your intitial request for suggestions it would have been worthwhile including the dimensions of the specimen and to have made it clear which measurements you wished to make.

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden



-----Original Message-----
From: Confocal Microscopy List on behalf of Guy Cox
Sent: Sat 18/08/2007 10:48
To: [hidden email]
Subject: Re: Software flattening
 
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Well, sure, but it's hard to get the topology of vascular branching
from a cross-section.
 
                                                                            Guy

________________________________

From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Thu 16/08/2007 5:51 PM
To: [hidden email]
Subject: Re: Software flattening


Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
a simple alternative is to transect the uterus and look at a cross section - no distortion whatsoever.
 
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden

________________________________

From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening


Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
One of our users is doing confocal / image analysis on
skink uterus - a cylinder of thin tissue which he dissects
out, opens and flattens on the slide.  However is is not
possible to get it completely flat, so it tends to get a bit
corrugated, which skews his quantification. It's east enough
to measure the curvature - does anyone know of software
which can computationally flatten the projection based on
this information?
 
                                                                  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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net <http://www.guycox.net/>  
 
George McNamara George McNamara
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Re: Software flattening

In reply to this post by Guy Cox
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Guy,

Try using Amira, http://www.tgs.com/products/amira.asp
Request the trial version ( http://www.tgs.com/download/trial_form.asp) and follow up with your rep to have all the modules activated. If you need more than the two week trial, you can likely get follow-up codes from the rep.

You didn't mention if the blood vessels are labeled intravitally (perfuse before sacrifice). I like fluorescent tomato lectin (specifically Vector Labs' biotin tomato lectin + Alexa 647 streptavidin). See also papers by Debbage in J Histochem Cytochem. I've seen others use DiI (someone here at UM), a red fluorescent dye in the resin used for vascular casts (image without digesting the tissue). McGrath and Daly have published several papers where they perfuse in a DNA counterstain to label all the endothelial and vascular smooth muscle cell nuclei.

George

At 04:48 AM 8/18/2007, you wrote:
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Well, sure, but it's hard to get the topology of vascular branching
from a cross-section.
 
                                                                            Guy


From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Thu 16/08/2007 5:51 PM
To: [hidden email]
Subject: Re: Software flattening

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
a simple alternative is to transect the uterus and look at a cross section - no distortion whatsoever.
 
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden


From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One of our users is doing confocal / image analysis on
skink uterus - a cylinder of thin tissue which he dissects
out, opens and flattens on the slide.  However is is not
possible to get it completely flat, so it tends to get a bit
corrugated, which skews his quantification. It's east enough
to measure the curvature - does anyone know of software
which can computationally flatten the projection based on
this information?
 
                                                                  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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
 




 

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office


Guy Cox Guy Cox
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Re: Software flattening

In reply to this post by Jeremy Adler
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: Software flattening
Jeremy,
 
           I guess I just haven't explained the problem very well 
since everyone who's replied on this topic seems to be
missing the point.  With the spread uterus we can get a
high-quality confocal 3D stack of the entire organ.  No problem
at all.  We can analyse it as a 3D volume too.  I'm sorry if I
didn't make this point clear.
 
         But what we'd like to do is analyse a 2D projection of the
flattened organ, since there is sophisticated software out there
which will measure things like branching topology.  Since it is
(unlike the mammalian uterus) a very thin layer of tissue this is
a very valid approach.  But since it doesn't lie flat the results will
be inaccurate.
 
          The corrugation which results from spreading it is really
easy to quantify - an XZ projection of the stack shows it very
accurately.  So we know just how much, and where, it is
corrugated - and it's only in one dimension.  So what we need
is software that will take our (e.g.) 512x512 projected image and
map it into (e.g.) a 512x768 image in accordance with our XZ
projection showing the corrugation.  But does such software
exist?
 
