I have some troubles about measuring fluorescence intensity in cells (timelapse experiment). My goal is “simple”: measure one fluorescence channel intensity in individual cells through time (for instance Hoechst and GFP stained cells).
I created a pipeline which give me a good segmentation of my cells (Primary object= Nuclei, Secondary object=Cells, Tertiary Object= Cytoplasm).
I used the function Track Object and I notice that this function works perfectly to attribute a specific number for individual cells (which never change during all the acquisition, despite cells motility, appearance of other cells etc).
But the problem appears when I export my data in spreadsheet: cell numbering change through the time. I noticed this problem by looking the intensity which highly differs sometimes between two time position. I upload 4 files to show you the problem: the two colored images show the “TrackedCells” which have a good numbering through all the time, I marked the cell n°41 with a white circle.
The two screenshots show the corresponding exported spreadsheet obtained, you will notice that the cell n°41 is perfectly measure for the first position (Intensity around 20) but not for the second position (Intensity= 0) and seams to be numbered as n°42 instead of 41…
Thus I have several questions:
- Is it possible to obtain the fluorescence intensity of “Tracked cells” (I can save a color image with cells numbered, but this image can’t be use for the “MeasureIntensity” function because it’s a color image instead of grayscale).
- Why is there a numbering difference between the function TrackedCells and the Spreadsheet export ?
- and obviously… how can I resolve these troubles !
In optimal way I would like to create a file (spreadsheet file) when all the cells are identify by an individual invariant number with the corresponding fluorescence intensity through the time.
I also uploaded the pipeline I use.
Thanks a lot for your consideration and help!
Fluorescence project.cpproj (774.9 KB)