12:00 pm Zoom
"Untethering single-molecule spectroscopy and capturing the dynamics of extracellular viruses"
Capturing dynamic processes occurring at high speeds in living systems is frequently limited by the acquisition time of existing live-cell microscopy methods. This limitation is particularly true in three dimensions, where extended axial sampling reduces the volumetric imaging rate. Here we introduce a new active-feedback 3D microscopy technique that overcomes this 3D imaging speed limit to capture the dynamics of rapidly diffusing single molecules in solution (3D Single-Molecule Active Real-time Tracking or 3D-SMART). This method "locks" target fluorophores in the focal volume of an optical microscope using real-time feedback to move the sample and compensate for molecular diffusion. 3D-SMART has been successfully applied to capture a wide range of targets, from single virus-like particles down to single proteins and nucleic acids at diffusive speeds up to 10 μm2/s. We will further describe how this microscope, when combined with a rapid volumetric imaging method, can capture the early events in the interactions between single viral particles and live cells in three dimensions with millisecond or better temporal resolution.