3D tracking of extracellular vesicles by holographic fluorescence imaging

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Researchers from The Barcelona Institute of Science and Technology report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. They expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, the researchers image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), they resolve near-isotropic 3D diffusion and directional transport. For longer lag times, they observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.

Proof-of-concept experiments

(A) A 200-nm fluorescent bead recorded 4.4 μm above focus (top) is computationally refocused (bottom). The inset shows an experimentally obtained in-focus image of the same particle alongside a cut through the respective PSFs (white dashed: in-focus; pink, solid: refocused). (B) Simultaneous 3D tracking of three 200-nm fluorescent beads by moving the sample with a piezo-stage along a known trajectory (pink: piezo movement; blue: reconstructed trajectories of individual beads; black: mean trajectory). The individual trajectories are overlaid in x/y for clarity; z = 0 μm corresponds to a particle being in focus. (C) Intended sub-diffraction–limited piezo-trajectories (pink) compared to a typical image obtained 900 nm above the focus (left). The resulting y/z and x/z mean trajectory projections (black) agree well with the piezo-trajectory (pink), and blue dots show all positions obtained by simultaneously tracking 17 individual fluorescent beads (right). Histogram-based analysis of the localization precisions yield σxy = 15 nm and σz = 21.5 nm, respectively (note S7). (D) Single ATTO647N molecules recorded out of focus (left) are successfully computationally focused (middle). The representative areas of fluorescence emission (pink, purple, and blue) show one-step photobleaching as expected for single emitters. (E) Photobleaching time traces of the three regions highlighted in (D); the dashed line indicates the background level.

Liebel M, Ortega Arroyo J, Beltrán VS, Osmond J, Jo A, Lee H, Quidant R, van Hulst NF. (2020) 3D tracking of extracellular vesicles by holographic fluorescence imaging. Sci Adv 6(45):eabc2508. [article]

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