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

[Pascal Lorentz]

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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