What open-source software did you use in the past and would like to use again?

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Cory Quammen Cory Quammen
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What open-source software did you use in the past and would like to use again?

Dear list,

Open-source and freely available software such as ImageJ, the Open
Microscopy Environment, and Micro-Manager are examples of popular,
actively-developed, and well-supported applications used within the
microscopy community. Presumably other popular software tools useful
in microscopy and image analysis have been created in the past, but
are no longer developed or supported because the original development
team lacks funding or interest. My question for the list is:

What abandoned or neglected microscopy and image analysis/processing
software have you used in the past and would love to use again if it
were under active development, but currently do not use because it
either does not run or does not do something you want it to? By active
development, I mean that a team of developers would release updated
versions of the software from time to time with new features and bug
fixes.

Thanks for your feedback,
Cory

--
Cory Quammen
Center for Computer Integrated Systems for Microscopy and Manipulation (CISMM)
Department of Computer Science
University of North Carolina at Chapel Hill
http://www.cs.unc.edu/~cquammen
Stephen Cody-2 Stephen Cody-2
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Re: What open-source software did you use in the past and would like to use again?

Dear Cory,
 
Confocal Assistant by Todd Berlji would fall into this category. It was developed for Win 3.1, had a nice GUI and was very popular. It will run uder XP but only supports short file names. You can download a copy at: http://www.ludwig.edu.au/confocal/Links.html
 
Cheers
Stephen Cody

2009/4/23 Cory Quammen <[hidden email]>
Dear list,

Open-source and freely available software such as ImageJ, the Open
Microscopy Environment, and Micro-Manager are examples of popular,
actively-developed, and well-supported applications used within the
microscopy community. Presumably other popular software tools useful
in microscopy and image analysis have been created in the past, but
are no longer developed or supported because the original development
team lacks funding or interest. My question for the list is:

What abandoned or neglected microscopy and image analysis/processing
software have you used in the past and would love to use again if it
were under active development, but currently do not use because it
either does not run or does not do something you want it to? By active
development, I mean that a team of developers would release updated
versions of the software from time to time with new features and bug
fixes.

Thanks for your feedback,
Cory

--
Cory Quammen
Center for Computer Integrated Systems for Microscopy and Manipulation (CISMM)
Department of Computer Science
University of North Carolina at Chapel Hill
http://www.cs.unc.edu/~cquammen



Tina Carvalho Tina Carvalho
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Re: What open-source software did you use in the past and would like to use again?

Hey, that was what I was going to say! Confocal Assistant. Really liked
it. Hated the short file names.

Aloha,
Tina

> Confocal Assistant by Todd Berlji would fall into this category. It was
> developed for Win 3.1, had a nice GUI and was very popular. It will run uder
> XP but only supports short file names. You can download a copy at:
> http://www.ludwig.edu.au/confocal/Links.html
>
> Cheers
> Stephen Cody
>
> 2009/4/23 Cory Quammen <[hidden email]>
>
> > Dear list,
> >
> > Open-source and freely available software such as ImageJ, the Open
> > Microscopy Environment, and Micro-Manager are examples of popular,
> > actively-developed, and well-supported applications used within the
> > microscopy community. Presumably other popular software tools useful
> > in microscopy and image analysis have been created in the past, but
> > are no longer developed or supported because the original development
> > team lacks funding or interest. My question for the list is:
> >
> > What abandoned or neglected microscopy and image analysis/processing
> > software have you used in the past and would love to use again if it
> > were under active development, but currently do not use because it
> > either does not run or does not do something you want it to? By active
> > development, I mean that a team of developers would release updated
> > versions of the software from time to time with new features and bug
> > fixes.
> >
> > Thanks for your feedback,
> > Cory
> >
> > --
> > Cory Quammen
> > Center for Computer Integrated Systems for Microscopy and Manipulation
> > (CISMM)
> > Department of Computer Science
> > University of North Carolina at Chapel Hill
> > http://www.cs.unc.edu/~cquammen
> >
>

****************************************************************************
* Tina (Weatherby) Carvalho               * [hidden email]           *
* Biological Electron Microscope Facility * (808) 956-6251                 *
* University of Hawaii at Manoa           * http://www.pbrc.hawaii.edu/bemf* 
****************************************************************************
Stephen Cody-2 Stephen Cody-2
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Re: What open-source software did you use in the past and would like to use again?

