Figure S1.
Parameter setups for AoC are compatible with live-imaging confocal microscopy and SIM to capture instant cellular responses during transverse stress. (A) Representative image illustrating flux-stressed axons of neurons co-expressing the F-actin marker Lifeact-GFP and the cytosolic marker mCherry. Bar = 10 µm. (B) Line profiles display normalized fluorescence intensity fluctuations in the stressed axons from A. Lifeact-GFP, labeling the axon cortex, exhibits lower fluctuations in significantly contracted or dilated axons than mCherry, which labels the cytosol. (C) The diagram illustrates key parameters for calculating the Ir, formation rate, and recovery rate of the beading process (see Materials and methods). (D) Representative time-lapse images show axonal deformation before (B), during (D), and after (A) the medium flux with indicated flow rates. Automatically detected beading regions (magenta) are shown in the bottom panels. Bar = 10 µm. (E) Representative high-temporal resolution images show the rapid beading formation stage during the initial 4.54 s following the onset of flux, which was set as 0 s. Bar = 5 μm. (F–H) Quantification of (F) peak bead number, (G) formation rate, and (H) recovery rate in response to different flux speeds (N = 11, 58, 20, 47 in F; 10, 66, 19, 59 in G; 11, 69, 20, 57 in H). (I) 3D-SIM images of axons from neurons expressing Lifeact-GFP were rendered into filaments using the Imaris software and displayed as multiple continuous spots with diameter fluctuation color-coded. Bar = 2 µm. (J) The mean axon diameter of I is shown (N = 5 axons). (K) Heatmap showing the frequency distribution of the diameter of an axon during the beading process. (L) The frequency distribution of axonal diameter before, during, and after flux is shown. (M) Quantification of normal axon segments with a diameter between 0.25 and 0.5 μm before, during, and after flux (N = 5 axons). Data represent mean ± SEM; in F and G, Mann-Whitney test; in J and M, paired two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001; n.s., non-significant.

Parameter setups for AoC are compatible with live-imaging confocal microscopy and SIM to capture instant cellular responses during transverse stress. (A) Representative image illustrating flux-stressed axons of neurons co-expressing the F-actin marker Lifeact-GFP and the cytosolic marker mCherry. Bar = 10 µm. (B) Line profiles display normalized fluorescence intensity fluctuations in the stressed axons from A. Lifeact-GFP, labeling the axon cortex, exhibits lower fluctuations in significantly contracted or dilated axons than mCherry, which labels the cytosol. (C) The diagram illustrates key parameters for calculating the Ir, formation rate, and recovery rate of the beading process (see Materials and methods). (D) Representative time-lapse images show axonal deformation before (B), during (D), and after (A) the medium flux with indicated flow rates. Automatically detected beading regions (magenta) are shown in the bottom panels. Bar = 10 µm. (E) Representative high-temporal resolution images show the rapid beading formation stage during the initial 4.54 s following the onset of flux, which was set as 0 s. Bar = 5 μm. (F–H) Quantification of (F) peak bead number, (G) formation rate, and (H) recovery rate in response to different flux speeds (N = 11, 58, 20, 47 in F; 10, 66, 19, 59 in G; 11, 69, 20, 57 in H). (I) 3D-SIM images of axons from neurons expressing Lifeact-GFP were rendered into filaments using the Imaris software and displayed as multiple continuous spots with diameter fluctuation color-coded. Bar = 2 µm. (J) The mean axon diameter of I is shown (N = 5 axons). (K) Heatmap showing the frequency distribution of the diameter of an axon during the beading process. (L) The frequency distribution of axonal diameter before, during, and after flux is shown. (M) Quantification of normal axon segments with a diameter between 0.25 and 0.5 μm before, during, and after flux (N = 5 axons). Data represent mean ± SEM; in F and G, Mann-Whitney test; in J and M, paired two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001; n.s., non-significant.

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