Intense, extremely short pulses of X-rays have been used for the first time to reconstruct 3D images of individual virus particles. Developed by an international team of physicists, the diffraction technique could make it possible to map the structure of infectious viruses such as HIV, influenza and herpes, shedding light on possible ways to combat diseases caused by these infectious agents.

Scientists have used X-ray diffraction to image live cells, viruses and simple nanostructures in 2D using a technique called nanocrystallography. This involves incorporating a number of identical particles (viruses, for example) into a crystal – a time-consuming and expensive process that does not work for some particles. Now, a team led by Janos Hajdu at Uppsala University in Sweden has tested a technique that avoids crystallization with the added benefit of delivering 3D images.

We outrun radiation damage but we only get one shot per sample
Janos Hajdu, Uppsala University

Brighter than the Sun

The team focused on the Acanthamoeba polyphaga mimivirus, which is about 450 nm across. The researchers injected virus particles into an aerosol stream, which was then subjected to high-energy pulses from an X-ray free-electron laser at Stanford University. Each pulse lasted just 70 fs and delivered a peak power density more than 1018 times that of sunlight hitting the Earth.

Such an extremely intense pulse will vaporize a mimivirus particle, but not before the X-rays scatter from the virus and create a diffraction pattern that is recorded by a bank of detectors. "We outrun radiation damage but we only get one shot per sample," explains Hajdu. Because X-rays primarily scatter off of electrons, the diffraction patterns that Hajdu and his team recovered can be used to calculate the distribution of electrons within the mimivirus particles.

Hajdu and his team scattered X-rays off nearly 200 identical mimivirus particles, collecting one 2D diffraction pattern from each event. To make sure that the particles were not altered by being injected into an aerosol, the team showed that mimivirus particles that did not intersect the laser pulses were still infectious.

Adding a dimension

With nearly 200 diffraction patterns in hand, Hajdu and his colleagues next set about adding the patterns together to produce a single 3D image. Combining data from different observations not only increases the signal – necessary for small viruses that scatter X-rays weakly – but also gives insights about viral structure that are only possible with 3D observations.

One important complication that the researchers had to overcome is that they were not all oriented in the same way with respect to the X-ray pulses. Instead, their orientations were randomly distributed. Therefore, it is necessary to retrieve the relative orientation of each mimivirus particle before adding the diffraction patterns together. Hajdu and his team did this using a mathematical optimization algorithm developed by another group of physicists in 2009. Because this algorithm presumes that the particles differ only in orientation, identical particles must be used. Fortunately, many pathogenic viruses that affect humans are reproducible, such as HIV, influenza and herpes.

The researchers achieved a spatial resolution of approximately 125 nm in their final reconstructed image of the mimivirus's electron density. Better resolutions have been recorded in other 2D studies, but this investigation is important because it shows that 2D diffraction patterns can be combined to produce 3D images of biologically important samples. "We now look forward both to pushing towards higher resolution and studying both smaller and larger samples," says Hajdu.

This research is described in Physical Review Letters.