Imaging of surface microdomains on individual extracellular vesicles in 3-D

Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single-particle studies under physiological conditions have been hampered.

Researchers from the University of North Carolina at Chapel Hill employed direct stochastic optical reconstruction microscopy (dSTORM) to visualize EVs in three-dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo-EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated.

3-D reconstruction of a single EV

(A) Outline of the geometric foundation, with “slide” indicating the focal plane at z = -max. The filled red circle and red radius depict how a 2D image would look like. Above, the red sphere indicates the ideal EV, and the orange ellipsoid indicates the actual data. The red arrow r = sqrt (x² + y²) depicts the radius at the equator, whereas “Norm” indicates the point vector from the centre to any point on the ellipsoid surface. Norm = sqrt (x² + y² +z ), which at z = 0 equals r. (B) 3-D representation of the data before transformation and (C) after transformation. (D) Principal component analysis (PCA) plot of an EV using 12 nm Z-axis binning of photoswitching events, showing hollowed core. (E) Same as D, but for a bin at one Z-axis increment shift. (F) Same as E, but for a bin at one Z-axis increment shift