importing .LSM files into ImageJ and losing 1 channel

[Holly Aaron]

Dear ImageJ Community - Hello! It has been a long time since I've been on
the list. I am happy to see it is still so active!

Anyway, one of my users recently ran into an unusual problem with importing
files from the Zeiss 710 Zen software.  She is doing large tile scans (6x8
or bigger) z-stacks (20+ z- planes), so the files are 0.5G++.  She is doing
them in 2 rounds to get 3 colors w/o any cross-talk:  first GFP & Cy5 and
then Cy3.  She then adds the Cy3 channel to the other image to create a
3-channel image. She's saving in the .lsm format. All the data (all 3
channels) are there if she re-opens in Zen. But when she imports the file
into ImageJ, there are only the first 2 channels.  Does anyone know what
could be happening to the 3rd channel?  Are there limits to the file sizes
that can be opened?

Thanks for any advice or help!

-Holly
___________________________
Holly L. Aaron
Molecular Imaging Center
Cancer Research Laboratory
University of California Berkeley
251 LSA #2751
Berkeley, CA  94720-2751
510.642.2901
510.642.5741 fax
[hidden email]
http://imaging.berkeley.edu

[Daniel James White]

Hi Holly.


On May 13, 2011, at 6:00 AM, IMAGEJ automatic digest system wrote:

>
> Date:    Thu, 12 May 2011 14:43:13 -0700
> From:    Holly Aaron <[hidden email]>
> Subject: importing .LSM files into ImageJ and losing 1 channel
>
> Dear ImageJ Community - Hello! It has been a long time since I've been on
> the list. I am happy to see it is still so active!
>
> Anyway, one of my users recently ran into an unusual problem with importing
> files from the Zeiss 710 Zen software.  She is doing large tile scans (6x8
> or bigger) z-stacks (20+ z- planes), so the files are 0.5G++.  She is doing
> them in 2 rounds to get 3 colors w/o any cross-talk:  first GFP & Cy5 and
> then Cy3.  She then adds the Cy3 channel to the other image to create a
> 3-channel image.

this is not the optimal way.

you should use 2 different "tracks"
1st track has GFP and Cy5
and the second track has Cy3.

then all the images get saved into the same .lsm file from the get go.

Imaging twice then merging the 2 files is an inefficient an derror prone way to do it.


> She's saving in the .lsm format. All the data (all 3
> channels) are there if she re-opens in Zen. But when she imports the file
> into ImageJ, there are only the first 2 channels.  

the most versatile way to open .lsm file
is probably the bio-formats importer plugin , part of the LOCI toos package.

If you use Fiji, ist already bundled and up to date and found in
plugins - loci - bio-formats importer

there is also the LSM toolbox plugins set.  


> Does anyone know what
> could be happening to the 3rd channel?  Are there limits to the file sizes
> that can be opened?

No, there is no limit... only in the RAM your computer....
and if that gets full, the java virtual machine running imageJ will complain loudly to you
via a nice error message.

I strongly sugest you use 2 or 3 "tracks"
in the Zen aodtware to do the imaging.

this works great for us and many others.

you can so a multi track (multi channel), z stack, tile scan, time series even ....
and alll gets saved in one huge .lsm file...

cheers

dan

>
> Thanks for any advice or help!
>
> -Holly
> ___________________________
> Holly L. Aaron
> Molecular Imaging Center
> Cancer Research Laboratory
> University of California Berkeley
> 251 LSA #2751
> Berkeley, CA  94720-2751
> 510.642.2901
> 510.642.5741 fax
> [hidden email]
> http://imaging.berkeley.edu

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Holly Aaron]

Hi, Dan - Yes, originally she was using 2 tracks, but this was causing
really long acquisition times => 40 minutes per image. By doing the 2
separately - these are fixed samples, so no worries about things crawling
around - it shortened the acquisition time to ~ 8 minutes per acquisition.

Anyway, thank you for the advice on the FIJI - I should have thought of
that! I'll have her get FIJI and also the bioformats.

