mb-FLIM: Model-based fluorescence lifetime imaging

We have developed a model-based, parallel procedure to estimate fluorescence lifetimes. Multiple frequencies are present in the excitation signal. Modeling the entire fluorescence and measurement process produces an analytical ratio of polynomials in the lifetime variable tau. A non-linear model-fitting procedure is then used to estimate tau. We have analyzed this model-based approach by simulating a 10 mu M fluorescein solution (tau = 4 ns) and all relevant noise sources. We have used real LED data to drive the simulation. Using 240 mu s of data, we estimate tau = 3.99 ns. Preliminary experiments on real fluorescent images taken from fluorescein solutions (measured tau = 4.1 ns), green plastic test slides (measured tau = 3.0 ns), and GFP in U2OS (osteosarcoma) cells (measured tau = 2.1 ns) demonstrate that this model-based measurement technique works.