Ultrafast X-ray imaging provides high resolution information on individual fragile specimens such as aerosols, metastable particles, superfluid quantum systems and live biospecimen, which is inaccessible with conventional imaging techniques. Coherent X-ray diffractive imaging, however, suffers from intrinsic loss of phase, and therefore structure recovery is often complicated and not always uniquely-defined. Here, we introduce the method of in-flight holography, where we use nanoclusters as reference X-ray scatterers in order to encode relative phase information into diffraction patterns of a virus. The resulting hologram contains an unambiguous three-dimensional map of a virus and two nanoclusters with the highest lat- eral resolution so far achieved via single shot X-ray holography. Our approach unlocks the benefits of holography for ultrafast X-ray imaging of nanoscale, non-periodic systems and paves the way to direct observation of complex electron dynamics down to the attosecond time scale.
