Label-free, ultrahigh-speed, 3D observation of bidirectional and correlated intracellular cargo transport by coherent brightfield microscopy

Abstract

The investigation of intracellular transport at the molecular scale requires measurements at high spatial and temporal resolutions. We demonstrate the label-free, direct imaging and tracking of native cell vesicles in live cells at an ultrahigh spatiotemporal resolution. Using coherent brightfield (COBRI) microscopy, we monitor individual cell vesicles traveling inside the cell with nanometer spatial precision in 3D at 30 000 frames per second. The stepwise directional motion of the vesicle on the cytoskeletal track is clearly resolved. We also observe the repeated switching of the transport direction of the vesicle in a continuous trajectory. Our high-resolution measurement unveils the transient pausing and subtle bidirectional motion of the vesicle, taking place over tens of nanometers in tens of milliseconds. By tracking multiple particles simultaneously, we found strong correlations between the motions of two neighboring vesicles. Our label-free ultrahigh-speed optical imaging provides the opportunity to visualize intracellular cargo transport at the nanoscale in the microsecond timescale with minimal perturbation.

Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking

“…In this study, by using scattering imaging of gold nanoparticle (AuNP), we visualized load-free fast stepping motion of artificially-dimerized chimeric dynein with 100 µs time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. Since AuNP strongly scatters light at its plasmon resonance wavelength without suffering from photobleaching and blinking, it has been used as a probe of single-molecule imaging of linear and rotary motor proteins in vitro [17][18][19][20][21][22][23][24][25] and imaging of intracellular cargo transport [26][27][28] . We analyzed trajectory of the stepping motion of the chimeric dynein in detail, including the step size, preference of the step direction, and dwell time, and revealed that this chimeric dynein moves with biased small stepping motion in which only backward steps are slightly suppressed compared to forward and side steps.…”

High-Precision Protein-Tracking With Interferometric Scattering Microscopy

“…To address the issue of intracellular transport more conclusively, one requires further investigation with a larger sample size to draw a more robust statistical analysis as well as measurement which harnesses fluorescence-based labeling to identify and visualize intracellular filaments and motor proteins in parallel with the iSPT tracking. Nonetheless, these initial efforts and recent similar in vivo iSPT tracking of cytoplasmatic vesicle transport (Huang et al, 2017b) demonstrate that iSPT is well-suited to investigate the nanoscopic minutia of intracellular transport events in living cells.…”

High-precision protein-tracking with interferometric scattering microscopy

“…To address the issue of intracellular transport more conclusively, one requires further investigation with a larger sample size to draw a more robust statistical analysis as well as measurement which harnesses fluorescence-based labelling to identify and visualise intracellular filaments and motor proteins in parallel with the iSPT tracking. Nonetheless, these initial efforts and recent similar in vivo iSPT tracking of cytoplasmatic vesicle transport (100) demonstrate that iSPT is well suited to investigate the nanoscopic minutia of intracellular transport events in living cells.…”