In reply to this post by Stephen Cody-2
Dear Cory,
 
Sorry, Confocal assistant is not open source, but is freely available. I misread your original message.
 
Steve

2009/4/23 Stephen Cody <[hidden email]>
Dear Cory,
 
Confocal Assistant by Todd Berlji would fall into this category. It was developed for Win 3.1, had a nice GUI and was very popular. It will run uder XP but only supports short file names. You can download a copy at: http://www.ludwig.edu.au/confocal/Links.html
 
Cheers
Stephen Cody

2009/4/23 Cory Quammen <[hidden email]>

Dear list,

Open-source and freely available software such as ImageJ, the Open
Microscopy Environment, and Micro-Manager are examples of popular,
actively-developed, and well-supported applications used within the
microscopy community. Presumably other popular software tools useful
in microscopy and image analysis have been created in the past, but
are no longer developed or supported because the original development
team lacks funding or interest. My question for the list is:

What abandoned or neglected microscopy and image analysis/processing
software have you used in the past and would love to use again if it
were under active development, but currently do not use because it
either does not run or does not do something you want it to? By active
development, I mean that a team of developers would release updated
versions of the software from time to time with new features and bug
fixes.

Thanks for your feedback,
Cory

--
Cory Quammen
Center for Computer Integrated Systems for Microscopy and Manipulation (CISMM)
Department of Computer Science
University of North Carolina at Chapel Hill
http://www.cs.unc.edu/~cquammen






--
Stephen H. Cody
Dale Callaham Dale Callaham
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Re: What open-source software did you use in the past and would like to use again?

In reply to this post by Tina Carvalho
Confocal Assistant was very nice for Biorad files in particular and the
things we tended to do with those images and stacks. But in addition to
the 8+3 filename limitation it seemed to also have a restricted space
allocated for the full path and filename string and if files were more
than a few directories deep with typical names it seemed to fail to open
the files - would show them in the browser but couldn't open; if the
files were simply moved closer to the root level, it opened them fine.

Dale

Tina Carvalho wrote:

> Hey, that was what I was going to say! Confocal Assistant. Really liked
> it. Hated the short file names.
>
> Aloha,
> Tina
>
>> Confocal Assistant by Todd Berlji would fall into this category. It was
>> developed for Win 3.1, had a nice GUI and was very popular. It will run uder
>> XP but only supports short file names. You can download a copy at:
>> http://www.ludwig.edu.au/confocal/Links.html
>>
>> Cheers
>> Stephen Cody
>>
>> 2009/4/23 Cory Quammen <[hidden email]>
>>
>>> Dear list,
>>>
>>> Open-source and freely available software such as ImageJ, the Open
>>> Microscopy Environment, and Micro-Manager are examples of popular,
>>> actively-developed, and well-supported applications used within the
>>> microscopy community. Presumably other popular software tools useful
>>> in microscopy and image analysis have been created in the past, but
>>> are no longer developed or supported because the original development
>>> team lacks funding or interest. My question for the list is:
>>>
>>> What abandoned or neglected microscopy and image analysis/processing
>>> software have you used in the past and would love to use again if it
>>> were under active development, but currently do not use because it
>>> either does not run or does not do something you want it to? By active
>>> development, I mean that a team of developers would release updated
>>> versions of the software from time to time with new features and bug
>>> fixes.
>>>
>>> Thanks for your feedback,
>>> Cory
>>>
>>> --
>>> Cory Quammen
>>> Center for Computer Integrated Systems for Microscopy and Manipulation
>>> (CISMM)
>>> Department of Computer Science
>>> University of North Carolina at Chapel Hill
>>> http://www.cs.unc.edu/~cquammen
>>>
>
> ****************************************************************************
> * Tina (Weatherby) Carvalho               * [hidden email]           *
> * Biological Electron Microscope Facility * (808) 956-6251                 *
> * University of Hawaii at Manoa           * http://www.pbrc.hawaii.edu/bemf* 
> ****************************************************************************
Guy Cox Guy Cox
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Re: What open-source software did you use in the past and would like to use again?

