Swinburne University of Technology - Melbourne Australia
Disease arms-race looks to powerful new X-ray tools
Story by Graeme O'Neill
If you slice a second into a million billion femtoseconds, then 20 femtoseconds would be proportionate to the duration of a single eye blink within the 300,000 years since humans diverged from Neanderthals. It is an unimaginably brief timespan.
Nonetheless, 20 femtoseconds is time enough for a pulse of soft X-rays produced by a high-powered laser to probe the structure of a single protein molecule - a development which, when completed, would reveal bioscientists' Holy Grail.
In the case of combating disease, knowing precisely how organic molecules are constructed equips scientists with a better understanding of how to fight a virus or bacterium. This is how the world's first anti-flu drug, Relenza, was developed. X-ray diffraction techniques identified the structure of the virus's neuraminidase protein, which allows newly minted virus particles to escape from their host cells and infect other cells. Relenza blocks the receptor molecule, destroying the ability of the virus to spread - it attacks the virus itself, not just the symptoms.
Now the bar has been raised, with researchers seeking to go deeper and deeper into the labyrinths of life and death.
Swinburne University of Technology laser physicists Professor Peter Hannaford - director of the Centre for Atom Optics and Ultrafast Spectroscopy - and his colleague Professor Lap Van Dao are members of an international research consortium that is developing a soft-X-ray laser light source, potentially capable of imaging single protein molecules.
The Swinburne centre is part of the Australian Research Council Centre of Excellence for Coherent X-ray Science (CXS). CXS's director is Professor Keith Nugent, of the University of Melbourne. The team is developing algorithms to reconstruct high-resolution images from the diffraction patterns created when organic structures are hit by high-intensity pulses of soft-X-ray laser light.
It is one more step in a long progression of developments that have provided increasingly precise views of the structure of proteins and other organic molecules. Scientists first started beaming X-rays into protein crystals and other organic crystals in the early 1950s, when James Watson and Francis Crick famously deduced the double-helix structure of DNA, with help from Rosalind Franklin's X-ray diffraction image of crystalline DNA.
In recent years, a synchrotron's brilliant, highly focused beams of partially coherent X-ray light have superseded conventional X-ray sources, but large crystals are still required to solve protein structures. And many proteins cannot be crystallised.
The hydrophobic receptor molecules that float in cell membranes are a case in point. Detergents can be used to solubilise the receptor proteins and neutralise their hydrophobicity, but many resist crystallisation because their hydrophobic nature prevents them forming ordered arrays.
La Trobe University malaria researcher and CXS deputy director Professor Leann Tilley has such a problem - and the femtosecond laser, the only one of its type in Australia, promises to resolve it by directly imaging protein molecules exported by the malaria parasite after it has infected its host's red blood cells.
The Swinburne Femtosecond High Power Laser facility effectively functions as a mini-synchrotron, generating a tightly focused, coherent beam of X-rays.
A biophysicist and microscopist by training, Professor Tilley studies the deadly agent of human malaria, Plasmodium falciparum.
She is trying to observe how the parasite remodels its host's red blood cells from the inside, causing them to form protein 'knobs' on their outer surface. These adhere to receptor proteins on the walls of blood cells in organs such as the brain.
Professor Tilley's efforts to observe fine details of intracellular structures have been frustrated by the limitations of light microscopes, which cannot resolve details smaller than about 200 nanometres, about half the wavelength of visible light. Professor Tilley needs to observe structural details 10 to 100 times smaller.
"For much higher resolution, you can use very short wavelengths in the X-ray range, but it's difficult to generate high-intensity X-rays at the wavelength you want - in the so-called 'water window'," she says.
(The so-called 'soft X-rays' have wavelengths between two and four nanometres, which allows them to pass readily through water, maximising the contrast between the watery background and solid structures within the cell.)
Professor Tilley says that synchrotron-based measurements have already yielded clear images of internal structures of Plasmodium-infected red blood cells. She expects to obtain similar images using the soft-X-ray laser source being developed at Swinburne.
The femtosecond laser will complement larger installations of another X-ray tool, X-ray free-electron lasers (XFELS), in Germany, the US and Japan. Swinburne's Professor Dao says the free-electron laser generates X-radiation at an intensity that would normally heat and vaporise any organic material in its path. But by employing ultra-short pulses of five to 10 femtoseconds duration, XFELS can deliver enough photons to generate a diffraction pattern, before the sample is heated to destruction.
"We aim eventually to get down to about 10 femtoseconds at a wavelength of two to four nanometres," Professor Dao says. "The world record of pulse duration in high harmonic generation is around 100 times shorter, at about 100 attoseconds, but it doesn't deliver enough photons to produce a diffraction pattern."
Currently, the Swinburne laser can deliver a 30-femtosecond pulse of very high energy, but the aim is to produce 10-femtosecond pulses within the water window.
The University of Melbourne's Professor Nugent says this capability will be achieved through a relatively new technology for generating powerful, coherent X-ray beams: the high harmonic generation of pulses from a femtosecond laser.
The principle involves shining a very brief pulse of infrared light into a gas. The excited atoms emit radiation at frequencies that are exact multiples, or harmonics, of the frequency of the input radiation, like the high-frequency 'overtones' of a single, pure musical note.
The shortest wavelengths extend well beyond the visible and ultraviolet parts of the spectrum into the soft X-ray region of the spectrum, creating coherent, high-energy beams of X-rays at wavelengths closest to the visible spectrum, which are less damaging to the specimen (hence the term 'soft').
The challenge, Professor Nugent says, is to use the new technique to generate coherent soft X-rays at an energy of around 500 eV (electron volts), at water-window wavelengths, to image biological specimens without damaging them.
Unlike electrons from an electron microscope, which do not penetrate far beyond the surface, the X-rays penetrate the full volume of the specimen, diffracting off internal features.The result - in theory - is a diffraction pattern that can be mathematically reconstituted into a high-resolution, three-dimensional image of the specimen, or, at the highest resolution, a single protein molecule, frozen in random orientation by a brief flash of laser light as it free-falls through the beam.
The diffraction patterns from non-crystalline biological specimens are very different to those obtained from protein crystals with conventional X-ray crystallography. In conventional crystallography, billions of protein molecules form an orderly 3-D pattern - a crystal - in which all of the molecules are presented in a particular orientation to the X-rays. The result is a highly ordered, symmetrical pattern of points on an X-ray detector screen. It records how the rays have been diffracted and focused as they pass through the crystal lattice.
By contrast, a diffraction pattern from a non-crystalline biological specimen such as a cell, or a single protein molecule, resembles a poached egg - dark at the centre, and progressively lighter toward the edges. Most of the fine detail is contained in patterns at the periphery of the image.
Researchers say that the technology, when perfected, will provide a new window into the internal structure and function of living cells. It will show how proteins bind to receptors on the surface of cells, providing invaluable information to chemists designing drugs to treat metabolic disorders or quell virus infections, or developing new antibiotics to defeat drug-resistant bacteria.
Professor Dao says generating short laser pulses within the water window, and synchronising and orienting them to study biological specimens, still involves technical challenges, but the project is making progress.
