Single virus particle detection and tracking
Using TIRF microscopy, we are able to detect single fluorescent viruses as they interact with the cell or its receptors. Using automated detection and tracking algorithms we can follow several hundred particles on the same surface, analyze when they land, how long they stay, and how they move on the surface. From this we can deduce mechanisms involved in viral entry and egress.
Highly sensitive fluorescence microscopy techniques make it possible to localize individual fluorescently labelled nanoparticles with nanometer precision and high temporal resolution. This allows us to detect and track individual fluorescent virus particles on both cell-surface mimics and living cells. Investigations on a single virus particle level provide the unprecedented possibility of quantifying the dynamics of the infectious entry pathway while directly seeing viral population heterogeneities and the stochastic nature of the process. Our lab harnesses this distinct vantage point to address previously intractable research questions related to viral entry and egress.
In our lab, we extensively use the surface sensitive total internal reflection fluorescence microscopy (TIRFM) technique to probe the attachment, detachment, and diffusion of individual virus particles on cell-surface mimics. Using TIRFM and an analytical approach termed equilibrium fluctuation analysis [1, 2] , we can quantify the attachment and detachment behavior of the viruses in real time and under equilibrium conditions. (Figure A) The arrival rate is directly proportional to the association rate constant (kon). In addition to this, the particle residence time, i.e. the time until the particle detaches again, can be used to estimate the dissociation rate constant (koff). The independent measure of the two rate constants allows for the determination of the apparent dissociation constant (KD) between the virus and the surface. Along with this, single particle tracking allows for the recording of the trajectories of individual particles and thus to characterize the particle’s diffusion behavior (diffusion coefficient, velocity, mobile fraction, type of motion). (Figure A)
Additionally, we work with single virus particle tracking in combination with live-cell microscopy to follow viruses from the very first moment they land on the cell surface to when they are taken up by the host cell (Figure B). In doing so, we can dissect and characterize the different steps leading to virus uptake and thereby better understand the role of each step in a successful infection. In addition, single virus tracking at the basal membrane using TIRFM can be used to look at egress.
Single particle tracking techniques applied to both cell-surface mimics and living cells allow us to comprehensively study virus-membrane interactions over a broad spectrum of system complexity. Live cell experiments describe in detail the dynamic behavior of a virus prior to entry, while well-controlled environments and cell-surface mimic compositions provide fundamental understanding on how processes occurring at the cell surface are modulated on a molecular level and how individual cellular components contribute to building up the complex, fine-tuned biomolecular interactions leading to cellular uptake and egress.
Key References