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 σ_x/σ_y = 15 nm and σ_z = 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.