High-Energy X-Ray Lasers Expose the Guts of Viruses from the Inside-Out

A team of Swedish physicists has for the first time imaged individual virus particles in truly three dimensions. Their x-ray technique was demonstrated on the Acanthamoeba polyphaga mimivirus, a molecular machine just 450 nanometers across, and offers potential a new way in to studying HIV and a whole range of infectious and potentially deadly viruses. The trick is in balancing the destructiveness of an x-ray laser—packing a power density of more than 10^18 times that of sunlight hitting the Earth—with its refractive potential. The method is described in the current Physical Review Letters.

The viruses targeted in said technique are indeed destroyed swiftly and brutally, vaporizing almost instantly. But not quite instantly. The x-ray pulses, each one lasting just 70 femtoseconds, are able to diffract just enough radiation just quickly enough to be captured by surrounding detectors. "We outrun radiation damage but we only get one shot per sample," Janos Hajdu, a biophysicist at Uppsala University, told Physics World.

The result is what Hajdu and his group call "diffraction without destruction."

"Free-electron lasers provide femtosecond x-ray pulses with a peak brilliance ten billion times higher than any previously available x-ray source," the study explains. "Such a large jump in one physical quantity is very rare and can have far-reaching implications for several areas of science. It has been suggested that such pulses could outrun key damage processes and allow structure determination without the need for crystallization."

Imaging very small stuff like viruses obviously hasn't been a total impossibility until now, and there's no really shortage of pretty good snapshots of viruses in action. The catch, however, is that the alternative, dominant method, in which particles are rendered in 2D, relies on crystallizing the target to be imaged. That's not always possible.

While x-ray crystallography has been one of the defining tools in structural biology over the past 50 years, there are still some extremely important molecules that resist being crystallized. One (huge) example are the proteins that interact with biological membranes ("membrane proteins") that are so crucial to the immune system—and, thus, life itself—that almost a third of the human genome is dedicated to their production.

Enter free-electron x-ray lasers.

This is a tricky method in its own right. Rather than what we'd normally consider a photograph—a single coherent image—here we're collecting and compiling diffraction patterns, which you can see above. The high-energy probe pulse smashes into the target molecule, which then scatters or reflects back the incoming electrons all over the place.

These electrons are registered by detectors, and the results can be pieced together into a pretty good picture of something very tiny, and very fragile. Imagine blasting a statue in the dark with a firehose, where the only way to glean information about that statue is by measuring each and every tiny water droplet that bounces back. And yet it's still more difficult than that: A single blast with the firehose won't yield enough information (as reflected water droplets) to usefully describe the statue. Complicating things further is that any given statue can only withstand a single blast of water, after which it's destroyed.

What's actually needed is a succession of identical statues, where each one is blasted once with our firehose and the information is recorded. Every new statue offers more information and, eventually, we have a good picture. "Can one collect diffraction patterns from tens, hundreds, thousand, or even millions of individual molecules and then put together these noisy data sets, by aligning the measured diffraction patterns to compensate for the random orientation of the molecules?," an Americal Physical Society synopsis asks. "The work of Hajdu's team demonstrates this key capability."

The group's success comes courtesy of an updated version of the so-called EMC algorithm. This is a three-step process of collecting diffraction patterns and verifying them against probabilistic data and then updating the final image. This is "iterative optimization." Eventually, an actual 3D image is constructed, layer by layer.

The scales imaged in the current study are in line with those of some familiar viruses, such as HIV, influenza, and herpes. The resolutions so far haven't beaten those achieved by more traditional crystallography methods, but it now seems possible. The nanoworld is soon to be exposed from the inside-out.