Image Processing
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We are working with some high content screening data and here is the problem:
1) We are labeling the membrane with WGA 2) The cells are dense so we have a honeycomb type structure 3) We are working with 96 well plates and we are trying to keep the exposure times low so our signal is about 300 intensity units with background of about 100 units.
We would like to use the WGA images to identify the plasma membrane at the periphery of the cells and use DAPI to identify the cells and get out cellular data.
I was wondering if anyone has any image processing tips for trying this. The problems are low signal to noise and that there is only one cell/cell junction for two or more DAPI nuclei so the algorithm has a hard time “splitting” up the cells.
Any thoughts would be appreciated.
We are using MetaMorph/MetaXpress software but are pretty adept in writing custom journals for this.
Claire M. Brown, PhD McGill University Life Sciences Complex Imaging Facility Director www.lifesciencescomplex.mcgill.ca/imaging
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Search the CONFOCAL archive at
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Having significant interest in such labeling and analytical tools, let me recommend a new dye that I have had reasonable success with from Molecular Probes. They just released a new "cell mask plasma membrane" dye that really does light up the cell surface (either on sparse or contacting cells). It is certainly best on live cells, but it does also fix (more or less). Doesn't work with any antibody permeabilization steps (that we've tried, yet). For the analysis, there are certainly excellent tools to mask the plasma membrane available, even for monolayers. the most direct (and affordable) one I know if is from a long time collaborator of mine, Jeff Price at Vala Sciences (www.valasciences.com). if you've a very, very large checkbook, Pipeline Pilot (accelrys.com) is also able to segment the honeycomb patterns you describe..... good luck! Mike Michael A. Mancini, Ph.D. Associate Professor Director, Integrated Microscopy Core Co-Director, Gulf Coast Consortium for Chemical Genomics Department of Molecular and Cellular Biology Baylor College of Medicine Houston, TX 77030 713 798 8952 voice 713 798 3017 fax 713 408 0179 cell On Nov 1, 2007, at 3:30 PM, Claire Brown wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Thanks again Mike. You are making my cursed IF existent more
tolerable. Have a nice weekend, Nik From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Mancini,
Michael A. Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Having significant interest in such labeling and analytical
tools, let me recommend a new dye that I have had reasonable success with from
Molecular Probes. They just released a new "cell mask plasma
membrane" dye that really does light up the cell surface (either on sparse
or contacting cells). It is certainly best on live cells, but it does
also fix (more or less). Doesn't work with any antibody permeabilization
steps (that we've tried, yet). For the analysis, there are certainly excellent tools to mask
the plasma membrane available, even for monolayers. the most direct (and
affordable) one I know if is from a long time collaborator of mine, Jeff Price
at Vala Sciences (www.valasciences.com).
if you've a very, very large checkbook, Pipeline Pilot (accelrys.com) is
also able to segment the honeycomb patterns you describe..... good luck! Mike ____________________ Michael A. Mancini, Ph.D. Associate Professor Director, Integrated Microscopy Core Co-Director, Gulf Coast Consortium for Chemical Genomics Department of Molecular and Cellular Biology Baylor College of Medicine Houston, TX 77030 713 798 8952 voice 713 798 3017 fax 713 408 0179 cell
On Nov 1, 2007, at 3:30 PM, Claire Brown wrote:
Search the CONFOCAL archive
at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We are working with some high content screening data and here is
the problem: 1) We are labeling the membrane with
WGA 2) The cells are dense so we have a
honeycomb type structure 3) We are working with 96 well
plates and we are trying to keep the exposure times low so our signal is about
300 intensity units with background of about 100 units. We would like to use the WGA images to identify the plasma
membrane at the periphery of the cells and use DAPI to identify the cells and
get out cellular data. I was wondering if anyone has any image processing tips for trying
this. The problems are low signal to noise and that there is only one cell/cell
junction for two or more DAPI nuclei so the algorithm has a hard time
“splitting” up the cells. Any thoughts would be appreciated. We are using MetaMorph/MetaXpress software but are pretty adept in
writing custom journals for this. Claire M. Brown, PhD McGill University Life Sciences Complex Imaging Facility Director |
 
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Alison J. North |
Membrane dye that works with multiphoton excitation?
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In reply to this post by Mancini, Michael A
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi all,
On a related note, we would like to find a general plasma membrane dye for living cells that is excited by multiphoton wavelengths around 860-890 nm but emits in the red region so that we can image it in GFP-labelled cells. One of our users has a big ball of cells (hundreds of microns thick, hence we need to use multiphoton), some of which are GFP-labelled, others not, so he needs a way to see all of the cells and yet distinguish the signal from the GFP. We tried Hoechst, but a plasma membrane dye would be much better as he'd like to see the outlines of the cells. I have tried searching online and calling the Probes hotline, but it seems that there is only limited information out there when it comes to multiphoton properties of dyes (please do prove me wrong, I would be very happy to hear of good sources!). So I was wondering - does anybody know about the multiphoton properties of any of these cool new dyes? The ideal situation would be to label the cells before they aggregate to form these big balls, so the dye would have to stay on there (without being toxic or affecting cell division) for days/weeks. Thanks in advance for any suggestions, and have a good weekend.... Alison Mancini, Michael A. wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- Alison J. North, Ph.D. Director of the Bio-Imaging Resource Center Research Assistant Professor The Rockefeller University 1230 York Avenue New York NY 10065 tel +1 212 327 7488 fax +1 212 327 7489 |
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