![]() The RNAi microsponge was introduced as one of effective delivery systems of small interference RNA (siRNA) molecules 24, 25, 26, 27, 28. We have utilized the unique advantages from each coherent X-ray radiation source, by virtue of their complementary characteristics, to establish a multimodal coherent X-ray analysis scheme, being applied to investigate biomolecular complexes, RNA interference (RNAi) microsponges. While the image resolution is more severely restricted by the radiation damage when using synchrotron X-rays, the radiation dose can be adjusted conveniently by limiting the exposure time 19, 20, 21, 22, 23. Synchrotrons, unlike XFELs, supply a quasi-continuous flow of X-ray photons at a lower flux, allowing visualization of the same specimen repeatedly within a manageable time frame. The samples are, however, damaged irreversibly after the single-shot exposure. This facilitates high-resolution imaging without being limited by radiation damage by capturing diffraction patterns before the onset of radiation-induced structural changes: ‘diffraction-before-destruction’ 17, 18. Intense, femtosecond X-ray laser pulses from XFELs have realized single-shot imaging 12, 13, 14, 15, 16. Here we introduce a multimodal coherent X-ray analysis scheme combining X-ray free-electron laser (XFEL) single-shot imaging of single particles, XFEL single-shot small-angle scattering, and coherent diffraction tomography, stereo-imaging and wet CDI using coherent X-rays from synchrotrons 11. A structural analysis scheme combining the advantages from each technique is essential for an ab initio understanding of macromolecular complexes, without tangible progress yet. X-ray-scattering techniques, on the other hand, provide faithful statistics, but the data interpretation is contingent on a pertinent model 10. Unless a very large number of images is collected, CDI, similar to other imaging probes, may lack in the ability to deliver statistical fidelity, which is particularly crucial to support new scientific discovery. Imaging of single objects has proven to be a powerful aide in the study of nanostructures, both non-invasively and quantitatively 8, 9. With its extension of crystallographic methodology to non-crystalline material, the coherent diffraction-imaging (CDI) technique lends support to the unveiling of structures in functional nanomaterial and biological cells and organelles 1, 2, 3, 4, 5, 6, 7. This newly introduced multimodal analysis with coherent X-rays can be applied to unveil nano-scale structural motifs from functional nanomaterials or biological nanocomplexes, without requiring a priori knowledge. Here we demonstrate a successful combination of femtosecond X-ray single-shot diffraction with an X-ray free-electron laser and coherent diffraction imaging with synchrotron X-rays to provide an insight into the nanostructure formation of a biological macromolecular complex: RNA interference microsponges. When combined judiciously with synchrotron X-rays of a complimentary nature, suitable for observing steady-state features, it is possible to perform ab initio structural investigation. Intense, femtosecond X-ray pulses from X-ray free-electron lasers enable single-shot imaging allowing for instantaneous views of nanostructures at ambient temperatures. New tools are required for their structural characterization. Nanostructures formed from biological macromolecular complexes utilizing the self-assembly properties of smaller building blocks such as DNA and RNA hold promise for many applications, including sensing and drug delivery.
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