A 640 nm monochromatic laser was used as an excitation fluorescence source.
These parameters can be obtained by directly reconstructing γ1− γ4, R γ 3 γ 5, and γ6; together they characterize the fluorescence source.
To model the kinetics of this process, we incorporate a compartment model into FODT, specifically in the description of the fluorescence source [ 22].
The fluorescence source S f in Step 4 follows a multi-exponential model and the exponential parameters (including magnitude and decay constants, shown later as γ1− γ6) are reconstructed directly through our inversion algorithm, giving the fluorescence source S f as a secondary (indirect) reconstructed result.
The steps used to relate X p DA, X t DA, and X t D to the fluorescence source S f in (2), which can be determined through FODT with optical measurements (fluorescence data), are as follows.
Due to the large separation between the OCT and fluorescence source wavelengths, the efficiency of the WDM for transmitting the fluorescence excitation light (488 nm) was sub-optimal, which was ~60%.
At λ m, using the reconstructed absorption (assuming μ a x = μ a m ), the coupled diffusion equations (1)– (2) are solved and γ1− γ4, R γ 3 γ 5, and γ6, which characterize the fluorescence source through (29), are reconstructed directly.
For the kinetic FODT reconstruction, we used (30) with k2 = 0.01 h−1 and R γ 3 γ 5 = 1.1 as the fluorescence source and reconstructed γ3 and γ4 (i.e., x in (31) represents γ3 and γ4).
The fluorescence source is then given by S f = η̃ DA(1 + jωτ DA)−1 + η̃ D(1 + jωτ D −1, with τ DA the donor lifetime in the presence of the acceptor and τ D the free donor lifetime.
If there is change in lifetime after cleavage (τ DA ≠ τ D), fluorescence data at multiple modulated frequencies can be used (thus a larger data set) for reconstruction through the frequency-dependent fluorescence source (17), and more accurate reconstructions can potentially be obtained due to the additional information in the large data set.
