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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
61
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.