Figure 1.

Pixel binning for particle phasor-FLIM. (A) Intensity image of simulated subresolution single particles with variety of intensities but a constant lifetime of 4 ns. (B) Phasor analysis of the simulated photon data results in a wide spread on the phasor plot where every pixel (threshold for pixels with more than five counts) is assigned one phasor. (C) In blue: scatterplot of the pixel counts against the phase lifetime determined from the phasor plot. In black: standard deviation and mean lifetime per bin. Bins are defined as 25 counts wide, mean and standard deviation are plotted in the center coordinate of each bin. (D) Intensity image color coded in red if the pixel counts >300, inset is a 2.5- × 2.5-μm box. (E) Schematic of a single particle consisting of pixels from the same subresolution origin and showing identical decay lifetime composition but different amplitudes. (F) Pixel binning improves the particle phasor. (G) Simulated data from (A–D) analyzed in phasor with particle-based pixel binning show a compact phasor distribution. (H) Particles color coded if the particle counts are >300 counts. Scale bars, 5 μm.