Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

33-POS Board 17 Label-Free, Ultrahigh-Speed, 3D Tracking Of Single Virus Particle on Cell Plasma Membranes by Coherent Brightfield (COBRI) Microscopy Yi-Fan Huang 1 , Cheng-Hao Lin 1 , Wen Chang 2 , Chia-Lung Hsieh 1 . 1 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan, 2 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan. Viral infection is a multistep process, starting with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. We used coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution[1]. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allowed us to track the motion of a single vaccinia virus particle with nanometer spatial precision (< 3 nm) in 3D and microsecond temporal resolution (up to 210,000 frames per second). Through image post-processing, relatively stationary background scattering of cellular structures was effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we revealed single virus particles exploring cell plasma membranes with unprecedented clarity. We found that immediately after attaching to the membrane (within a second), the virus particle was locally confined within hundreds of nanometers. Surprisingly, within this confinement, the virus particle diffuses laterally with a very high diffusion coefficient (~1 μm2/s) in microsecond-timescale. We also observed numerous transient confinements of the virus particle in nano-sized zones on the membrane, presumably due to the rapid interaction between the virus and the cell membrane receptors. In order to elucidate the virus-membrane interaction with specificity, we further manipulated cell membrane receptors and the viral membrane proteins via molecular biological approaches. Critical interplays between viral membrane proteins and cell receptors that were thought to determine endocytic pathways (e.g., endocytosis and membrane fusion) were investigated at the molecular scales. Keywords: coherent brightfield microscopy; single-virus tracking; early-stage infection; digital background removal, virus-membrane interaction. References: Yi-Fan Huang et al., ACS Nano, Article ASAP.

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