Todd Brelje, not Berlji!

According to the grapevine, he was working on a later version which would work under NT and have an interface more like the later Biorad software.  (For those who don't know, Todd also wrote the first version of COMOS, then called CM).  However Bio-Rad refused to allow him to continue with it, for unfathomable reasons of their own.  Since he had been given access to Bio-Rad source code and had signed the appropriate agreements, he had no option but to comply.  

Maybe someone else out there knows more details.

                                         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 Dale Callaham
Sent: Thursday, 23 April 2009 1:20 PM
To: [hidden email]
Subject: Re: What open-source software did you use in the past and would like to use again?

Confocal Assistant was very nice for Biorad files in particular and the things we tended to do with those images and stacks. But in addition to the 8+3 filename limitation it seemed to also have a restricted space allocated for the full path and filename string and if files were more than a few directories deep with typical names it seemed to fail to open the files - would show them in the browser but couldn't open; if the files were simply moved closer to the root level, it opened them fine.

Dale

Tina Carvalho wrote:

> Hey, that was what I was going to say! Confocal Assistant. Really
> liked it. Hated the short file names.
>
> Aloha,
> Tina
>
>> Confocal Assistant by Todd Berlji would fall into this category. It
>> was developed for Win 3.1, had a nice GUI and was very popular. It
>> will run uder XP but only supports short file names. You can download a copy at:
>> http://www.ludwig.edu.au/confocal/Links.html
>>
>> Cheers
>> Stephen Cody
>>
>> 2009/4/23 Cory Quammen <[hidden email]>
>>
>>> Dear list,
>>>
>>> Open-source and freely available software such as ImageJ, the Open
>>> Microscopy Environment, and Micro-Manager are examples of popular,
>>> actively-developed, and well-supported applications used within the
>>> microscopy community. Presumably other popular software tools useful
>>> in microscopy and image analysis have been created in the past, but
>>> are no longer developed or supported because the original
>>> development team lacks funding or interest. My question for the list is:
>>>
>>> What abandoned or neglected microscopy and image analysis/processing
>>> software have you used in the past and would love to use again if it
>>> were under active development, but currently do not use because it
>>> either does not run or does not do something you want it to? By
>>> active development, I mean that a team of developers would release
>>> updated versions of the software from time to time with new features
>>> and bug fixes.
>>>
>>> Thanks for your feedback,
>>> Cory
>>>
>>> --
>>> Cory Quammen
>>> Center for Computer Integrated Systems for Microscopy and
>>> Manipulation
>>> (CISMM)
>>> Department of Computer Science
>>> University of North Carolina at Chapel Hill
>>> http://www.cs.unc.edu/~cquammen
>>>
>
> ****************************************************************************
> * Tina (Weatherby) Carvalho               * [hidden email]           *
> * Biological Electron Microscope Facility * (808) 956-6251                 *
> * University of Hawaii at Manoa           * http://www.pbrc.hawaii.edu/bemf* 
> **********************************************************************
> ******

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Eric Scarfone Eric Scarfone
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Re: What open-source software did you use in the past and would like to use again?

hej
For a totally free software , CAS worked surprisingly well!
It was particularly usefull to split and merge the infamous original Biorad split image format! To my knowledge it is the only software able to do that, even on stacks that can then be saved as tiff series.
Brings back good mems!

