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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] |
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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. |
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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 |
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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 |
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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. |
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In reply to this post by Guy Cox
Search the CONFOCAL archive at
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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 |
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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 |
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In reply to this post by Stephen Cody
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
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 |
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In reply to this post by Guy Cox
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
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. From: 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 |
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In reply to this post by Guy Cox
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 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 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 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 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information. |
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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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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 No virus found in this incoming message. Checked by AVG Free Edition. Version: 7.5.484 / Virus Database: 269.12.1/963 - Release Date: 20/08/2007 5:44 PM No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.484 / Virus Database: 269.12.1/965 - Release Date: 21/08/2007 4:02 PM |
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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/> |
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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 George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office |
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In reply to this post by Jeremy Adler
Search the CONFOCAL archive at
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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 |
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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 |
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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/> > |
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