Virology studies with SML
Vero cells

The Vutara single molecule localization microscope for viral research:

The Vutara single molecule localization system has been a critical instrument for understanding viral particles. Viral particles are typically much smaller than the diffraction limit of light (<200 nm), making single molecule localization microscopy the best suited fluorescence technique for resolving virus particle structural details or determining localization of virus components with the cellular machinery. Below we highlight the key features of the Vutara for virus research.

 

  • Super resolved images using proprietary biplane single molecule localization, achieving at least 20 nm XY and 50 nm Z precision.
  • The only 3D single molecule system capable of imaging multiple sample types, from purified virions to tissue sections and whole model organisms.
  • High speed acquisition: ideal for live imaging, particle tracking and rapid data acquisition.
  • Integrated fluidics: multiplexed imaging of the proteome, genome or live cell applications.
  • Powerful acquisition software with real time single molecule localization.
  • Powerful visualization and analysis software package provides a complete statistics tool set.

Vutara Virus Applications

Below we highlight virus research performed on the Vutara. The unique ability to perform single molecule localization of virus samples at both the coverslip and deep within tissue sections makes the Vutara the only system capable of imaging virus particle structures, virus particle host cell interactions, and effects of virus infection on cell biology on the same microscope. At the bottom of the page you can find some highlighted virus research papers performed with the Vutara super resolution microscope.

Vutara Viral Studies

  • Viral Particle Structure

  • Virus Host Interactions

  • Virus Pathology

Vesicular stomatitis virus particle
Vesicular stomatitis virus particle. Red – VSV-G protein tagged with Alexa Fluor 647

Virus Particle Structure

Alonas, E., Lifland, A.W., Gudheti, M., Vanover, D., Jung, J., Zurla, C., Kirschman, J., Fiore, V.F., Douglas, A., Barker, T.H., Yi, H., Wright, E.R., Crowe, J.E., Santangelo, P.J., 2014. Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells. ACS Nano 8, 302–315. doi.org/10.1021/nn405998v

 

The authors developed tools to study the early infectivity and replication of enveloped viruses.

  • The authors developed MTRIPs (multiply labeled tetravalent RNA imaging probes). A method to live label hRSV viral genomes.
  • The MTRIP technique enabled the simultaneous super resolution imaging of proteins and the viral genome; not possible with conventional fluorescence in situ hybridization techniques (FISH).
  • The authors used the Vutara 352 to determine the distribution of viral proteins along the viral gRNA. Only single molecule localization microscopy gives the resolution to image these sub-300 nm particles

Host Cell Interactions

Tiwari, P.M., Vanover, D., Lindsay, K.E., Bawage, S.S., Kirschman, J.L., Bhosle, S., Lifland, A.W., Zurla, C., Santangelo, P.J., 2018. Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection. Nature Communications 9, 1–15. doi.org/10.1038/s41467-018-06508-3

 

The authors used the Vutara 352 microscope to determine the mechanism of action of the therapeutic antibody, palivizumab, on RSV infections.

  • Using the Vutara’s ability to image cultured cells in 3D, the authors were able to visualize viral particles on the cell membrane.
  • When the cells expressed palivizumab on their cell surface the virions could be observed adjacent, but outside, of the cell membrane (virion size of ~100-300 nm).
  • This suggested the mechanism of action of palivizumab is to prevent infection by stopping fusion and cytosolic uptake of RSV.
Tiwari et al 2018 Nature Communications
Palizumab (green) expressing Vero cells exposed to RSV virions (magenta). 3D single molecule localization microscopy with the Vutara revealed that the cells expressing palizumab were incapable of being infected with RSV the cell and the virions were instead trapped on the membrane outside of the cell.
Figure taken from: Tiwari et al. 2018 Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection. Nature Communications.

