Seeing viruses by both light and electron microscopy

January 6, 2017 by Quinn Eastman, Emory University
Seeing viruses by both light and electron microscopy
An example of the images of viruses obtainable with cryo-CLEM. These images show pseudotyped HIV-1 particles being taken into the cell, with viral membrane as light blue, mature core as yellow, and clathrin cages as purple. Credit: Hampton et al Nat. Protocols (2016)

Advances in both light and electron microscopy are improving scientists' ability to visualize viruses such as HIV, respiratory syncytial virus (RSV), measles, influenza, and Zika in their native states. Researchers from Emory University School of Medicine and Children's Healthcare of Atlanta developed workflows for cryo-correlative light and electron microscopy (cryo-CLEM), which were published in the January 2017 issue of Nature Protocols.

Previously, many electron microscopy images of well-known viruses were obtained by studying purified virus preparations. Yet the process of purification can distort the structure of enveloped viruses, says Elizabeth R. Wright, PhD, associate professor of pediatrics at Emory University School of Medicine.

Wright and her colleagues have refined techniques for studying viruses in the context of the they infect. That way, they can see in detail how viruses enter and are assembled in cells, or how genetic modifications alter viral structures or processing.

"Much of what is known about how some viruses replicate in cells is really a black box at the ultrastructural level," she says. "We see ourselves as forming bridges between light and electron microscopy, and opening up new realms of biological questions."

Wright is director of Emory's Robert P. Apkarian Integrated Electron Microscopy Core and a Georgia Research Alliance Distinguished Investigator. The co-first authors of the Nature Protocols paper are postdoctoral fellows Cheri Hampton, PhD. and Joshua Strauss, PhD, and graduate students Zunlong Ke and Rebecca Dillard.

The Wright lab's work on cryo-CLEM includes collaborations with Gregory Melikyan in Emory's Department of Pediatrics, Phil Santangelo in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory, and Paul Spearman, now at Cincinnati Children's.

For this technique, virus-infected or transfected cells are grown on fragile carbon-coated gold grids and then "vitrified," meaning that they are cooled rapidly so that ice crystals do not form. Once cooled, the cells are examined by cryo-fluorescent light microscopy and cryo-electron tomography.

Extremely low temperatures (well below -150° C) are necessary for cryo-fluorescent light microscopy and cryo-electron tomography. Performing the light microscopy steps after sample vitrification prevents the cells from continuing to grow and shift in position in between live-cell light and cryo-. The cryo-fluorescent is facilitated by use of a heat-insulated, short working distance ceramic objective lens, Wright says. Other technological advances that have enabled CLEM include computer software for combining data from the two imaging modes, and a variety of fluorescent proteins and probes.

With the data, it is possible to obtain images of individual intact viruses and viral proteins at high resolution – in some cases, close to atomic resolution—via sub-volume averaging, Wright says. This approach was used in a recent Nature Communications paper on RSV, demonstrating that a live attenuated vaccine candidate resembles RSV structurally.

The cryo-CLEM technique works best with cells that can grow flat, because a standard electron beam can't penetrate cell bodies more than about 1 micron (a millionth of a meter) thick. Mammalian cells are usually several microns wide, while such as HIV are around 0.1 microns across.

Potentially, the cryo-CLEM technique could be extended to study many systems including neuronal cells or bacterial biofilms, Wright says.

Explore further: Threading the RSV vaccine needle

More information: Cheri M Hampton et al. Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells, Nature Protocols (2016). DOI: 10.1038/nprot.2016.168

Related Stories

Threading the RSV vaccine needle

December 21, 2016

Crafting a vaccine against RSV (respiratory syncytial virus) has been a minefield for 50 years, but scientists believe they have found the right balance.

Human aichi virus atomic structure identified

September 7, 2016

Using cryo-electron microscopy, an international group of scientists has solved the atomic structure of the human aichi virus (AiV), a rather unusual but poorly characterized picornavirus that is very common and can cause ...

Recommended for you

How quinoa plants shed excess salt and thrive in saline soils

September 21, 2018

Barely heard of a couple of years ago, quinoa today is common on European supermarket shelves. The hardy plant thrives even in saline soils. Researchers from the University of Würzburg have now determined how the plant gets ...

Basking sharks can jump as high and as fast as great whites

September 20, 2018

A collaborative team of marine biologists has discovered that basking sharks, hundreds of which are found off the shores of Ireland, Cornwall, the Isle of Man and Scotland, can jump as fast and as high out of the water as ...


Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.