Fig 1.
IRF curves for each nominal gate width mode available in the time-gated FLIM instrument, with laser power set to 12.53 mW.
Table 1.
Camera parameters used in the simulations.
Fig 2.
Comparison between the mean values of the decay parameters τ1, τ2 and α2/α1 ratio, obtained from simulated and experimental data using the RLD algorithm.
Results are presented for three values of Δt (1, 2 and 3 times the gate width). Error bars correspond to ± SD (standard deviation).
Fig 3.
Accuracy and precision of endogenous fluorescence lifetimes obtained by simulation as a function of the gate separation Δt, for the different gains of the gated intensified camera (k).
The accuracy is measured by the relative mean error (RME) and the precision by the relative standard deviation (RSD).
Fig 4.
Accuracy of endogenous fluorescence decay parameters α1 and α2 and of the free to protein-bound ratio α2/α1 obtained by simulation as a function of the gate separation Δt, for the different gains of the gated intensified camera (k).
The accuracy is measured by the relative mean error (RME).
Fig 5.
Precision of endogenous fluorescence decay parameters α1 and α2 and of the free to protein-bound ratio α2/α1 obtained by simulation as a function of the gate separation Δt, for the different gains of the gated intensified camera (k).
The precision is measured by the relative standard deviation (RSD).
Fig 6.
Accuracy (a) and precision (b) of the free to protein-bound ratio α2/α1 for k = 2.41; Δt = 1800 ps, as a function of gate separation Δt, for a free to protein-bound ratio range between 0.25 and 2.846. On the precision graph, the ratio decreases from top to bottom. On the accuracy graph, the sequence of ratio curves is not regular.
Fig 7.
Evaluation of the best gating scheme for a solution sample.
Simulations used as expected lifetimes τ1 = 500 ps and τ2 = 4000 ps. The α1 value was varied from 0.7 to 0.9. The continuous lines correspond to the accuracy and precision ranges obtained for the decay parameters. The dots indicate the accuracy and the precision errors measured experimentally for the interval of gate separations that were considered to provide the best overall measurement performance (1600 to 2000 ps).