The challenge in observing the viral infection process in its entirety rests in the difficulty of simultaneously addressing the multiple spatial and temporal scales associated with the process. An extracellular virion can move with speeds up to and exceeding 10 m2/sec. Meanwhile, these nanoscale particles interact with cells on the 10s of microns scale. These interactions are dictated by the subtle molecular motions of single proteins within the virus and at cellular interfaces at the nanometer and microsecond time scales. Current imaging methods cannot successfully synthesize information at these disparate scales. Cellular imaging methods were developed with intention to focus on a cell or cells and image as rapidly as possible. The overhead associated with imaging such large areas diminishes temporal precision. With this proposal, we aim to develop and apply virus-targeted imaging. These new methods will lock on to single viral particles starting in the extracellular space, following them with photon-limited temporal resolution throughout the entirety of the viral infection pathway. These unprecedented measurements will uncover the transient interactions which underlie the critical infection points, such as the moment of viral landing, the association of ligands and receptors, viral envelope fusion and associated cellular membrane fluctuations responsible for viral uptake. Research Direction 1 will describe the development and implementation of a highly sensitive real-time 3D single particle tracking technique capable of locking on to a single virus particle lightly stained with a fluorescent dye or fluorescent protein fusion. The technique will utilize a rapidly moving 3D laser spot generated by a 2D electro- optic deflector and a tunable acoustic gradient (TAG) lens. The rapidly moving laser spot will tag each photon received from the viral particle with position information. These rapid position measurements will be used to drive a piezoelectric stage to lock on to the viral particle's position, holding it in the objective focal volume while exerting zero force on the particle. This will yield continuous photon-limited observation of the viral particle. Research Direction 2 will merge this continuous observation with 3D volumetric imaging to correlate the behavior of the viral particle with the larger cell environment, enabling measurement of the exact moment of viral landing, as well as the interaction of viral particles with the extracellular matrix. Research Direction 3 will extend this volumetric imaging down to the submillisecond time scale local to the viral particle. This will enable highly rapid measurement of ligand-receptor interactions at the cell surface. Research Direction 4 will probe the viral envelope fusion process to pinpoint the spatiotemporal details of the viral envelope at different stages of the viral life-cycle for pH independent viruses. Taken together, these research directions will carve a new path into the study of viral infection. By discarding the measurement overhead associated with measuring a large 3D cellular surface, these new approaches will probe into uncharted spatiotemporal resolution of the viral infection process.
The study of viral infection has been impeded by lack of spatiotemporal precision in live cell studies. This proposal aims to develop virus-locked 3D imaging methods to enable continuous observation throughout the viral lifecycle. These methods will investigate the interactions of viral particles with the extracellular matrix, the cell membrane and cell surface receptors with unprecedented spatiotemporal resolution.
| Hou, Shangguo; Welsher, Kevin (2018) A Protocol for Real-time 3D Single Particle Tracking. J Vis Exp : |
