Cell level changes in endogenous fluorescence intensity and lifetime with loss of pluripotency of mESCs.

A. GFP intensity of MPLSM single optical section of live mESCs expressing the pOct4:GFP transgene at 5 days post-EB formation using 890 nm excitation and 520/35 nm emission filter. Right image shows regions of interest (ROI), roughly corresponding to single cells, with high intensity GFP (GFP(H)) outlined with red; medium intensity GFP (GFP(M)) outlined with orange; low intensity GFP (GFP(L)) outlined with blue. B. Quantitative assessment of ROIs of GFP intensity. C. Intensity images of the same MPLSM single optical section as (A) using 780 nm excitation and 457/50 nm emission, corresponding primarily to NADH endogenous fluorescence. Right images show same ROIs as (A). D. Quantitative assessment of ROIs of endogenous intensity. E. Color mapped mean lifetime (τ_m) images of endogenous fluorescence showing the same ROIs, with quantitative assessment in F. G. Color mapped long lifetime (τ₂) images of endogenous fluorescence with quantitative assessment of ROIs (H). Scale bars = 20 µm. Color bar indicates lifetime values for τ_m (E) or τ₂ (G). * indicates significant difference between GFP(H) ROIs and ROIs of medium or low GFP intensity (P≤0.001; ANOVA on ranks followed by Tukey multiple pairwise comparison test at P<0.05).