Analysis of organelle membrane thickness heterogeneity within an exemplifying tomogram. (A) Central slice from a denoised C. reinhardtii tomogram (Kelley et al., 2024, Preprint) (EMPIAR-11830, 2140.mrc, 7.84 Å/pix at bin4), showing the ER, IMM, OMM, thylakoid, and chloroplast membranes. Scale bar, 100 nm. (B) Instance segmentation generated by MemBrain-seg (Lamm et al., 2025, Preprint), with distinct colors marking different membrane instances. (C) Membrane thickness map, where color intensity represents differences in local membrane thickness. (D) Detailed view of the surface point extraction and normal vector orientation step, shown for a region of the chloroplast membranes (marked by black boxes in A and B). Points assigned to surface one in dark green, and to surface two in light gray. Black lines indicate normal vectors from each point to the opposite segmentation surface. (E) Comparative thickness distribution plots across membranes (color-coded as in B). Box plots show median (center line), interquartile range (box), and whiskers extending to 1.5× the interquartile range. (F) Co-localization of local membrane thickness with macromolecular complexes in a single mitochondrial crista (black box in C). ATP synthase particle positions were obtained from Righetto et al. (2025) as determined by subtomogram-averaging by Kelley et al. (2024, Preprint), with the resulting subtomogram average map (EMD-52802) used for visualization.
Figure 2.

Analysis of organelle membrane thickness heterogeneity within an exemplifying tomogram. (A) Central slice from a denoised C. reinhardtii tomogram (Kelley et al., 2024, Preprint) (EMPIAR-11830, 2140.mrc, 7.84 Å/pix at bin4), showing the ER, IMM, OMM, thylakoid, and chloroplast membranes. Scale bar, 100 nm. (B) Instance segmentation generated by MemBrain-seg (Lamm et al., 2025, Preprint), with distinct colors marking different membrane instances. (C) Membrane thickness map, where color intensity represents differences in local membrane thickness. (D) Detailed view of the surface point extraction and normal vector orientation step, shown for a region of the chloroplast membranes (marked by black boxes in A and B). Points assigned to surface one in dark green, and to surface two in light gray. Black lines indicate normal vectors from each point to the opposite segmentation surface. (E) Comparative thickness distribution plots across membranes (color-coded as in B). Box plots show median (center line), interquartile range (box), and whiskers extending to 1.5× the interquartile range. (F) Co-localization of local membrane thickness with macromolecular complexes in a single mitochondrial crista (black box in C). ATP synthase particle positions were obtained from Righetto et al. (2025) as determined by subtomogram-averaging by Kelley et al. (2024, Preprint), with the resulting subtomogram average map (EMD-52802) used for visualization.

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