Figure 4 | Scientific Reports

Figure 4

From: Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy

Figure 4

Quantitative validation of organization of the MPS in sensory axons as obtained by ExM compared to STED nanoscopy. (a) Selected images from βII-spectrin stained DRG sensory axons obtained by either STED or ExM. Scale bar: 500 nm. (b) The Gollum software enabled quantitative assessment of the global correlation values (as shown in (c)) and estimation of the abundance (as shown in d). Briefly, the algorithm first recognizes axons and lays consecutive 1 µm × 1 µm segments. A modeled MPS pattern is superimposed on the image (b1) and a Pearson correlation is obtained for each segment (b2). MPS abundance is calculated by the proportion of segments with a correlation value above a defined threshold correlation that would correspond to a visible MPS, as predefined by the user (b3). See Materials and methods for more details. (c) Histogram showing the global correlation values obtained by STED (green line, n = 300) compared to ExM (red line, n = 140). (d) Graph showing the MPS abundance as observed by STED or ExM. Mean ± SEM, n = 7. (e) Mean correlation values obtained from control sensory axons (NGF), compared to axons subjected to NGF withdrawal for 12 hours. Mean ± SEM. n = 20.

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