ACS Nano. 2017 Mar 28;11(3):2575-2585. doi: 10.1021/acsnano.6b05601.

Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells.

Huang YF1, Zhuo GY1, Chou CY1, Lin CH1, Chang W2, Hsieh CL1.

1 Institute of Atomic and Molecular Sciences, Academia Sinica , Taipei 10617, Taiwan.
2 Institute of Molecular Biology, Academia Sinica , Taipei 11529, Taiwan.



Viral infection starts 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. Here, we use coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allows 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 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image postprocessing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (∼1 μm2/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.


3D localization; coherent brightfield microscopy; diffusion; digital background removal; early stage infection; high-speed imaging; interferometric imaging; local delivery of virus particle; single-virus tracking; virus−membrane interaction

PMID: 28067508



Despite the great advance in biomedical sciences, unexpected disease outbreaks via viral infection still threaten our lives and cause significant economic loss. Meanwhile, virus has also been used as vaccine vector in the battle against many diseases, including recombinant HIV vaccine and vectors for cancer therapy. Toward the development of better anti-viral strategy and vaccine with optimal efficacy, it is necessary to understand how virus infects host cells. Viral infection is an extremely complex process. A lot about viral infection have been learned by biochemical and molecular biological approaches. With fluorescence optical microscopy, it is now possible to monitor the process of viral infection in live cells at single virus level in real time. It has been observed that in a viral infection, a virus particle needs to attach to the cell surface, moves laterally, interacts with cellular receptors, and then penetrates through the cell plasma membrane. Dynamic interaction between the virus particle and the cell membrane receptor is crucial for the success of viral infection. Due to the small size of the virus particle and the membrane receptor, it is difficult to inspect their interaction with sufficient spatial and temporal resolution. As a result, little is known about how a virus particle finds the receptor and how their interaction leads to membrane penetration.


A research team at Academia Sinica, the national research institute in Taiwan, recently demonstrated an ultrahigh-speed optical microscope technique that shows promise of resolving the rapid virus-receptor interaction with molecular clarity in live cells. This new technique, called Coherent Brightfield (COBRI) microscopy (Fig. 1), exploits the linear scattering of the native virus particle as the signal. Compared to the fluorescence signal that has been popularly used for virus imaging, linear scattering signal is stable, making it more suitable for high speed, high precision, and long-term observation. In addition, this technique does not require any label and thus avoids possible artifacts due to the labeling. It allows the researchers to investigate the viral infection in its most native condition.



Figure 1. Coherent brightfield (COBRI) microscopy allows researchers to investigate rapid virus-host interaction through direct visualization.


Although the merit of scattering signal has been well recognized, imaging and tracking virus particles in live cells via scattering has been thought difficult (if not impossible) because the virus particle is very small. The scattering signal of the virus is much weaker than the scattering background created by the cell structures. The team led by Dr. Chia-Lung Hsieh at Academia Sinica tried to overcome this difficulty by two approaches. First, they detected the scattering signal by imaging-based interferometry, which greatly enhances the imaging sensitivity for detecting individual virus particles. Then, they used image post-processing to separate the relatively stationary cell background from the highly dynamic virus signal. This approach becomes particularly useful in high-speed imaging where the cell barely moves in a few seconds while the virus constantly diffuses over the space because of its small size. Using their methods, background-free virus image can be captured even in the live cellular environment at a very high acquisition rate.


The research team successfully recorded the 3D motion of single vaccinia virus particle attaching to the cell membrane in a continuous manner. In order to capture the landing process, the virus particles were locally delivered to the microscope observation area through a micropipette. The video was recorded at high speed of 100,000 frames per second. In each frame, the virus position is determined with a precision better than 3 nm in all three dimensions. From the technology point of view, this is the first demonstration of label-free imaging and tracking of single virus particle in live cells. Moreover, the measurement was performed at ultrahigh spatiotemporal resolution (nanometer spatial precision and microsecond temporal resolution). The spatiotemporal resolution provided by this new technique allows the researchers to investigate virus-membrane interaction at the molecular scale.


It was found that, immediately after attaching to the membrane, the virus particle was confined in a zone of hundreds of nanometers. Surprisingly, within this confined area, the virus laterally explored the membrane with a very high diffusion coefficient. During the local exploration, the virus was trapped in numerous nano-sized zones that could possibly reflect the transient virus-receptor interaction. This project is still actively ongoing and the goal is to identify and resolve the interplay between the virus particle and membrane receptors.


Link to the research team:


More studies of ultrahigh-speed optical microscope imaging and tracking:

  1. Huang YF, Zhuo GY, Chou CY, Lin CH, Hsieh CL, “Label-free, ultrahigh-speed, 3D observation of bidirectional and correlated intracellular cargo transport by coherent brightfield microscopy,” Nanoscale, 9, pp. 6567-6574 (2017).
  2. Huang YF, Zhuo GY, Chou CY, Lin CH, Chang W, Hsieh CL, “Coherent brightfield microscopy provides the spatiotemporal resolution to study early stage viral infection in live cells,” ACS Nano, 11(3), pp. 2575-2585 (2017).
  3. Wu HM, Lin YH, Yen TC, Hsieh CL, “Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking,” Scientific Reports 6:20542 (2016).
  4. Lin YH, Chang WL, Hsieh CL, ”Shot-noise limited localization of single 20 nm gold particles with nanometer spatial precision within microseconds,” Optics Express, 22(8), pp. 9159-9170 (2014).