                                                                                Guy


From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Wed 22/08/2007 6:26 PM
To: [hidden email]
Subject: Re: Software flattening

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

your basic ploy of opening up the specimen effectively reduces its thickness by 50%, at the expense of heterogenous distortion.
you could deal with the distortion if you can relate the position of a large number of points in the final images to their location in the original specimen ~ except that this really requires images of the original, you can of course estimate/guess the original positions but there goes the accuracy

However, assuming that you can satifactorily image through half the speciemen depth, simply make one Z series of the unopened specimen, turn it over and make a second Z series, then align the two Z series. Distortion free, no assumptions. clearly you need to be able to turn the tissue over without distorting it.


1) transecting the tissue. having made a transection you can obviously make a Z series at the cut surface to produce an undistorted 3D dataset to whatever depth you can image.

2) if you want high res undistorted images of the whole organ, the whole specimen can be mounted on a microscope equipped with microtome and run through a section, image, section cycle / imaging the fresh surface of the tissue block / which ensures alignment. A few years ago a US based company offered a commercial version of this idea (I forget who) but similar approches have been implemented by others.

3) casting the vessels is a very good idea, you then mount them in an RI matched media. The advantage is that most the causes of image degradation with depth have been eliminated.

4) if you only want to make a topological measurement of blood vessels ~ topology, at least with the normal mathematical meaning,  is not altered by distortion.

5) stereology / 3D answers from 2D sections.

in your intitial request for suggestions it would have been worthwhile including the dimensions of the specimen and to have made it clear which measurements you wished to make.

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden



-----Original Message-----
From: Confocal Microscopy List on behalf of Guy Cox
Sent: Sat 18/08/2007 10:48
To: [hidden email]
Subject: Re: Software flattening

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

Well, sure, but it's hard to get the topology of vascular branching
from a cross-section.

                                                                            Guy

________________________________

From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Thu 16/08/2007 5:51 PM
To: [hidden email]
Subject: Re: Software flattening


Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
a simple alternative is to transect the uterus and look at a cross section - no distortion whatsoever.

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden

________________________________

From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening


Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One of our users is doing confocal / image analysis on
skink uterus - a cylinder of thin tissue which he dissects
out, opens and flattens on the slide.  However is is not
possible to get it completely flat, so it tends to get a bit
corrugated, which skews his quantification. It's east enough
to measure the curvature - does anyone know of software
which can computationally flatten the projection based on
this information?

                                                                  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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net <http://www.guycox.net/

Chris Tully Chris Tully
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Re: Software flattening

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

This may also miss the point entirely but I wanted to pass the idea along anyway...  (I am not aware of any software that will do what you describe :(   ).

Have you considered using the confocal as a line scanner?

Mount the uterus on an appropriately thin rod and then incrementally rotate the rod under the microscope.
Take an image or Z-stack at each rotation and then stitch the narrow bands of in focus information together into a single stack.

The basics of this approach are nicely illustrated here:
http://www.mediacy.com/index.aspx?page=ImageContest1st2002

Although Dr. Gabriel Corkidi Blanco's contact info is not listed on the page linked above, I am sure that Customer Service at Media Cybernetics would be able to put you in contact with him.

Chris Tully
Applications Engineer
Vashaw Scientific, Inc.
[hidden email]
800-874-9986 x339

On 8/22/07, Guy Cox <[hidden email]> wrote:
Search the CONFOCAL archive at <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Jeremy,
 
           I guess I just haven't explained the problem very well 
since everyone who's replied on this topic seems to be
missing the point.  With the spread uterus we can get a
high-quality confocal 3D stack of the entire organ.  No problem
at all.  We can analyse it as a 3D volume too.  I'm sorry if I
didn't make this point clear.
 
         But what we'd like to do is analyse a 2D projection of the
flattened organ, since there is sophisticated software out there
which will measure things like branching topology.  Since it is
(unlike the mammalian uterus) a very thin layer of tissue this is
a very valid approach.  But since it doesn't lie flat the results will
be inaccurate.
 
          The corrugation which results from spreading it is really
easy to quantify - an XZ projection of the stack shows it very
accurately.  So we know just how much, and where, it is
corrugated - and it's only in one dimension.  So what we need
is software that will take our (e.g.) 512x512 projected image and
map it into (e.g.) a 512x768 image in accordance with our XZ
projection showing the corrugation.  But does such software
exist?
 
