Feasibility and parameter requirements for multiplexed fluorescence intensity and lifetime imaging.
(A) Schematic illustrating the motivation for the question. Sensor 1 and sensor 2 are intensity- based sensors with the same color but different fluorescence lifetimes (with single exponential decay constants τ1 and τ2, respectively). The fluorescence signals are combined, and then mathematically deconvolved into individual components. Dotted lines: true fluorescence lifetime histograms of the two sensors; solid lines: simulated fluorescence lifetime histograms with standard deviation (gray shading) before and after deconvolution.
(B) Schematic illustrating the question under investigation: when sensor 1 and sensor 2 change fluorescence intensities and their combined fluorescence is collected simultaneously, can the respective intensities be deconvoluted from the combined measurements?
(C) Deconvoluted number of sensor 1 photons based on different simulated sensor 1 photons. Sensor 2 was simulated with a photon count of 500k. The left and right graphs are based on either 0.5 or 4 ns differences in the single exponential decay constants between the two sensors. The left and right graphs show that the variance in deconvoluted sensor 1 photons is less when the lifetime separation between sensor 1 and sensor 2 is greater. The data are represented as means and standard deviations.
(D) Schematic illustrating the calculation of the statistical power for detecting a specific change in sensor 1 photon counts.
(E) Heatmaps showing the statistical power of detecting specific changes in the intensity of sensor 1 with different sensor 1 baseline photons, different sensor 2 photons, and different lifetime separations between the two sensors. The red line denotes the change in intensity that provides a statistical power of 80%.
(F) Relationship between the minimum detectable intensity change in sensor 1 to reach 80% statistical power and the lifetime difference between the two sensors. Curves were plotted with different sensor 1 baseline photons (left, 500k sensor 2 photons) and different sensor 2 photons (right, 200k sensor 1 baseline photons).