(A) Same as main Figure 2A, but now with logarithmic scaling. Shown are representative whole-cell fluorescence lifetime decay curves of dendritic cells expressing Stx3-mCitrine (red curves; left graphs), Stx3-mCitrine with VAMP3-mCherry (green; middle graphs), or Stx3 conjugated to both mCitrine and mCherry (Stx3-mCitrine-mCherry; cyan; right graphs). Dashed lines: fits with mono-exponential decay functions convoluted with the instrument response function (IRF; gray; residuals from the fits shown). Graphs are normalized to the maximum photon counts (depicted in each graph). (B) Same as main Figure 2B, but now with logarithmic scaling. Shown is the overlap of the fluorescence lifetime decay curves from panel A. (C) Representative fluorescence lifetime decay curve of rhodamine B in methanol (about 1 µM concentration; magenta curves). Dashed line: fit with a mono-exponential decay function. The fit is shown with a linear (left) and a logarithmic (right) scaling. A fluorescence lifetime was obtained of 2.19 ± 0.04 ns (mean ± SEM from four independent measurements), close to the reported lifetime of 2.32 ns (Kristoffersen et al., 2014). (D) Same as main Figure 2C, but now the average whole-cell apparent fluorescence lifetimes of individual donors are shown (n: number of donors; mean ± SEM shown; one-way ANOVA with Bonferroni correction). (E) Same as main Figure 2D for three more donors. Shown are whole-cell apparent fluorescence lifetimes of Stx3-mCitrine from fits with mono-exponential decay functions for individual cells co-expressing Stx3-mCitrine with VAMP3-mCherry. Dashed lines: linear regression (top: β = −0.001, R2 = 0.756; middle: β = −0.001, R2 = 0.702; bottom: β = −0.001, R2 = 0.869). (F) Representative confocal microscopy and FLIM images of a dendritic cell co-expressing Stx3-mCitrine (green in merge) and VAMP3-mCherry (magenta). The intracellular (I; light blue in lower right panel) and peripheral (P; white) regions of the imaged cell area were manually selected. BF: bright-field. Scale bar, 10 µm. (G) Whole-cell fluorescence lifetime decay curves of the intracellular (I) and peripheral (P) cell regions for the cell from panel F. The apparent fluorescence lifetimes obtained from mono-exponential fits were 2.41 ns (I) and 2.24 ns (P).