Design of a liquid cell toward three-dimensional imaging of unidirectionally-aligned particles in solution using X-ray free-electron lasers

X-ray free-electron lasers (XFELs) opened up a possibility for molecular-scale single particle imaging (SPI) without the need for crystallization. In SPI experiments, the orientation of each particle has to be determined from the measured diffraction pattern. Preparing unidirectionally-aligned particles can facilitate the determination of the sample orientation. Here, we show the design principles of a liquid cell for three-dimensional imaging of unidirectionally-aligned particles in solution with XFELs. The liquid cell was designed so that neither incident X-rays nor diffracted X-rays are blocked by the substrate of the liquid cell even at high tilt angles. As a feasibility evaluation, we performed coherent diffraction measurements using the cells with a 1 mm focused XFEL beam. We successfully measured coherent diffraction patterns of a nano-fabricated metal pattern at 701 tilt angle and obtained the reconstructed image by applying iterative phase retrieval. The liquid cell will be usefully applied to molecular-scale SPI by using more tightly focused XFELs. In particular, imaging of membrane proteins embedded in lipid membranes is expected to have an enormous impact on life science and medicine

High-fluence and high-gain multilayer focusing optics to enhance spatial resolution in femtosecond X-ray laser imaging

With the emergence of X-ray free-electron lasers (XFELs), coherent diffractive imaging (CDI) has acquired a capability for single-particle imaging (SPI) of non-crystalline objects under non-cryogenic conditions. However, the single-shot spatial resolution is limited to similar to 5 nanometres primarily because of insufficient fluence. Here, we present a CDI technique whereby high resolution is achieved with very-high-fluence X-ray focusing using multilayer mirrors with nanometre precision. The optics can focus 4-keV XFEL down to 60 nm x 110 nm and realize a fluence of >3 x 10(5) J cm(-2) pulse(-1) or >4 x 10(12) photons mu m(-2) pulse(-1) with a tenfold increase in the total gain compared to conventional optics due to the high demagnification. Further, the imaging of fixed-target metallic nanoparticles in solution attained an unprecedented 2-nm resolution in single-XFEL-pulse exposure. These findings can further expand the capabilities of SPI to explore the relationships between dynamic structures and functions of native biomolecular complexes

Micro-liquid enclosure array and its semi-automated assembling system for x-ray free-electron laser diffractive imaging of samples in solution

We developed micro-liquid enclosure arrays (MLEAs) for holding solution samples in coherent diffractive imaging (CDI) using x-ray free-electron lasers (XFELs). Hundreds of fully isolated micro-liquid enclosures are arranged in a single MLEA chip for efficient measurement, where each enclosure is destroyed after exposure to a single XFEL pulse. A semi-automated MLEA assembling system was also developed to enclose solution samples into MLEAs efficiently at high precision. We performed XFEL-based CDI experiments using MLEAs and imaged in-solution structures of self-assembled gold nanoparticles. The sample hit rate can be optimized by adjusting solution concentration, and we achieved a single-particle hit rate of 31%, which is not far from the theoretical upper limit of 37% derived from the Poisson statistics. MELAs allow us to perform CDI measurement under controlled solution conditions and will help reveal the nanostructures and dynamics of particles in solution

Micro-liquid enclosure array and its semi-automated assembling system for x-ray free-electron laser diffractive imaging of samples in solution

We developed micro-liquid enclosure arrays (MLEAs) for holding solution samples in coherent diffractive imaging (CDI) using x-ray free-electron lasers (XFELs). Hundreds of fully isolated micro-liquid enclosures are arranged in a single MLEA chip for efficient measurement, where each enclosure is destroyed after exposure to a single XFEL pulse. A semi-automated MLEA assembling system was also developed to enclose solution samples into MLEAs efficiently at high precision. We performed XFEL-based CDI experiments using MLEAs and imaged in-solution structures of self-assembled gold nanoparticles. The sample hit rate can be optimized by adjusting solution concentration, and we achieved a single-particle hit rate of 31%, which is not far from the theoretical upper limit of 37% derived from the Poisson statistics. MELAs allow us to perform CDI measurement under controlled solution conditions and will help reveal the nanostructures and dynamics of particles in solution