Eric  

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

Postal Address:
CFH, M1:02
Karolinska Hospital,
SE-171 76 Stockholm, Sweden

Work: +46 (0)8-517 79343,
Cell: +46 (0)70 888 2352
Fax: +46 (0)8-301876

email: [hidden email]
http://www.ki.se/cfh/


----- Original Message -----
From: Guy Cox <[hidden email]>
Date: Thursday, April 23, 2009 6:52 am
Subject: Re: What open-source software did you use in the past and would like to use again?
To: [hidden email]

> Todd Brelje, not Berlji!
>
> According to the grapevine, he was working on a later version
> which would work under NT and have an interface more like the
> later Biorad software. (For those who don't know, Todd also wrote
> the first version of COMOS, then called CM). However Bio-Rad


> refused to allow him to continue with it, for unfathomable reasons
> of their own. Since he had been given access to Bio-Rad source
> code and had signed the appropriate agreements, he had no option
> but to comply.
>
> Maybe someone else out there knows more details.
>
> 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 Dale Callaham
> Sent: Thursday, 23 April 2009 1:20 PM
> To: [hidden email]
> Subject: Re: What open-source software did you use in the past and
> would like to use again?
>
> Confocal Assistant was very nice for Biorad files in particular
> and the things we tended to do with those images and stacks. But
> in addition to the 8+3 filename limitation it seemed to also have
> a restricted space allocated for the full path and filename string
> and if files were more than a few directories deep with typical
> names it seemed to fail to open the files - would show them in the
> browser but couldn't open; if the files were simply moved closer
> to the root level, it opened them fine.
>
> Dale
>

> Tina Carvalho wrote:
> > Hey, that was what I was going to say! Confocal Assistant.
> Really
> > liked it. Hated the short file names.
> >
> > Aloha,
> > Tina
> >
> >> Confocal Assistant by Todd Berlji would fall into this
> category. It
> >> was developed for Win 3.1, had a nice GUI and was very popular.
> It
> >> will run uder XP but only supports short file names. You can
> download a copy at:
> >> http://www.ludwig.edu.au/confocal/Links.html
> >>
> >> Cheers
> >> Stephen Cody
> >>
> >> 2009/4/23 Cory Quammen <[hidden email]>
> >>
> >>> Dear list,
> >>>
> >>> Open-source and freely available software such as ImageJ, the
> Open
> >>> Microscopy Environment, and Micro-Manager are examples of

> popular,
> >>> actively-developed, and well-supported applications used
> within the
> >>> microscopy community. Presumably other popular software tools
> useful
> >>> in microscopy and image analysis have been created in the
> past, but
> >>> are no longer developed or supported because the original
> >>> development team lacks funding or interest. My question for
> the list is:
> >>>
> >>> What abandoned or neglected microscopy and image
> analysis/processing
> >>> software have you used in the past and would love to use again
> if it
> >>> were under active development, but currently do not use
> because it
> >>> either does not run or does not do something you want it to?
> By

> >>> active development, I mean that a team of developers would
> release
> >>> updated versions of the software from time to time with new
> features
> >>> and bug fixes.
> >>>
> >>> Thanks for your feedback,
> >>> Cory
> >>>
> >>> --
> >>> Cory Quammen
> >>> Center for Computer Integrated Systems for Microscopy and
> >>> Manipulation
> >>> (CISMM)
> >>> Department of Computer Science
> >>> University of North Carolina at Chapel Hill
> >>> http://www.cs.unc.edu/~cquammen
> >>>
> >
> >
> ****************************************************************************> * Tina (Weatherby) Carvalho * [hidden email] *
> > * Biological Electron Microscope Facility * (808) 956-6251

> *
> > * University of Hawaii at Manoa *
> http://www.pbrc.hawaii.edu/bemf*
> >
> **********************************************************************> ******
>
> No virus found in this incoming message.
> Checked by AVG.
> Version: 7.5.557 / Virus Database: 270.12.3/2075 - Release Date:
> 22/04/2009 5:25 PM
>
>
> No virus found in this outgoing message.
> Checked by AVG.
> Version: 7.5.557 / Virus Database: 270.12.3/2075 - Release Date:
> 22/04/2009 5:25 PM
>
>

Dale Callaham Dale Callaham
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Re: What open-source software did you use in the past and would like to use again?

