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The direct FEL pulse passes through the sample window and exits the camera through a hole in the multilayer mirror. The mirror reflects only the diffracted light from the nanoscale object onto a CCD detector that records a continuous diffraction pattern. An algorithm converts this pattern into an image of the object: two small cowboys in the sun.
A Perfect Image from a Single FEL Shot
Theory had predicted it; a model experiment at FLASH delivered the proof of principle. A nanoscale object can be imaged by a single femtosecond FEL pulse before the sample turns into a plasma and explodes.
One of the key problems facing structural biology is the difficulty in determining the structures of biological macromolecules that cannot be crystallized; some examples are pathogenic viruses such as HIV, and the majority of human membrane proteins, which are the most important drug targets.
Thus, if scientists could use the extremely intense and coherent femtosecond pulses from future hard X-ray FELs to record atomic structures from non-crystalline samples consisting of a few or even single biomolecules, the impact on biological and pharmaceutical research can hardly be overestimated.
But is it really possible to record a diffraction pattern before the powerful radiation destroys the sample? Theoretical calculations had suggested it, and now a model experiment at FLASH strongly supports the view. This experiment was performed by researchers from Lawrence Livermore National Laboratory, University of California, SLAC, and Spiller X-ray Optics, all in the USA, as well as Uppsala University in Sweden, Technische Universität Berlin, and DESY.
With a single 25-femtosecond FEL pulse the researchers recorded a continuous diffraction pattern of a nanoscale object with a CCD camera before the image-forming pulse destroyed the sample. What is particularly impressive is the fact that a perfect image of the object – two small cowboys – could be reconstructed from this diffraction pattern using an algorithm requiring no a priori knowledge about the object.
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A coherent diffraction pattern of the object recorded from a single 25-femtosecond FEL pulse. |
Imaging of Biological Specimens
The research group concludes: “Our apparatus provides a new and unique tool at FLASH to perform imaging of biological specimens beyond the conventional radiation damage limits and to acquire images of ultrafast processes initiated by an FEL pulse or another laser.”
Nevertheless, many obstacles remain before scientists will be able to churn out structures of large biomolecules at atomic resolution. Such atomic structures can only be resolved using hard X-rays from e.g. the planned European XFEL facility and not by the soft X-rays from FLASH. Furthermore, the structure of these macromolecules will have to be stable at near-atomic dimensions during the exposure, and the diffraction patterns will be weaker than in the model experiment. Overcoming these hurdles may not be easy, but the potential payoff, especially in terms of advances in biology, medicine and material science, is certainly worth the effort.
Ultimately structural biologists may be able to uncover the extremely fast steps in biochemical reactions such as enzymatic processes and drug–receptor interactions using the single-shot approach.
The idea is to start a reaction with a pump pulse, e.g. from an optical laser, and to record snapshots of the system with atomic resolution using a femtosecond hard X-ray free-electron laser beam. These snapshots could be taken on reproducible samples at different time intervals after initiation of the reaction and assembled into a molecular movie. Further experiments at FLASH will be indispensable to realize this potential at the European XFEL and its American and Japanese counterparts.
Reconstructed image of the cowboys, which shows no signs of radiation damage caused by the pulse. |
Diffraction pattern from the subsequent pulse showing that the first pulse destroyed the object after recording the image. |
Coherent Diffractive Imaging
If the single-shot method can be applied to solve the structures of biological macromolecules at the XFEL, it will have significant advantages compared to the generally applied method using crystals and X-rays from synchrotron radiation sources.
In contrast to macromolecular crystallography where the scattering from many unit cells interferes to give discrete Bragg spots, an exposure of a non-periodic sample to a coherent FEL pulse produces a continuous landscape of peaks and troughs.
This diffraction pattern can be sampled in principle on an arbitrarily fine scale that opens the way for solving the phase problem by iterative algorithms and finally inverting the pattern to yield an image of the object. A three-dimensional data set may be assembled from such images when copies of reproducible samples are exposed to the beam one by one.
