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. 2017 Aug 18;6:e24820. doi: 10.7554/eLife.24820

Figure 5. Fat4-citrine and Ds1-mCherry fluorescence at the boundary between cells are shifted by 100–200 nm.

(A) A high resolution image of a boundary exhibiting a ‘rainbow’ feature (composed of three stripes green, yellow and red; white arrow) indicating a shift between red and green fluorescence. Scale bar - 5 μm. (B) An illustration of the observed ‘rainbow’ feature. (C) A straightened version of the boundary shown in A (top). Fluorescence profiles (bottom) of Fat4-citrine (green) and Ds1-mCherry (red) along lines perpendicular to the boundary. Mean gap size for this boundary is as indicated (D) Probability distribution function of the distance between the peaks in the fluorescence profiles. Mean gap width for 61 boundaries as indicated. Supplementary figure (Figure 5—figure supplement 1) shows control experiments in MCF7 cells, super resolution STED images, and rainbows after correction of chromatic aberrations.

Figure 5.

Figure 5—figure supplement 1. The rainbow feature is observed in other cell types and with super resolution microscopy.

Figure 5—figure supplement 1.

(A–C) A rainbow feature observed in a confocal image of a co-culture of Fat4-citrine and Ds1-mCherry in MCF7 cells. Scale bar - 10 μm. (D) A zoom in on the boundary in (C). Scale bar - 2 μm. (E–G) A super-resolution image of the rainbow feature in a stimulated emission depletion (STED) microscope (Here, the co-culture is the same as Figure 5A). Scale bar - 2 μm. (H) A zoom in on the boundary in (G). Scale bar - 0.5 μm. (I–N) High resolution confocal images of a rainbow next to a 100 nm tetraspeck bead (marked by an arrow). (I) A raw image of the boundary without any correction. Scale bar - 2.5 μm. (J) A zoom in on the bead in (I). (K) A zoom in on the boundary in (I). Scale bar - 0.5 μm. (L) The image in (I) after correction of chromatic aberration. The correction of chromatic aberration was performed by applying global conformal transformation over the whole image (not shown) that minimizes the distances between the centers of the beads in the two channels. While the correction eliminates the shift between the two channels on the bead (M), the rainbow feature in the boundary is maintained (L,N).