Hi Eric,

I have written some macros that split the Biorad dual-channel
split-image files; also one that does split-image Z-stacks. These are
basically modifications to the "Biorad Reader" plugin with some code
added to do the task at hand.

I would be happy to send or put the java and class files on my website
for download.

Below is an example for a single frame dual image. The others are longer.

/* Author:  Dale A. Callaham
*  email: [hidden email]
*
* Splits an OPEN (!) 2-channel Biorad Mrc-600 confocal image into the "Red"
* and "Green" channels. Assumes 768x512 dual-channel (side-by-side) image.
*
*/

import ij.*;
import ij.process.*;
import ij.gui.*;
import java.awt.*;
import ij.plugin.*;

public class BioRad_Splitter implements PlugIn {

        public void run(String arg) {
                IJ.run("Rename...", "title=Untitled");
                IJ.selectWindow("Untitled");
                IJ.makeRectangle(0, 0, 383, 511);
                IJ.run("Copy");
                IJ.run("New...", "name=Green type='8-bit Unsigned' fill=Black
width=384 height=512 slices=1");
                IJ.run("Paste");
                IJ.run("Select None");
                IJ.selectWindow("Untitled");
                IJ.makeRectangle(384, 0, 767, 511);
                IJ.run("Copy");
                IJ.run("New...", "name=Red type='8-bit Unsigned' fill=Black width=384
height=512 slices=1");
                IJ.run("Paste");
                IJ.run("Select None");
                IJ.selectWindow("Untitled");
                IJ.run("Select None");
        }

}

##########################################
Eric Scarfone wrote:

