Figure 6.
sEVs bind to laminin on the PMs of living MRC-5 cells, as revealed by pseudo real-time super-resolution movie observation. (A) Schematic diagram for generating merged movies of dSTORM images of the ECM structures and single-particle images of sEVs. Data acquisition was performed by observing single-fluorescent molecules of ECM components immunostained with SF650B-conjugated antibodies at 200 frames/s (3,504 frames), and dSTORM images were reconstructed using the data acquired every 5.0 s (=1,002 frames). The first dSTORM image was reconstructed using frames 1–1,002, and the process was then repeated by shifting the initial frames backward by 6 frames; thus, 417 dSTORM images were obtained. These dSTORM still images were connected to construct the dSTORM movie. The single-particle movies of sEVs were subjected to a rolling average for 6 frames and synchronously merged with the dSTORM movie. (B–D) Conventional immunofluorescence images (top-left) and dSTORM images (bottom-left) of the ECM structures (B: fibronectin, C: collagen type I, and D: laminin) on the cells. dSTORM images of the ECM structures (magenta) and single-particle images of sEV–CD63Halo7-TMR particles (green) were merged (right). sEVs localized near (<100 nm) the boundary of ECM structures and sEVs localized alone are indicated by yellow and white arrowheads, respectively. (E) The image sequence (every 0.3 s) of laminin obtained by dSTORM (magenta) and a single sEV–CD63Halo7-TMR particle (green) on the living MRC-5 cell membrane. sEVs colocalized with laminin, as indicated by the yellow arrowhead. (F) Colocalization analysis method. We measured the nearest distance from an edge of the ECM structure to a centroid of the sEV spot and performed this measurement for all pairs of ECM structures and sEV particles. The normalized relative frequency was defined as the ratio of the average value of the spatial pair correlation function of the actual image to that of randomly distributed spots generated by a computer. We obtained histograms showing the distribution of the normalized relative frequency of sEVs at each distance from the edge of the ECM structures. Zero on the x axis indicates the contour of the ECM structures in the dSTORM images determined by the KDE method. When sEVs are enriched near the ECM structures, the normalized relative frequency is >1. (G) Probability density analysis of the sEVs and ECM structures. The colored areas indicate regions within the ECM structures. The sEV–CD63Halo7-TMR particles localized near the contour of laminin (n = 20 cells).

sEVs bind to laminin on the PMs of living MRC-5 cells, as revealed by pseudo real-time super-resolution movie observation. (A) Schematic diagram for generating merged movies of dSTORM images of the ECM structures and single-particle images of sEVs. Data acquisition was performed by observing single-fluorescent molecules of ECM components immunostained with SF650B-conjugated antibodies at 200 frames/s (3,504 frames), and dSTORM images were reconstructed using the data acquired every 5.0 s (=1,002 frames). The first dSTORM image was reconstructed using frames 1–1,002, and the process was then repeated by shifting the initial frames backward by 6 frames; thus, 417 dSTORM images were obtained. These dSTORM still images were connected to construct the dSTORM movie. The single-particle movies of sEVs were subjected to a rolling average for 6 frames and synchronously merged with the dSTORM movie. (B–D) Conventional immunofluorescence images (top-left) and dSTORM images (bottom-left) of the ECM structures (B: fibronectin, C: collagen type I, and D: laminin) on the cells. dSTORM images of the ECM structures (magenta) and single-particle images of sEV–CD63Halo7-TMR particles (green) were merged (right). sEVs localized near (<100 nm) the boundary of ECM structures and sEVs localized alone are indicated by yellow and white arrowheads, respectively. (E) The image sequence (every 0.3 s) of laminin obtained by dSTORM (magenta) and a single sEV–CD63Halo7-TMR particle (green) on the living MRC-5 cell membrane. sEVs colocalized with laminin, as indicated by the yellow arrowhead. (F) Colocalization analysis method. We measured the nearest distance from an edge of the ECM structure to a centroid of the sEV spot and performed this measurement for all pairs of ECM structures and sEV particles. The normalized relative frequency was defined as the ratio of the average value of the spatial pair correlation function of the actual image to that of randomly distributed spots generated by a computer. We obtained histograms showing the distribution of the normalized relative frequency of sEVs at each distance from the edge of the ECM structures. Zero on the x axis indicates the contour of the ECM structures in the dSTORM images determined by the KDE method. When sEVs are enriched near the ECM structures, the normalized relative frequency is >1. (G) Probability density analysis of the sEVs and ECM structures. The colored areas indicate regions within the ECM structures. The sEV–CD63Halo7-TMR particles localized near the contour of laminin (n = 20 cells).

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