I’m using TrackMate to capture fluorescence data from a z-stack of vesicles ranging in size from 5-20um in diameter. I’m wondering how TrackMate calculates mean pixel intensity.
First, below are three points from http://imagej.net/TrackMate_Algorithms. I think these say that the mean pixel intensity is measured within R? Given the range of vesicle sizes I generally set R to be 11um to capture the range of sizes, but if R is used to calculate mean pixel intensity I would also be averaging pixels outside the smaller vesicles.
"R the spot radius, also in physical units. The current detectors only set this radius value to be the one specified by the user."
"Estimated diameter This feature estimates an optimal diameter for each spot, based on contrast calculation."
"Mean, Median, Min, Max, Total intensity and its Standard Deviation The plain statistical estimates are simply calculated from all the values for pixels within the physical radius from the spot center."
Second, I’m tracking vesicles in a z-stack. Does TrackMate find spots in every z slice? Are these spots linked in the z dimension to get a spherical volume? And finally, if volumes are identified, are all the pixels in the volume used to calculate mean pixel intensity?
Thank you for you time,