                                                                                Guy


From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Wed 22/08/2007 6:26 PM

To: [hidden email]
Subject: Re: Software flattening

Search the CONFOCAL archive at
<a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

your basic ploy of opening up the specimen effectively reduces its thickness by 50%, at the expense of heterogenous distortion.
you could deal with the distortion if you can relate the position of a large number of points in the final images to their location in the original specimen ~ except that this really requires images of the original, you can of course estimate/guess the original positions but there goes the accuracy

However, assuming that you can satifactorily image through half the speciemen depth, simply make one Z series of the unopened specimen, turn it over and make a second Z series, then align the two Z series. Distortion free, no assumptions. clearly you need to be able to turn the tissue over without distorting it.


1) transecting the tissue. having made a transection you can obviously make a Z series at the cut surface to produce an undistorted 3D dataset to whatever depth you can image.

2) if you want high res undistorted images of the whole organ, the whole specimen can be mounted on a microscope equipped with microtome and run through a section, image, section cycle / imaging the fresh surface of the tissue block / which ensures alignment. A few years ago a US based company offered a commercial version of this idea (I forget who) but similar approches have been implemented by others.

3) casting the vessels is a very good idea, you then mount them in an RI matched media. The advantage is that most the causes of image degradation with depth have been eliminated.

4) if you only want to make a topological measurement of blood vessels ~ topology, at least with the normal mathematical meaning,  is not altered by distortion.

5) stereology / 3D answers from 2D sections.

in your intitial request for suggestions it would have been worthwhile including the dimensions of the specimen and to have made it clear which measurements you wished to make.

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden



-----Original Message-----
From: Confocal Microscopy List on behalf of Guy Cox
Sent: Sat 18/08/2007 10:48
To: [hidden email]
Subject: Re: Software flattening

Search the CONFOCAL archive at
<a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Well, sure, but it's hard to get the topology of vascular branching
from a cross-section.

                                                                            Guy

________________________________

From: Confocal Microscopy List on behalf of Jeremy Adler
Sent: Thu 16/08/2007 5:51 PM
To: [hidden email]
Subject: Re: Software flattening


Search the CONFOCAL archive at <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
a simple alternative is to transect the uterus and look at a cross section - no distortion whatsoever.

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden

________________________________

From: Confocal Microscopy List on behalf of Guy Cox
Sent: Thu 16/08/2007 09:11
To: [hidden email]
Subject: Software flattening


Search the CONFOCAL archive at <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One of our users is doing confocal / image analysis on
skink uterus - a cylinder of thin tissue which he dissects
out, opens and flattens on the slide.  However is is not
possible to get it completely flat, so it tends to get a bit
corrugated, which skews his quantification. It's east enough
to measure the curvature - does anyone know of software
which can computationally flatten the projection based on
this information?

                                                                  Guy

________________________________

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
   <a href="http://www.guycox.com/optical.htm" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
<a href="http://www.guycox.net" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://www.guycox.net <<a href="http://www.guycox.net/" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> http://www.guycox.net/> 


Ricardo Figueroa Ricardo Figueroa
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Re: Software flattening

In reply to this post by Guy Cox
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Guy,
This seam like a job for ImageJ, I wrote a plugin to see if I remember
the things I learnt at the EMBO course it should work as a starting
point it has some flaws but it will do the job, I will send it to you
off list. If any body else is interested e-mail me.

