Figure 2.

Morphologic Analysis of the Cellular Changes in the Early Stage of Cell Death by AFM

(A–D) Four-panel morphological characterization: top left, 3D AFM image; top right, whole-cell 2D AFM image; bottom left, AFM close up of RCD modality-specific characteristics; bottom right, profiles indicating geometries of typical characteristics noticed for each RCD. (A) Intrinsic apoptosis is characterized by shrinkage while maintaining adhesion points (white arrows), height profiles indicate diameters of the remaining focal adhesion points, close-up (bottom-left panel) shows observed surface irregularities in the intrinsic apoptosis. (B) Extrinsic apoptosis is characterized by total cell shrinkage and extensive blebbing (white arrows), height profiles: the average diameters of blebs. (C) Necroptosis is characterized by the overall shrinkage accompanied by the appearance of sub-micron pore-like membrane disruptions (white arrows in the close-up [bottom left panel]), height profiles: the diameter and depth of the pores. (D) Ferroptosis is characterized by the shrinkage and the presence of uniform circular membrane protrusions (indicated by white arrows), height profiles: dimensions of these protrusions.

(E) Bar graph comparing three roughness parameters extracted from AFM images: Ra, the average roughness; Rq, the RMS roughness; Rt, the maximum height of the profile. For all RCD modalities, a significant increase is noted (black stars) compared with control cells (orange bars), which allows to quantitatively distinguish dead cells from viable cells based on morphological data. Both ferroptotic and extrinsic apoptotic cells also exhibit a significantly higher roughness compared with that for necroptotic cells (red stars). Error bars represent the standard deviation (n = 15). The statistical analysis was determined by a Kruskal-Wallis test followed by a pairwise Wilcoxon test with Benjamini-Hochberg adjusted p values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S2.