REAL-TIME 3D SINGLE-PARTICLE TRACKING AND 3D MULTI-RESOLUTION IMAGING

The interior of a cell is highly heterogeneous where molecular and nanoscale dynamics span multiple time and length scales. Furthermore, these dynamics all occur in three dimensions (3D). For example: How does a viral particle or a vaccine-delivery vehicle approach a living cell and become internalized? What are the dynamic molecule-level interactions that lead to a (un)successful entry? How does the intracellular cargo get delivered to its destination? To understand the molecule-nanoscale dynamics in the live-cell context, time must be explicitly considered. My lab recognized this problem very early on and has developed a real-time 3D single-particle tracking (RT-3DSPT) technology which enables us to follow a tagged nanoscale probe with 10 μs time resolution and ~10 nm spatial localization precision. We are also developing new ways to integrate this RT-3DSPT technology to a variety of imaging microscopy modalities as well as single-particle spectroscopy of a freely moving nanoparticle such as the measurement of single-particle 3D reorientation dynamics.

3D Multi-Resolution Microscopy

We have used our high-resolution RT-3DSPT combined with a lower-resolution imaging modality that gives the surrounding larger cell context to study the early stages of cellular uptake of a virus-like nanoparticle. With this we were able to observe correlation between small membrane terrain structures and the nanoparticle dynamics.

Links:
https://doi.org/10.1038/nnano.2014.12
https://doi.org/10.1039/c5fd00090d

Multi-focal Microscopy

We have implemented a multi-color, multi-focal microscope to acquire 3D volume images more quickly. We are currently using this to understand diffusion in confined environments.

Links:
https://doi.org/10.1371/journal.pone.0230217
https://doi.org/10.1364/JOSAB.430594

Lifetime Gating

We have implemented hardware-based lifetime gating to improve our signal-to-background ratio in RT-3DSPT. We are currently using this new technology to study interfacial enzyme catalysis in high-background biological environments, in situ.

Links:
https://doi.org/10.1063/5.0063634