New research paves the way for nano-movies of biomolecules; Scientists use X-ray laser as ultra slow-motion camera

An international team has caught a light sensitive biomolecule at work with an X-ray laser. The study proves that X-ray lasers can capture the fast dynamics of biomolecules in ultra slow-motion.

An international team, including scientists from DESY, has caught a light sensitive biomolecule at work with an X-ray laser. The study demonstrates that X-ray lasers can capture the fast dynamics of biomolecules in ultra slow-motion, as the scientists led by Prof. Marius Schmidt from the University of Wisconsin-Milwaukee write in the journalScience. “Our study paves the way for movies from the nano world with atomic spatial resolution and ultrafast temporal resolution,” says Schmidt.\

The researchers used the photoactive yellow protein (PYP) as a model system. PYP is a receptor for blue light that is part of the photosynthetic machinery in certain bacteria. When it catches a blue photon, it cycles through various intermediate structures as it harvests the energy of the photon, before it returns to its initial state. Most steps of this PYP photocycle have been well studied, making it an excellent candidate for validating a new method.

For their ultra-fast snapshots of the PYP dynamics, the scientists first produced tiny crystals of PYP molecules, most measuring less than 0.01 millimetres across. These microcrystals were sprayed into the focus of the world’s most powerful X-ray laser, LCLS at the SLAC National Accelerator Laboratory in the US, as their photocycle was kicked off with a meticulously synchronised blue laser pulse. Thanks to the incredibly short and intense X-ray flashes of the LCLS, the researchers could watch how PYP changes its shape at different time steps in the photocycle, by taking snapshot X-ray diffraction patterns.

With a resolution of 0.16 nanometres, these are the most detailed images of a biomolecule ever made with an X-ray laser. A nanometre is a millionth of a millimetre. The diameter of the smallest atom, hydrogen, is about 0.1 nanometres.

Beyond reproducing known aspects of the PYP photocycle, thereby validating the new method, this investigation revealed much finer details. Also, thanks to the high temporal resolution, the X-ray laser could, in principle, study steps in the cycle that are shorter than 1 picosecond (a picosecond is a trillionth of a second) — too fast to be caught with previous techniques. The ultrafast snapshots can be assembled into a movie, showing the dynamics in ultra slow-motion.

“This is a real breakthrough,” emphasises co-author Prof. Henry Chapman from the Center for Free-Electron Laser Science at DESY, who is also a member of the Hamburg Centre for Ultrafast Imaging. “Our study is opening the door for time resolved studies of dynamic processes with atomic resolution.”

Compared to other methods, X-ray lasers, like the LCLS or the European XFEL that is currently being built from the DESY campus in Hamburg to the neighbouring town of Schenefeld, offer several advantages for the investigation of ultrafast dynamics of molecules. They produce the most brilliant X-ray flashes on earth, offering femtosecond time resolution. A femtosecond is a quadrillionth of a second. While 40 femtosecond X-ray flashes were used for this experiment, the pulse duration can be made even shorter down to just a few femtoseconds.

“You need a short pulse to resolve the steps of these fast processes,” underlines co-author Dr. Anton Barty, also from DESY. “The short flashes also overcome the problem of damaging the often delicate samples with the intense X-rays.” Although the powerful pulses usually vaporise the sample, they are so short that they produce a high-quality diffraction signal on the detector before the sample disintegrates. This principle, called diffraction before destruction, was proven a few years ago by an international collaboration led by DESY.

X-ray lasers use a fresh sample for every shot, which also avoids radiation damage that can accumulate in the samples in other types of investigations. And X-ray lasers typically investigate very small crystals that often are much easier to fabricate than larger crystals. In fact, some biomolecules are so hard to crystallise that they can only be investigated with an X-ray laser. The small crystal size is also an advantage when it comes to kick-starting molecular dynamics uniformly across the sample. In larger samples, the initiating optical laser pulse is often quickly absorbed in the sample, which excites only a thin layer and leaves the bulk of the crystal unaffected. The PYP microcrystal dimensions were perfectly matched to the optical absorption so that all molecules in the crystal were undergoing the same dynamics, which in turn allowed sensitive measurements of the molecular changes by snapshot X-ray diffraction.