Thank you, thank you!!
-Holly
___________________________
Holly L. Aaron
Molecular Imaging Center
Cancer Research Laboratory
University of California Berkeley
251 LSA #2751
Berkeley, CA  94720-2751
510.642.2901
510.642.5741 fax
[hidden email]
http://imaging.berkeley.edu

On Fri, May 13, 2011 at 1:57 AM, Daniel James White <[hidden email]>wrote:

> Hi Holly.
>
>
> On May 13, 2011, at 6:00 AM, IMAGEJ automatic digest system wrote:
>
> >
> > Date:    Thu, 12 May 2011 14:43:13 -0700
> > From:    Holly Aaron <[hidden email]>
> > Subject: importing .LSM files into ImageJ and losing 1 channel
> >
> > Dear ImageJ Community - Hello! It has been a long time since I've been on
> > the list. I am happy to see it is still so active!
> >
> > Anyway, one of my users recently ran into an unusual problem with
> importing
> > files from the Zeiss 710 Zen software.  She is doing large tile scans
> (6x8
> > or bigger) z-stacks (20+ z- planes), so the files are 0.5G++.  She is
> doing
> > them in 2 rounds to get 3 colors w/o any cross-talk:  first GFP & Cy5 and
> > then Cy3.  She then adds the Cy3 channel to the other image to create a
> > 3-channel image.
>
> this is not the optimal way.
>
> you should use 2 different "tracks"
> 1st track has GFP and Cy5
> and the second track has Cy3.
>
> then all the images get saved into the same .lsm file from the get go.
>
> Imaging twice then merging the 2 files is an inefficient an derror prone
> way to do it.
>
>
> > She's saving in the .lsm format. All the data (all 3
> > channels) are there if she re-opens in Zen. But when she imports the file
> > into ImageJ, there are only the first 2 channels.
>
> the most versatile way to open .lsm file
> is probably the bio-formats importer plugin , part of the LOCI toos
> package.
>
> If you use Fiji, ist already bundled and up to date and found in
> plugins - loci - bio-formats importer
>
> there is also the LSM toolbox plugins set.
>
>
> > Does anyone know what
> > could be happening to the 3rd channel?  Are there limits to the file
> sizes
> > that can be opened?
>
> No, there is no limit... only in the RAM your computer....
> and if that gets full, the java virtual machine running imageJ will
> complain loudly to you
> via a nice error message.
>
> I strongly sugest you use 2 or 3 "tracks"
> in the Zen aodtware to do the imaging.
>
> this works great for us and many others.
>
> you can so a multi track (multi channel), z stack, tile scan, time series
> even ....
> and alll gets saved in one huge .lsm file...
>
> cheers
>
> dan
>
>
>
> >
> > Thanks for any advice or help!
> >
> > -Holly
> > ___________________________
> > Holly L. Aaron
> > Molecular Imaging Center
> > Cancer Research Laboratory
> > University of California Berkeley
> > 251 LSA #2751
> > Berkeley, CA  94720-2751
> > 510.642.2901
> > 510.642.5741 fax
> > [hidden email]
> > http://imaging.berkeley.edu
>
> Dr. Daniel James White BSc. (Hons.) PhD
> Senior Microscopist / Image Visualisation, Processing and Analysis
> Light Microscopy and Image Processing Facilities
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108
> 01307 DRESDEN
> Germany
>
> [phone] (German Mobile)
> [phone] (Work phone at MPI-CBG)
> [phone] (Fax MPI-CBG LMF)
>
> http://www.bioimagexd.net       BioImageXD
> http://pacific.mpi-cbg.de               Fiji -  is just ImageJ (Batteries
> Included)
> http://www.chalkie.org.uk               Dan's Homepages
> https://ifn.mpi-cbg.de                  Dresden Imaging Facility Network
> dan (at) chalkie.org.uk
> ( white (at) mpi-cbg.de )
>
>
>
>
>
>
>
>
>
>
>
>