Fig. 5: Volumetric distribution of cytoskeletal components from correlative SICM-3D-SOFI images. | Nature Communications

Fig. 5: Volumetric distribution of cytoskeletal components from correlative SICM-3D-SOFI images.

From: Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

Fig. 5

Vertical cross-sections of actin (a) and tubulin (b) in a fixed COS-7 cell marked in Figs. 3c and 4a overlaid with the SICM topography displayed as a semi-transparent contour. Correlative imaging of different cellular components resolved by SICM: filopodia overlaid with fluorescently labeled f-actin from 3D-SOFI (c), microvilli overlaid with fluorescently labeled f-actin from 3D-SOFI (d) and microtubules of chemically unroofed COS-7 cells overlaid with fluorescently labeled tubulin from 3D-SOFI (e). Normalized intensity profiles are displayed below each image. The arrows in c–e are guides for the eye to indicate the locations of cross sections. For overlays only, the intensity values of SOFI images were projected on spatially filtered SICM images for a better structural representation. f Normalized Pearson cross-correlation coefficients of normalized height and intensity cross-sections for both SICM topography and fluorescence channels. Cross-sections (N = 30) were manually selected (Supplementary Fig. 13) from the SICM image for each of the features in 3 different correlative images. Correlation coefficients were measured on filipodia structures, microvilli structures and microtubules. Significantly higher (p < 0.05, two-sided t-test) correlation values were identified for filopodia(SICM)/actin (2D-SOFI), microvilli (SICM)/actin (3D-SOFI) and microtubules (SICM)/tubulin (3D-SOFI). For all comparisons, p values are shown in the upper part of f panel. The lower and upper bounds of the box plot correspond to the first and third quartiles, while the middle line shows the median. The upper and lower whiskers extend no further than 1.5 times of the interquartile range (IQR) from upper and lower bounds. Points outside this range are defined as outliers. Raw source data are provided in the Source Data File. This figure is representative of more than 10 correlative SICM and 2-color 2D/3D SOFI single-cell images obtained from at least two independent experiments.

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