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Incoherent diffractive imaging (IDI) promises structural analysis with atomic resolution based on intensity interferometry of pulsed X‐ray fluorescence emission. However, its experimental realization is still pending and a comprehensive theory of contrast formation has not been established to date. Explicit expressions are derived for the equal‐pulse two‐point intensity correlations, as the principal measured quantity of IDI, with full control of the prefactors, based on a simple model of stochastic fluorescence emission. The model considers the photon detection statistics, the finite temporal coherence of the individual emissions, as well as the geometry of the scattering volume. The implications are interpreted in view of the most relevant quantities, including the fluorescence lifetime, the excitation pulse, as well as the extent of the scattering volume and pixel size. Importantly, the spatiotemporal overlap between any two emissions in the sample can be identified as a crucial factor limiting the contrast and its dependency on the sample size can be derived. The paper gives rigorous estimates for the optimum sample size, the maximum photon yield and the expected signal‐to‐noise ratio under optimal conditions. Based on these estimates, the feasibility of IDI experiments for plausible experimental parameters is discussed. It is shown in particular that the mean number of photons per detector pixel which can be achieved with X‐ray fluorescence is severely limited and as a consequence imposes restrictive constraints on possible applications., Starting from a simple model of stochastic fluorescence emission, a theory is derived of contrast formation and signal‐to‐noise ratio for incoherent diffractive imaging; its feasibility for plausible experimental parameters is discussed. image

Viscous sample delivery that decreases the net protein consumed in serial femtosecond crystallography is described. The agarose stream has a low background, is compatible with membrane proteins and can be used at a wide range of temperatures., Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

The application of serial femtosecond crystallography to naturally occurring peroxisomal protein crystals within yeast cells is described. The concept of utilizing peroxisomes for the production of protein nanocrystals is outlined., The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.