> hej
> For a totally free software , CAS worked surprisingly well!
> It was particularly usefull to split and merge the infamous original
> Biorad split image format! To my knowledge it is the only software able
> to do that, even on stacks that can then be saved as tiff series.
> Brings back good mems!
>
> Eric  
>
> Eric Scarfone, PhD, CNRS,
> Center for Hearing and communication Research
> Department of Clinical Neuroscience
> Karolinska Institutet
>
> Postal Address:
> CFH, M1:02
> Karolinska Hospital,
> SE-171 76 Stockholm, Sweden
>
> Work: +46 (0)8-517 79343,
> Cell: +46 (0)70 888 2352
> Fax: +46 (0)8-301876
>
> email: [hidden email]
> http://www.ki.se/cfh/
>
>
> ----- Original Message -----
> From: Guy Cox <[hidden email]>
> Date: Thursday, April 23, 2009 6:52 am
> Subject: Re: What open-source software did you use in the past and would
> like to use again?
> To: [hidden email]
>
>  > Todd Brelje, not Berlji!
>  >
>  > According to the grapevine, he was working on a later version
>  > which would work under NT and have an interface more like the
>  > later Biorad software. (For those who don't know, Todd also wrote
>  > the first version of COMOS, then called CM). However Bio-Rad
>  > refused to allow him to continue with it, for unfathomable reasons
>  > of their own. Since he had been given access to Bio-Rad source
>  > code and had signed the appropriate agreements, he had no option
>  > but to comply.
>  >
>  > Maybe someone else out there knows more details.
>  >
>  > 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 Dale Callaham
>  > Sent: Thursday, 23 April 2009 1:20 PM
>  > To: [hidden email]
>  > Subject: Re: What open-source software did you use in the past and
>  > would like to use again?
>  >
>  > Confocal Assistant was very nice for Biorad files in particular
>  > and the things we tended to do with those images and stacks. But
>  > in addition to the 8+3 filename limitation it seemed to also have
>  > a restricted space allocated for the full path and filename string
>  > and if files were more than a few directories deep with typical
>  > names it seemed to fail to open the files - would show them in the
>  > browser but couldn't open; if the files were simply moved closer
>  > to the root level, it opened them fine.
>  >
>  > Dale
>  >
>  > Tina Carvalho wrote:
>  > > Hey, that was what I was going to say! Confocal Assistant.
>  > Really
>  > > liked it. Hated the short file names.
>  > >
>  > > Aloha,
>  > > Tina
>  > >
>  > >> Confocal Assistant by Todd Berlji would fall into this
>  > category. It
>  > >> was developed for Win 3.1, had a nice GUI and was very popular.
>  > It
>  > >> will run uder XP but only supports short file names. You can
>  > download a copy at:
>  > >> http://www.ludwig.edu.au/confocal/Links.html
>  > >>
>  > >> Cheers
>  > >> Stephen Cody
>  > >>
>  > >> 2009/4/23 Cory Quammen <[hidden email]>
>  > >>
>  > >>> Dear list,
>  > >>>
>  > >>> Open-source and freely available software such as ImageJ, the
>  > Open
>  > >>> Microscopy Environment, and Micro-Manager are examples of
>  > popular,
>  > >>> actively-developed, and well-supported applications used
>  > within the
>  > >>> microscopy community. Presumably other popular software tools
>  > useful
>  > >>> in microscopy and image analysis have been created in the
>  > past, but
>  > >>> are no longer developed or supported because the original
>  > >>> development team lacks funding or interest. My question for
>  > the list is:
>  > >>>
>  > >>> What abandoned or neglected microscopy and image
>  > analysis/processing
>  > >>> software have you used in the past and would love to use again
>  > if it
>  > >>> were under active development, but currently do not use
>  > because it
>  > >>> either does not run or does not do something you want it to?
>  > By
>  > >>> active development, I mean that a team of developers would
>  > release
>  > >>> updated versions of the software from time to time with new
>  > features
>  > >>> and bug fixes.
>  > >>>
>  > >>> Thanks for your feedback,
>  > >>> Cory
>  > >>>
>  > >>> --
>  > >>> Cory Quammen
>  > >>> Center for Computer Integrated Systems for Microscopy and
>  > >>> Manipulation
>  > >>> (CISMM)
>  > >>> Department of Computer Science
>  > >>> University of North Carolina at Chapel Hill
>  > >>> http://www.cs.unc.edu/~cquammen
>  > >>>
>  > >
>  > >
>  >
> ****************************************************************************>
> * Tina (Weatherby) Carvalho * [hidden email] *
>  > > * Biological Electron Microscope Facility * (808) 956-6251
>  > *
>  > > * University of Hawaii at Manoa *
>  > http://www.pbrc.hawaii.edu/bemf*
>  > >
>  >
> **********************************************************************>
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Cameron Nowell Cameron Nowell
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Multiple Fluorophores and Two Photon Imaging

Howdy List,

Has anyone out there had any experience with imaging multiple
fluorescent constructs in the one sample using two photon microscopy? I
have a user that already has a zebrafish with GFP and DsRed in it and
would like to add a third fluorophore if possible.

Does anyone know if this is possible to image, or have any ideas on a
good choice for the third fluorophore?

A blue protein like CFP would be ideal as its 2PE is ~800nm, leaving GFP
at 920nm and DsRed at ~980nm. But my experience in the past with CFP
under single photon and widefiled fluorescence has been that it is quite
dim. Has anyone used CFP under 2PE before?


Any advice would be greatly appreciated.


Thanks


Cam



Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website




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 Ltd 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 Ltd.

