Chemistry Faculty Colloquium Featuring Professor Kevin Welsher: "Locking on" to single molecules and the extracellular phase of viral infection

Single-molecule measurements have the power to uncover heterogeneity and dynamics obscured by bulk methods, but are severely limited to surface-tethered phenomena. Over the past seven years, our group has sought to overcome this barrier by developing high-speed, active-feedback 3D single-molecule microscopy methods. In the first part of this talk, we introduce a new active-feedback 3D microscope to capture the dynamics of rapidly diffusing single molecules in solution (3D Single-Molecule Active Real-time Tracking or 3D-SMART). 3D-SMART "locks" single target fluorophores in the focal volume of an optical microscope using real-time feedback to move the sample and compensate for molecular diffusion at diffusive speeds up to 10 μm2/s. 3D-SMART serves to "untether" single-molecule spectroscopy measurements, dramatically expanding the application scope of these measurements to solution-phase chemical processes. In the second part of the talk, we will discuss how we have advanced this tool to capture the earliest critical events in the viral infection process: penetration of virions through the epithelial layer. When combined with a rapid volumetric imaging method (3D Tracking and Imaging, 3D-TrIm), we show that active-feedback single-virus tracking can capture the previously unobserved early events in the interactions between single viral particles and live cells. Critically, this new method can propel single-virus tracking from simple monolayer culture towards more tissue-like conditions by tracking single virions in tightly packed epithelial cells, leading to new insights into this earliest stage of the infectious cycle.