Capturing the extracellular phase of viral infection
While viruses pose a persistent threat to human health, the ability to measure the behavior of a single virus particle through the entire infection process is elusive. The critical steps in viral infection have long been studied by indirect biochemical assays, live-cell fluorescence microscopy, and electron microscopy. These measurements have shed light on the final stages of viral infection, where the virus has already attached to the tissue and has started the process of replicating its genetic material. However, this ignores the critical first stages, where the virus searches the complex extracellular space to find an infection site. Advances in real-time 3D microscopy open a new possibility of tracking the entire viral infection process, allowing us to probe the long-range interactions between the virus and the cell, the dynamics of viral attachment to the cell surface, and the role of receptors in internalization.
Real-time 3D tracking of a YFP labeled lentivirus using 3D Single-Molecule Active Real-time Tracking (3D-SMART)
Real-time 3D view of the binding of a virus-like particle with the cell surface
Coming soon – 3D Tracking and Imaging (3D-TrIm)! Capturing the extracellular phase of viral infection in full, 4D detail!
3D-TrIm Method Animation Demo
VSV-G VLP exploring the extracellular matrix
3D reconstruction of real-time VSV-G VLP trajectory in the extracellular matrix of live GM701 cells (stained with f-actin label SiR650-actin), from a 4D data set covering 10 local volumes, at 16 FPV. Trajectory (~ 162 sec) is segmented into 25 segments per second (25 frames per second when playback rate is 1×) and color mapped by time. The progress bar shows how the trajectory is further categorized: (1) Free diffusion period (playback rate: 2×): 0-14 sec, 18-38 sec, 44-62 sec, 70-108 sec. (2) Skimming period (playback rate: 1×): 14-18 sec, 38-44 sec, 62-70 sec, 108-122 sec. (3) Detachment (playback rate: 2×): 122-162 sec. Sphere represents the VLP position in the current frame (refreshing rate is consistent with the trajectory, i.e., 25 fps at 1× playback rate). Image volumes formed from maximum intensity projection over time from local volumes acquired over 16 frame-times. In (A) cells are color-coded by imaging intensity while in (B) cells are color-coded depending on distance of the virus from the cell surface. Panels (A and B) share the same trajectory color scale, camera angle and camera path, however, (A) is magnified compared to (B).
VSV-G VLP diffusing through multi-layered epithelial cells
3D reconstruction of suspended HT29-MTX cells grown on inverted matrix support (stained with f-actin label SiR650-actin) co-rendered with virus trajectory. Trajectory (~ 166 sec) is segmented into 25 segments per second (25 frames per second) and color mapped by time. Playback rate is 4×(real time). This movie shows how the global volume intensity is accumulated from local volumes acquired over 4 frame-times. Cells are color-coded by image intensity. Axes grid represents approximate position of matrix support on which the cells are suspended.