Paul Herzmark Paul Herzmark
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Re: Multiple Fluorophores and Two Photon Imaging

We do 2P microscopy with samples containing CFP, GFP,YFP and TD tomato. Excitation wavelength is around 910-920 nm.
You can sort of separate the emission channels with dichroics like 495, 515 and 560. 
You might also try these bandpass filters 525/30 and 645/75 as necessary to minimize crossover.


Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA  94720-3200
(510) 643-9603
(510) 643-9500 fax


On Thu, Apr 23, 2009 at 7:20 PM, Cameron Nowell <[hidden email]> wrote:
Howdy List,

Has anyone out there had any experience with imaging multiple
fluorescent constructs in the one sample using two photon microscopy? I
have a user that already has a zebrafish with GFP and DsRed in it and
would like to add a third fluorophore if possible.

Does anyone know if this is possible to image, or have any ideas on a
good choice for the third fluorophore?

A blue protein like CFP would be ideal as its 2PE is ~800nm, leaving GFP
at 920nm and DsRed at ~980nm. But my experience in the past with CFP
under single photon and widefiled fluorescence has been that it is quite
dim. Has anyone used CFP under 2PE before?


Any advice would be greatly appreciated.


Thanks


Cam



Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website




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 Ltd 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 Ltd.


Adrian Smith-6 Adrian Smith-6
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Re: Multiple Fluorophores and Two Photon Imaging

In reply to this post by Cameron Nowell
Hi Cameron,

I know of several publications with CFP under MPE. For example Paulus Mrass who just moved here from the US is using it - search PubMed for Paulus Mrass and Wolfgang Weninger.

Can't comment on how bright it is compared to other alternatives.

Regards,

Adrian Smith
Centenary Institute, Sydney, Australia


Mrass, P., & Weninger, W. (2006). Immune cell migration as a means to control immune privilege: lessons from the CNS and tumors. Immunological reviews, 213, 195-212.
Mrass, P., Kinjyo, I., Ng, L. G., Reiner, S. L., Puré, E., & Weninger, W. (2008). CD44 mediates successful interstitial navigation by killer T cells and enables efficient antitumor immunity. Immunity, 29, 971-85.
Mrass, P., Takano, H., Ng, L. G., Daxini, S., Lasaro, M. O., Iparraguirre, A., et al. (2006). Random migration precedes stable target cell interactions of tumor-infiltrating T cells. J Exp Med, 203, 2749-61.




On 24/04/2009, at 9:20 AM, Cameron Nowell wrote:

Howdy List,

Has anyone out there had any experience with imaging multiple
fluorescent constructs in the one sample using two photon microscopy? I
have a user that already has a zebrafish with GFP and DsRed in it and
would like to add a third fluorophore if possible.

Does anyone know if this is possible to image, or have any ideas on a
good choice for the third fluorophore?

A blue protein like CFP would be ideal as its 2PE is ~800nm, leaving GFP
at 920nm and DsRed at ~980nm. But my experience in the past with CFP
under single photon and widefiled fluorescence has been that it is quite
dim. Has anyone used CFP under 2PE before?


Any advice would be greatly appreciated.


Thanks


Cam



Shivaprasad Bhuvanendran Shivaprasad Bhuvanendran
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Re: Multiple Fluorophores and Two Photon Imaging

In reply to this post by Cameron Nowell
Hi Cameron,

We have used ECFP, EGFP, EYFP and DsRed at the same time for intravital multiphoton imaging of Lymph nodes using the Olympus Fluoview 1000MPE with a SpectraPhysics MaiTai laser set to 910nm.  
Dichroic Mirrors 505 and 570 were used along with bandpass filters 460-510, 515-560 and 570-625 for CFP, YFP and DsRed respectively. The GFP was collected in both the CFP and YFP channels and could be distinguished both by the morphology and the distinct color.

If you need further details, you can look up the publication cited here or contact me offline.
 