Viral Infections Effects on Host Cells

Milrot, E., Shimoni, E., Dadosh, T., Rechav, K., Unger, T., Etten, J.L.V., Minsky, A., 2017. Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting Paramecium bursaria chlorella virus-1. PLOS Pathogens 13, e1006562. doi.org/10.1371/journal.ppat.1006562

 

The authors used the Vutara to determine the effects of viral infection on cytoskeletal structure. From this they determined that the actin cytoskeleton plays a critical role in viral infectivity.

  • The authors used super-resolution imaging to monitor how the microtubule and actin cytoskeleton changed over the course of viral infection.
  • During infection, the microtubule network became more fragmented and disappeared from the center of the cell.
  • During infection, the actin cytoskeleton loses its fine structure at the periphery of the cell and forms a shell around the rounded edge of the cell.
  • Pharmacological experiments and other experiments revealed that disruption of the microtubule network had little effect on virion production, while disruption of the actin cytoskeleton reduced virion production.

Live Cell Imaging

Virus researchers may also be interested in the live cell and single molecule particle tracking capabilities of the Vutara. The Vutara 352 is fully capable of both live cell single molecule imaging of cellular structures such as organelles and single molecule particle tracking. Uniquely, the Vutara is also capable of combining these two techniques to do particle tracking in conjunction with cellular structure imaging.

 

Please refer to the live cell web page and the Vutara live cell webinar to learn more about live cell imaging with the Vutara microscope and SRX software.

Live Mitochondria
Live combined particle
Top Live imaging of mitochondria using glitter bomb dyes.
Bottom Live combined particle tracking of individual tom20 proteins (left) and mitochondria imaging (right).

Highlighted Virus Research Publications:

  • Akiyama, H., Ramirez, N.-G.P., Gudheti, M.V., Gummuluru, S., 2015. CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies. PLoS Pathog 11. https://doi.org/10.1371/journal.ppat.1004751
  • Alonas, E., Lifland, A.W., Gudheti, M., Vanover, D., Jung, J., Zurla, C., Kirschman, J., Fiore, V.F., Douglas, A., Barker, T.H., Yi, H., Wright, E.R., Crowe, J.E., Santangelo, P.J., 2014. Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells. ACS Nano 8, 302–315. https://doi.org/10.1021/nn405998v
  • Hodges, J., Tang, X., Landesman, M.B., Ruedas, J.B., Ghimire, A., Gudheti, M.V., Perrault, J., Jorgensen, E.M., Gerton, J.M., Saffarian, S., 2013. Asymmetric packaging of polymerases within vesicular stomatitis virus. Biochemical and Biophysical Research Communications 440, 271–276. https://doi.org/10.1016/j.bbrc.2013.09.064
  • Kiss, G., Holl, J.M., Williams, G.M., Alonas, E., Vanover, D., Lifland, A.W., Gudheti, M., Guerrero-Ferreira, R.C., Nair, V., Yi, H., Graham, B.S., Santangelo, P.J., Wright, E.R., 2014. Structural Analysis of Respiratory Syncytial Virus Reveals the Position of M2-1 between the Matrix Protein and the Ribonucleoprotein Complex. Journal of Virology 88, 7602–7617. https://doi.org/10.1128/JVI.00256-14
  • Milrot, E., Shimoni, E., Dadosh, T., Rechav, K., Unger, T., Etten, J.L.V., Minsky, A., 2017. Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting Paramecium bursaria chlorella virus-1. PLOS Pathogens 13, e1006562. https://doi.org/10.1371/journal.ppat.1006562
  • Tiwari, P.M., Vanover, D., Lindsay, K.E., Bawage, S.S., Kirschman, J.L., Bhosle, S., Lifland, A.W., Zurla, C., Santangelo, P.J., 2018. Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection. Nature Communications 9, 1–15. https://doi.org/10.1038/s41467-018-06508-3
  • Yaakov, L.B., Mutsafi, Y., Porat, Z., Dadosh, T., Minsky, A., 2019. Kinetics of Mimivirus Infection Stages Quantified Using Image Flow Cytometry. Cytometry Part A 95, 534–548. https://doi.org/10.1002/cyto.a.23770