Fig. 5: Classification of five progressive stages of apoptosis based on chromatin structure.

a Live confocal images (left, time-stamped images) followed by SMLM (images with typical details highlighted by white boxes) after fixation of the same nucleus from a cultured primary cortical neuron expressing H2B::mCherry; scale: 5 µm in both. Time stamps on the confocal images refer to time elapsed since the beginning of the acquisition. Note, in cells identified to be at stages 4 and 5, a transient caspase-3 activation (indicated by NucView*) was detectable during the confocal acquisition. Representative details highlighted in super resolution images (white boxes, scale 0.5 µm) show the compaction of chromatin increasing from stage 1 to stage 5. b Intensity plots of lines in representative detail views for stages 1, 2, and 4 show a stepwise increase in signal intensity, which indicates an increase in the compaction of chromatin clusters in neuronal nuclei. c Theoretical model describing chromatin appearance during the five identified stages of apoptosis based on the observed morphological changes presented by neuronal nuclei before and during the apoptotic process. d–g Quantitative comparison of average nuclear size, edge count, H2B::mCherry and NucView signal intensities of neuronal nuclei categorized into stages 1–5 (n = 13/6/4/3/2 cells). Data are represented as mean ± SEM. One-way ANOVA was applied for comparison of nuclear size F(4, 23)=27.02 p < 0.0001 and edge count F(4, 23) = 4.11 p = 0.0118, Kruskal-Wallis test for comparison of H2B::mCherry signal intensity H(5) = 12.49, p = 0.0141, and caspase activation H(5) = 16.75, p = 0.002 across nuclei at different stages.