Temporal or Class Variation in Populations with the assumption that lifetimes are constant
Here we assume that we have bi-exp data and the lifetimes are constant where each image represents a time point or a particular class (i.e. dose point or treatment class). It is better to get a good estimate of the donor lifetime in FRET by using control cells and then fixing that value in the following fits.
Do a multi-image invariant fit. To do a multi-image invariant fit, add all the images together (In TRI2 use menu Macros->Add All Images) and then do an invariant fit on that one sum image with your control lifetime fixed (if you have one). This will give you the FRET lifetime or both lifetimes.
Use these values to fix the lifetimes in bi-exponential invariant fits of each image in turn (in TRI2 (version 2.3.6 or later) use “all pixels in mask” and the “Save Values – All Images”). Make sure the TOTAL count in each image is >B* (say 10,000).