On 21st November 2024, we'll have an on-line seminar by dr Sam Peng from MIT, who will speak about engineering photostable probes for ultralong single-molecule imaging in live cells.
Single-particle tracking (SPT) is a powerful technique to unveil molecular behaviors crucial to the understanding of many biological processes, but it is limited by factors such as probe photostability and spectral orthogonality. To overcome these limitations, we develop upconverting nanoparticles (UCNPs), which are photostable over several hours at the single-particle level, enabling long-term multicolor SPT. I will present one biological application where we investigate the dynamics of cytoplasmic dynein that is essential for intracellular transport. Despite extensive in vitro characterizations, how the dynein motors work collectively to transport vesicles and how they step in live cells remains elusive because of the much faster speed of dynein under physiological conditions. Using long-term SPT with high spatiotemporal resolutions, we found that the number of active dynein motors transporting the cargo switched stochastically from one to five pairs during the long-range transport. Moreover, our results from temperature-dependent dwell time measurements suggested that two ATP molecules were hydrolyzed sequentially during each dynein step. Our observations shed new light on the chemomechanical cycle of dynein in living cells. Finally, to enable more biological applications, we engineered 10 nm UCNPs with different colors. We found that UCNPs doped with 2% Tm3+/30% Yb3+, 10% Er3+/90% Yb3+, and 15% Tm3+/85% Yb3+ represent the optimal probes for blue, green, and near-infrared emission, respectively. The multiplexed 10 nm probes enable three-color single-particle tracking on live HeLa cells for tens of minutes. These photostable and multiplexed probes open new avenues for numerous biological applications.
Link: seminar
