The connection between the protein structure and protein function is one central topic in modern biology. Knowledge of the protein sequence and composition is not enough: the three-dimensional structure is a crucial parameter to enable the protein function. X-ray crystallography has already provided us with the full 3D conformation of hundreds of macroproteins, but its limitations are increasingly obvious: The big protein crystals necessary for X-ray crystallography are often not available and crystallization turned into a serious bottleneck for structure determination. Smaller crystals (nanocrystals) are easier to produce, but, as we will see later, the X-ray dose required to measure a high-quality signal is too large and the small crystal is damaged before the diffraction signal can be collected. (With a larger crystal the dose is spread over many replicas of the protein. The molecular dose is consequently reduced). Alternative imaging techniques, allowing to reach Angström spatial resolution, are needed. Third-generation synchrotron sources provide us with high-brilliance beams at Angström wavelength; hence one could consider duplicating traditional microscopy techniques to image the sample under study directly. High-quality optics is required for this approach. Soft X-ray zone plates have been developed, for example. However, the maximal spatial resolution is limited by the outermost period of the zone plate [1]. Nowadays, the most advanced zone plates have an outermost zone width of 15.1 nm, hence offering a spatial resolution down to approx. 12 nm [2]. Those results are very interesting, but atomic resolution seems to be out of reach. Moreover, the diffraction efficiency of such state-of-the-art zone plates is very low (less than a percent). To acquire good images, they must be accumulated over a long time or very high-radiation doses are required, the latter which is often forbidden by the sample tolerance. As we will see in this chapter, coherent diffractive imaging (CDI) of hard X-ray free-electron laser radiation has the potential to allow such an experiment [3,4].Reaching the atomic spatial resolution is not the only reason to use CDI with EUV or X-ray light. In this energy domain, the penetration depth of radiation is Attosecond and XUV Physics, First Edition. Edited by Thomas Schultz and Marc Vrakking.
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