Science April 2009 Liu et al., pp. 392 - 397
http://www.sciencemag.org/cgi/rapidpdf/1170540.pdf
http://www.sciencemag.org/cgi/data/1170540/DC1/1


Regards,
Shiva


Shivaprasad Bhuvanendran
Research Support Specialist
Bio-Imaging Resource Center
The Rockefeller University
1230 York Avenue
Box 209 (DWB 201)
New York NY 10065

tel +1 212 327 7487
fax +1 212 327 7489


On Thu, Apr 23, 2009 at 7:20 PM, Cameron Nowell <[hidden email]> wrote:
Howdy List,

Has anyone out there had any experience with imaging multiple
fluorescent constructs in the one sample using two photon microscopy? I
have a user that already has a zebrafish with GFP and DsRed in it and
would like to add a third fluorophore if possible.

Does anyone know if this is possible to image, or have any ideas on a
good choice for the third fluorophore?

A blue protein like CFP would be ideal as its 2PE is ~800nm, leaving GFP
at 920nm and DsRed at ~980nm. But my experience in the past with CFP
under single photon and widefiled fluorescence has been that it is quite
dim. Has anyone used CFP under 2PE before?


Any advice would be greatly appreciated.


Thanks


Cam



Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website




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 Ltd 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 Ltd.


Cameron Nowell Cameron Nowell
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Re: Multiple Fluorophores and Two Photon Imaging

Hi Shiva,
 
Thanks for the references, this is exactly what i was looking for. We even have the same microscope system:)
 
 
Cam
 
 

________________________________

From: Confocal Microscopy List on behalf of Shivaprasad Bhuvanendran
Sent: Sat 25/04/2009 2:42 AM
To: [hidden email]
Subject: Re: Multiple Fluorophores and Two Photon Imaging


Hi Cameron,

We have used ECFP, EGFP, EYFP and DsRed at the same time for intravital multiphoton imaging of Lymph nodes using the Olympus Fluoview 1000MPE with a SpectraPhysics MaiTai laser set to 910nm.  
Dichroic Mirrors 505 and 570 were used along with bandpass filters 460-510, 515-560 and 570-625 for CFP, YFP and DsRed respectively. The GFP was collected in both the CFP and YFP channels and could be distinguished both by the morphology and the distinct color.

If you need further details, you can look up the publication cited here or contact me offline.
 
Science April 2009 Liu et al., pp. 392 - 397
http://www.sciencemag.org/cgi/rapidpdf/1170540.pdf
http://www.sciencemag.org/cgi/data/1170540/DC1/1


Regards,
Shiva



Shivaprasad Bhuvanendran
Research Support Specialist
Bio-Imaging Resource Center
The Rockefeller University

1230 York Avenue
Box 209 (DWB 201)
New York NY 10065

tel +1 212 327 7487
fax +1 212 327 7489


On Thu, Apr 23, 2009 at 7:20 PM, Cameron Nowell <[hidden email]> wrote:


        Howdy List,
       
        Has anyone out there had any experience with imaging multiple
        fluorescent constructs in the one sample using two photon microscopy? I
        have a user that already has a zebrafish with GFP and DsRed in it and
        would like to add a third fluorophore if possible.
       
        Does anyone know if this is possible to image, or have any ideas on a
        good choice for the third fluorophore?
       
        A blue protein like CFP would be ideal as its 2PE is ~800nm, leaving GFP
        at 920nm and DsRed at ~980nm. But my experience in the past with CFP
        under single photon and widefiled fluorescence has been that it is quite
        dim. Has anyone used CFP under 2PE before?
       
       
        Any advice would be greatly appreciated.
       
       
        Thanks
       
       
        Cam
       
       
       
        Cameron J. Nowell
        Microscopy Manager
        Centre for Advanced Microscopy
        Ludwig Institute for Cancer Research
        PO Box 2008
        Royal Melbourne Hospital
        Victoria, 3050
        AUSTRALIA
        Office: +61 3 9341 3155
        Mobile: +61422882700
        Fax: +61 3 9341 3104
        Facility Website
       
       
       
       
        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 Ltd 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 Ltd.