Fig. 5: Phasor FLIM event counting approach resolves heterogeneous MNP populations in intestinal organoids.
a Distinct positions of internalized NP D, NP B and B/D mixture clusters on a phasor plot. From left to right: comparison of average S and G coordinates of phasor cluster groups and their position on a phasor plot. Box plots represent 25 and 75 percentiles, while whiskers show standard deviation. Each point corresponds to average G or S values of individual organoids. ND = 7, NB = 10, NDB = 28. Asterisks indicate statistical difference between groups detected with Kruskal-Wallis ANOVA and following Dunn’s post-hoc test (*p < 0.05; **p < 0.0005; ***p < 0.0000005). b–d Illustration of the main steps of the fingerprint phasor FLIM event counting approach applied for tracking of heterogeneous MNP in organoids. b Typical examples of NP D and NP B loaded BO organoids with corresponding phasor plots reconstructed in LAS X software. c Phasor plot with fingerprint clusters of NP D (light magenta) and NP B (cyan) in organoids reconstructed in Excel from exported G and S coordinates. Yellow color indicates average cluster coordinates, magenta and dark blue color indicate the range of Gave ± SD. d Analysis of NP uptake from the mixture of NP D and NP B in individual organoids with apical-basal topology. Right: phasor plots of individual organoids, reconstructed in Excel. The numbers show the percentage of total cluster points in NP D, D/B and B lifetime zones. Magenta and cyan color indicate cluster points in the range of corresponding Gave +/- SD; yellow indicates D/B zone. All MNP intensity images have pseudo color masks (magenta, yellow and cyan) based on the circular ROIs of the LAS X-produced phasor plots (NP D zone with τϕ = 2.1 ns in magenta, intermediate D/B zone with τϕ = 1.5 ns in yellow, and NP B zone with τϕ = 1.0 ns in cyan, circle radius range is 46-36). Grayscale (bottom right) inserts show WGA labeling in the corresponding organoids. The scale bar is 100 µm
