Coherent diffractive imaging (CDI) and scanning transmission x-ray microscopy (STXM) are two popular microscopy techniques that have evolved quite independently. CDI promises to reach resolutions below 10 nanometers, but the reconstruction procedures put stringent requirements on data quality and sample preparation. In contrast, STXM features straightforward data analysis, but its resolution is limited by the spot size on the specimen. We demonstrate a ptychographic imaging method that bridges the gap between CDI and STXM by measuring complete diffraction patterns at each point of a STXM scan. The high penetration power of x-rays in combination with the high spatial resolution will allow investigation of a wide range of complex mesoscopic life and material science specimens, such as embedded semiconductor devices or cellular networks.

The solution and crystal structures of a bacterial phytochrome photosensory core in both its resting and activated states are determined; switching between closed (resting) and open (activated) forms is found to be mediated by a conserved ‘tongue’, and the structures indicate that smaller changes in the vicinity of the chromophore are amplified in scale as they are transmitted through the tongue and beyond. The crystal structure of the photosensory core of a bacterial phytochrome in both the resting and the active (illuminated) state has now been solved. Working with the phytochrome from the extremophile Deinococcus radiodurans, Sebastian Westenhoff and colleagues demonstrate that toggling between resting and active forms is mediated by a conserved 'tongue' that contacts the chromophore. Atomic-scale structural changes in the vicinity of the chromophore are amplified as they are transmitted through the tongue and beyond, culminating in a nanometre-scale conformational signal that feeds into the rest of the cellular signalling network. Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light-sensing kinases that control diverse cellular functions in plants, bacteria and fungi1,2,3,4,5,6,7,8,9. Bacterial phytochromes consist of a photosensory core and a carboxy-terminal regulatory domain10,11. Structures of photosensory cores are reported in the resting state12,13,14,15,16,17,18 and conformational responses to light activation have been proposed in the vicinity of the chromophore19,20,21,22,23. However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here we report crystal and solution structures of the resting and activated states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures show an open and closed form of the dimeric protein for the activated and resting states, respectively. This nanometre-scale rearrangement is controlled by refolding of an evolutionarily conserved ‘tongue’, which is in contact with the chromophore. The findings reveal an unusual mechanism in which atomic-scale conformational changes around the chromophore are first amplified into an ångstrom-scale distance change in the tongue, and further grow into a nanometre-scale conformational signal. The structural mechanism is a blueprint for understanding how phytochromes connect to the cellular signalling network.

X-ray ptychography is a scanning variant of coherent diffractive imaging with the ability to image large fields of view at high resolution. It further allows imaging of non-isolated specimens and can produce quantitative mapping of the electron density distribution in 3D when combined with computed tomography. The method does not require imaging lenses, which makes it dose efficient and suitable to multi-keV X-rays, where efficient photon counting, pixelated detectors are available. Here we present the first highly resolved quantitative X-ray ptychographic tomography of an extended object yielding 16 nm isotropic 3D resolution recorded at 2 Å wavelength. This first-of-its-kind demonstration paves the way for ptychographic X-ray tomography to become a promising method for X-ray imaging of representative sample volumes at unmatched resolution, opening tremendous potential for characterizing samples in materials science and biology by filling the resolution gap between electron microscopy and other X-ray imaging techniques.