[ome-users] Increase in intensity in every second frame in Leica lif files

[Melissa Linkert]

Hi Pascal,

> We still have problems with the intensity changes in every second
> frame after opening the mentioned lif files. I used the latest Fiji.
> I can again send you the file if you need it.
> 
> I think the problem that has been solved under the following link
> was something different.
> 
> https://github.com/openmicroscopy/bioformats/pull/804
> 
> 
> Could you please have a look at the problem again.

Have you followed the instructions for updating Fiji to Bio-Formats 5 from
http://fiji.sc/Bio-Formats#Daily_builds?  Could you please verify that
"Help > About Plugins > LOCI Plugins" shows a recent date (within the
last couple of days) and a version number similar to 5.0.0-DEV?

Regards,
-Melissa

On Fri, Jan 31, 2014 at 11:16:09AM +0100, Pascal Lorentz wrote:
> Dear Melissa
> 
> We still have problems with the intensity changes in every second
> frame after opening the mentioned lif files. I used the latest Fiji.
> I can again send you the file if you need it.
> 
> I think the problem that has been solved under the following link
> was something different.
> 
> https://github.com/openmicroscopy/bioformats/pull/804
> 
> 
> Could you please have a look at the problem again.
> 
> Thanks a lot and best regards
> 
> Pascal
> 
> 
> 
> 
> 
> Am 22.11.2013 03:27, schrieb Melissa Linkert:
> >Hi Pascal,
> >
> >>I just uploaded the zip folder "lif file problem" containing the
> >>four files on the specified website.
> >>
> >>I am curious what the problem is.
> >Thank you for uploading an example dataset.
> >
> >The problem is that the reported order of channels and timepoints does
> >not match the actual order.  We are reviewing a fix for the problem
> >here:
> >
> >https://github.com/openmicroscopy/bioformats/pull/804
> >
> >Once that shows as being closed, enabling the "Bio-Formats 5" update
> >site (in Fiji) or downloading the latest trunk build (in ImageJ) from:
> >
> >http://downloads.openmicroscopy.org/bio-formats/5.0.0-beta1/
> >
> >should solve the problem.
> >
> >Regards,
> >-Melissa
> >
> >On Thu, Nov 21, 2013 at 03:33:34PM +0100, Pascal Lorentz wrote:
> >>Dear Curtis
> >>
> >>Thanks a lot for your reply.
> >>I just uploaded the zip folder "lif file problem" containing the
> >>four files on the specified website.
> >>
> >>I am curious what the problem is.
> >>
> >>Best regards
> >>
> >>Pascal
> >>
> >>Am 21.11.2013 10:18, schrieb Curtis Rueden:
> >>>Hi Pascal,
> >>>
> >>>>I also tried with Fiji. I updated Fiji through the menu and also
> >>>>manually updated the loci plugin there. I am not sure if it takes the
> >>>>trunk build in that case.
> >>>Fiji ships with the 4.4.9 release of Bio-Formats. If you want to
> >>>enable 4.4.x or 5.x development builds, you can use the
> >>>"Bio-Formats 4" or "Bio-Formats 5" update sites. For more details,
> >>>please see the instructions at:
> >>>
> >>>http://fiji.sc/Bio-Formats#Daily_builds
> >>>
> >>>With Fiji, it should not be necessary to download loci_tools.jar
> >>>manually, nor to ever run the "Update LOCI Plugins" command (that
> >>>command is disabled in Fiji anyway).
> >>>
> >>>>Anyway it is always the same. We see the change in intensity in every
> >>>>second time frame.
> >>>Indeed, it sounds like a bug.
> >>>
> >>>>I can provide you with 4 files but I need an ftp access
> >>>Thanks. If you ZIP up the four files, you can upload them using this link:
> >>>
> >>>http://qa.openmicroscopy.org.uk/qa/upload/
> >>>
> >>>Unless your archive is >500MB? In which case, let us know and we
> >>>will provide alternative upload instructions off-list.
> >>>
> >>>Regards,
> >>>Curtis
> >>>
> >>>
> >>>On Thu, Nov 21, 2013 at 2:38 AM, Pascal Lorentz
> >>><Lorentz.Pascal at gmail.com <mailto:Lorentz.Pascal at gmail.com>>
> >>>wrote:
> >>>
> >>>    Dear Bio Formats team
> >>>
> >>>    First of all I would like to thank you for your impressive work.
> >>>    This plugin is really great.
> >>>
> >>>    We came across a problem yesterday.
> >>>    We have a lif file that has been acquired with a Leica confocal
> >>>    SP5 with the LAS AF Software Version 2.7.3. It is a timelaps
> >>>    dataset with 12 timepoints in 2 channels. If we look at these
> >>>    cells in the Leica software the intensity over time stays in the
> >>>    same range.
> >>>    If we open this dataset as a hyperstack with the bio formats
> >>>    importer we see an increase in intensity in every second time
> >>>    frame. It is not just a display effect since we can really measure
> >>>    the intensity change.
> >>>    As a work around we exported single tifs from the lif file out of
> >>>    the Leica software. So we end up with 24 images for the 12
> >>>    timepoints for the two channels. If we then import these images as
> >>>    an image sequence and if we measure the exact same area, the
> >>>    intensity stays in a similar range over the whole timelaps.
> >>>
> >>>    I can provide you with 4 files but I need an ftp access to upload
> >>>    them:
> >>>    1. The original lif file “Series 7.lif”
> >>>    2. The image sequence after exporting from the Leica Software and
> >>>    importing as sequence in ImageJ saved as tiff “image_sequence.tif”
> >>>    3. The results table of the intensity measurement of an area of
> >>>    the lif dataset opened with the loci importer
> >>>    “Results_loci_import.xls.
> >>>    4. The results table of the intensity measurement of the same area
> >>>    as in point 3 after importing the single tifs as an image sequence.
> >>>
> >>>    My user saw that problem on a Mac with Fiji but we also saw it on
> >>>    my PC as well.
> >>>    I downloaded the latest stable release from
> >>>    http://downloads.openmicroscopy.org/bio-formats/4.4.9/#tools
> >>>    I also tried to update the plugin to the trunk build with Plugins
> >>>    -> Loci -> Update loci plugins but it always fails with the
> >>>    message “an error occurred while downloading the plugins”
> >>>    I did all that that with ImageJ 1.48g
> >>>    I also tried with Fiji. I updated Fiji through the menu and also
> >>>    manually updated the loci plugin there. I am not sure if it takes
> >>>    the trunk build in that case.
> >>>    Anyway it is always the same. We see the change in intensity in
> >>>    every second time frame.
> >>>    I also tried an older version of the loci plugin from 2010 but it
> >>>    shows the same effect.
> >>>
> >>>    We never so that problem with any lif files before. But our
> >>>    acquisition software has been updated a few weeks ago and I am not
> >>>    sure if Leica changed something on the file format.
> >>>
> >>>    So I was using ImageJ 1.48g with the loci plugin 4.4.9 on a
> >>>    windows 7 64bit machine with Java Version 7 Update 45 (build
> >>>    1.7.0_45-b18).
> >>>
> >>>    We would be more than happy if you can have a look at our problem.
> >>>    If you need more information just let me know.
> >>>
> >>>
> >>>    Many thanks and best regards
> >>>
> >>>    Pascal
> >>>
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> >>>
> >>>
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