Fig. 5: Visualization of NK-cell degranulation by ExM and computation analysis of granule size pre-/post-ExM.

Non-degranulated NK cells (a) that remain negative for LAMP1 surface marker protein (cyan) show perforin structures (magenta) in a ring-like shape (see a magnification of the indicated box for details). Degranulated NK cells (b, c) show a high concentration of LAMP1 protein at the surface during degranulation (b) and perforin surrounded by LAMP1 is frequently observed (c). Note the differences in the size of perforin accumulation in a and b, c. NK cells were cocultured with A. fumigatus hyphae for 2.5 h and then treated with BFA for 2 h. All images shown represent maximum intensity z-projections of all slices with exception (magn. in b, z projection of slices 9–14). Dotted lines indicate borders of NK cells (gray) and fungal hyphae (yellow). Scale bars, 10 µm (a–c) and 2 µm in magnifications (magn.). Representative images of two biological replicates (a–c). d Computational analysis workflow. Volumes of interest (VOI) were identified in the perforin channel by a connected component analysis and compared with the LAMP1 signal. Volumes that had more than 10% correlating voxels in the LAMP1 channel were judged as degranulated. VOI were further segmented by cellpose. The volume was computed using a convex hull algorithm. For diameter calculation, a spherical shape of particles was assumed. e, f Histogram showing the diameter (top) and volume (bottom) distribution of degranulated and non-degranulated particles before (d) and after (e) expansion. e Before the expansion, there is no noticeable difference in volume or diameter. Note that diameters are close to the resolution limit, especially regarding the sampling rate in the z-direction. f After expansion, degranulated particles exhibit lower volume and lower diameter than non-degranulated particles. Clusters are drawn apart and can be separated due to higher resolution.