Taken together, X-ray laser investigations can offer previously inaccessible new insights into the dynamics of the molecular world, complementing other methods. Using the ultra slow-motion, the scientists next plan to elucidate the fast steps of the PYP photocycle that are too short to be seen with previous methods.

Scientists line up unruly gas molecules for X-rays.

It’s hard to study individual molecules in a gas because they tumble around chaotically and never sit still. Researchers at SLAC overcame this challenge by using a laser to point them in the same general direction, like compass needles responding to a magnet, so they could be more easily studied with an X-ray laser.

The experiment with SLAC’s Linac Coherent Light Source (LCLS), reported Dec. 6 in Physical Review A, is a key step toward producing movies that show how a single molecule changes during a chemical reaction. Understanding the many stages of a reaction could help scientists design more efficient, controllable reactions for important industrial processes, many of which rely on gases that react with solids.

“This is the ‘trailer’ for the molecular movie – these are the first frames,” said Daniel Rolles of the Center for Free-Electron Laser Science at DESY national laboratory in Germany, who led the experiment. “People know, theoretically, that molecules do all kinds of weird things. If you can see the changes and check the theory, then you can understand how it’s happening and have a handle on controlling it.”

In the experiment, researchers jetted a thin stream of fluorocarbon gas into the path of two intersecting lasers: an optical laser that polarized the molecules – aligning them along a common axis, like a spinning top with a slight wobble – and the LCLS X-ray laser.

Fluorocarbon molecules were chosen because their chemical makeup allows them to be polarized by the electric field of a laser and because they are somewhat complex; each features a ringed structure and a tail-like spur and contains more than a dozen atoms. This makes them a good test case for future studies of even larger molecules.
This diagram shows the setup for an experiment at SLAC’s Linac Coherent Light Source that positioned fluorocarbon molecules along a common axis with an optical laser and then used X-ray laser pulses to explore their structural details. The molecules were first channeled into a narrow molecular beam. The optical laser and X-ray laser intersected the path of this gas beam (center). The ball-and-stick structure of the molecule is shown at the upper left. Detectors captured the fingerprint of fluorine atoms and electrons that were ejected from the molecules by the X-ray pulses, which were used to understand the original shape of the molecules. Credit: Phys. Rev. A 88, 061402(R), 2013

The X-ray laser was carefully tuned so it would eject electrons mostly from the fluorine atoms in the sample before bursting the molecules into charged fragments. Scientists measured the freed fluorine electrons and charged fluorine fragments with sensitive detectors, and sorted and analyzed this data to reconstruct the original shape and structure of the molecules. Even though each X-ray laser pulse hit many molecules, the angles of the ejected electrons revealed details about the structure of individual molecules.

The LCLS X-ray is uniquely suited for this type of atomic-scale chemical study because it allows scientists to pinpoint a particular element in a molecule that they want to study, Rolles said.

“This is element and site specific: We can pick one place in a molecule and image that environment,” he said.” It’s like singling out one type of tree that otherwise would be hidden by the forest around it.” That selectivity can allow scientists to zero in on areas of particular interest in a chemical reaction.

Laser alignment of molecules, first demonstrated in 1999, is still a very young field, and the ultrafast X-ray pulses from the LCLS could allow scientists to study changes in aligned molecules that occur in quadrillionths of a second – a far shorter timescale than possible with other research tools. While not all molecules can be aligned with lasers, the researchers note that a rich assortment of molecules is suited to the technique.

If researchers could achieve fuller, three-dimensional alignment of molecules – like stopping a spinning top with your finger and rotating it to face you in a certain way – they would have an even easier time measuring their properties and determining their structure. “You could solve the structure of an individual molecule even without prior knowledge of its shape,” said John Bozek, an LCLS staff scientist who participated in the experiment, adding that this could be useful for studying the intermediate stages of a chemical reaction.