/Ricardo Figueroa

Guy Cox wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Jeremy,
>  
>            I guess I just haven't explained the problem very well
> since everyone who's replied on this topic seems to be
> missing the point.  With the spread uterus we can get a
> high-quality confocal 3D stack of the entire organ.  No problem
> at all.  We can analyse it as a 3D volume too.  I'm sorry if I
> didn't make this point clear.
>  
>          But what we'd like to do is analyse a 2D projection of the
> flattened organ, since there is sophisticated software out there
> which will measure things like branching topology.  Since it is
> (unlike the mammalian uterus) a very thin layer of tissue this is
> a very valid approach.  But since it doesn't lie flat the results will
> be inaccurate.
>  
>           The corrugation which results from spreading it is really
> easy to quantify - an XZ projection of the stack shows it very
> accurately.  So we know just how much, and where, it is
> corrugated - and it's only in one dimension.  So what we need
> is software that will take our (e.g.) 512x512 projected image and
> map it into (e.g.) a 512x768 image in accordance with our XZ
> projection showing the corrugation.  But does such software
> exist?
>  
>                                                                                
> Guy
>
> ------------------------------------------------------------------------
> *From:* Confocal Microscopy List on behalf of Jeremy Adler
> *Sent:* Wed 22/08/2007 6:26 PM
> *To:* [hidden email]
> *Subject:* Re: Software flattening
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> your basic ploy of opening up the specimen effectively reduces its
> thickness by 50%, at the expense of heterogenous distortion.
> you could deal with the distortion if you can relate the position of a
> large number of points in the final images to their location in the
> original specimen ~ except that this really requires images of the
> original, you can of course estimate/guess the original positions but
> there goes the accuracy
>
> However, assuming that you can satifactorily image through half the
> speciemen depth, simply make one Z series of the unopened specimen,
> turn it over and make a second Z series, then align the two Z series.
> Distortion free, no assumptions. clearly you need to be able to turn
> the tissue over without distorting it.
>
>
> 1) transecting the tissue. having made a transection you can obviously
> make a Z series at the cut surface to produce an undistorted 3D
> dataset to whatever depth you can image.
>
> 2) if you want high res undistorted images of the whole organ, the
> whole specimen can be mounted on a microscope equipped with microtome
> and run through a section, image, section cycle / imaging the fresh
> surface of the tissue block / which ensures alignment. A few years ago
> a US based company offered a commercial version of this idea (I forget
> who) but similar approches have been implemented by others.
>
> 3) casting the vessels is a very good idea, you then mount them in an
> RI matched media. The advantage is that most the causes of image
> degradation with depth have been eliminated.
>
> 4) if you only want to make a topological measurement of blood vessels
> ~ topology, at least with the normal mathematical meaning,  is not
> altered by distortion.
>
> 5) stereology / 3D answers from 2D sections.
>
> in your intitial request for suggestions it would have been worthwhile
> including the dimensions of the specimen and to have made it clear
> which measurements you wished to make.
>
> Jeremy Adler
> Cell Biology
> The Wenner-Gren Inst.
> Arrhenius Laboratories E5
> Stockholm University
> Stockholm 106 91
> Sweden
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List on behalf of Guy Cox
> Sent: Sat 18/08/2007 10:48
> To: [hidden email]
> Subject: Re: Software flattening
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Well, sure, but it's hard to get the topology of vascular branching
> from a cross-section.
>
>                                                                            
> Guy
>
> ________________________________
>
> From: Confocal Microscopy List on behalf of Jeremy Adler
> Sent: Thu 16/08/2007 5:51 PM
> To: [hidden email]
> Subject: Re: Software flattening
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> a simple alternative is to transect the uterus and look at a cross
> section - no distortion whatsoever.
>
> Jeremy Adler
> Cell Biology
> The Wenner-Gren Inst.
> Arrhenius Laboratories E5
> Stockholm University
> Stockholm 106 91
> Sweden
>
> ________________________________
>
> From: Confocal Microscopy List on behalf of Guy Cox
> Sent: Thu 16/08/2007 09:11
> To: [hidden email]
> Subject: Software flattening
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> One of our users is doing confocal / image analysis on
> skink uterus - a cylinder of thin tissue which he dissects
> out, opens and flattens on the slide.  However is is not
> possible to get it completely flat, so it tends to get a bit
> corrugated, which skews his quantification. It's east enough
> to measure the curvature - does anyone know of software
> which can computationally flatten the projection based on
> this information?
>
>                                                                   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
> ______________________________________________
> Phone +61 2 9351 3176     Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net <http://www.guycox.net/>
>