Leica FLIM: Incorrect lifetime values

[dgault]

This issue was raised on forum thread https://forum.image.sc/t/import-of-leica-flim-data-does-not-display-correct-lifetimes/40508/5

A sample dataset has been provided and the issue can be reproduced with Bio-Formats 6.12.0.

Details of the issue copied from the original post:

In the dataset, you will find the lif file (FLIM_testdata.lif) with one image (sample1_slice1) and a folder with tifs exported with LAS X (LASXv3.5.6_exported/tifs) and a few supplementary screenshots.

To re-iteratee: the issue is that the lifetime image exported with LAS X has very different pixel values that the one imported with BioFormats and I can’t quite figure out what conversion/compression is performed on the file and why the values are different. From what I know about the lifetime of the probe that I am using, the values exported with LAS X make more sense (there should be two peaks, one around 1.5 ns and one in the range 4-6 ns).

I export the lifetime image in LAS X (LAS X FLIM FACS Offline, this image was exported with v3.5.6, but I had same issues with v4.6) by right clicking on the image and selecting “Export Raw Image”, two following tif files are saved (LASXv3.5.6_exported/tifs):

filename_ch0 - intensity [cnts] filename_ch1 - lifetime [ns] These files are uint16 which is fine for counts but bad for lifetime (they have float values). My workaround is to use a conversion factor of 0.001 for lifetime to not loose information (see LASX_ExportRawImage.png). I have exported one file with conversion and one without (“no scaling”). When I import the FLIM_testdata.lif to Fiji using Bio-Formats plugin, I see the following list of files:

‘sample1_slice1’,
‘sample1_slice1/FLIM Compressed/Phasor Plot’,
‘sample1_slice1/FLIM Compressed/Pattern Matching Scatter Plot Channel 1’,
‘sample1_slice1/FLIM Compressed/Intensity’,
‘sample1_slice1/FLIM Compressed/Fast Flim’,
‘sample1_slice1/FLIM Compressed/Standard Deviation’,
‘sample1_slice1/FLIM Compressed/Phasor Real’,
‘sample1_slice1/FLIM Compressed/Phasor Imaginary’,
‘sample1_slice1/FLIM Compressed/Phasor Intensity’,
‘sample1_slice1/FLIM Compressed/Phasor Mask’

I believe that the ‘sample1_slice1/FLIM Compressed/Fast Flim’ is the file that should contain the lifetime data. The pixel values that I see for this image (see ‘LASXv3.5.6_exported/Histograms/Histogram_lif_ImageJ_FLIM_testdata_sample1_slice1_FLIMCompressed_FastFlim.png’) are completely different from the ones I see for the ch1 exported with LAS X (‘LASXv3.5.6_exported/Histograms/Histogram_LASXexported_sample1_slice1_ch1.png’).

[imagesc-bot]

This issue has been mentioned on Image.sc Forum. There might be relevant details there:

https://forum.image.sc/t/import-of-leica-flim-data-does-not-display-correct-lifetimes/40508/7