Quantitative, in situ visualization of intracellular insulin vesicles in pancreatic beta cells.

Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.

Design and first-round commissioning result of the SASE beamline at the Shanghai Soft X-ray FEL facility.

The Shanghai Soft X-ray Free-Electron Laser (SXFEL) is the first X-ray free-electron laser facility in China. The SASE beamline, which consists of a pink-beam branch and a mono-beam branch, is one of the two beamlines in the Phase-I construction. The pink-beam branch opened for users in 2023 after successful first-round beamline commissioning. In this paper, the design of the beamline is presented and the performance of the pink-beam branch is reported. The measured energy-resolving power of the online spectrometer is over 6000 @ 400 eV. The focusing spot size of the pink beam is less than 3 µm in both the horizontal and vertical at the endstation.

Atomic-Level Imaging of Zeolite Local Structures Using Electron Ptychography.

Zeolites are among the most important heterogeneous catalysts, widely employed in separation reaction, fine chemical production, and petroleum refining. Through rational design of the frameworks, zeolites with versatile functions can be synthesized. Local imaging of zeolite structures at the atomic scale, including the basic framework atoms (Si, Al, and O) and extra-framework cations, is necessary to understand the structure-function relationship of zeolites. Herein, we implemented electron ptychography into direct imaging of local structures of two zeolites, Na-LTA and ZSM-5. Not only all the framework atoms but also extra-framework Na+ cations with only 1/4 occupation probabilities in Na-LTA were directly observed. Local structures of ZSM-5 zeolites having guest molecules among channels with different orientations were also unraveled using different reconstruction algorithms. The approach presented here provides a new way to locally image zeolites structure, and it is expected to be an essential key for further studying and tuning zeolites active sites at the atomic level.

Single-pulse characterization of the focal spot size of X-ray free-electron lasers using coherent diffraction imaging.

The characterization of X-ray focal spots is of great significance for the diagnosis and performance optimization of focusing systems. X-ray free-electron lasers (XFELs) are the latest generation of X-ray sources with ultrahigh brilliance, ultrashort pulse duration and nearly full transverse coherence. Because each XFEL pulse is unique and has an ultrahigh peak intensity, it is difficult to characterize its focal spot size individually with full power. Herein, a method for characterizing the spot size at the focus position is proposed based on coherent diffraction imaging. A numerical simulation was conducted to verify the feasibility of the proposed method. The focal spot size of the Coherent Scattering and Imaging endstation at the Shanghai Soft X-ray Free Electron Laser Facility was characterized using the method. The full width at half-maxima of the focal spot intensity and spot size in the horizontal and vertical directions were calculated to be 2.10 ± 0.24 µm and 2.00 ± 0.20 µm, respectively. An ablation imprint on the silicon frame was used to validate the results of the proposed method.

Three-Dimensional Quantitative Coherent Diffraction Imaging of Staphylococcus aureus Treated with Peptide-Mineralized Au-Cluster Probes.

Characterizing interactions between microbial cells and their specific inhibitory drugs is essential for developing effective drugs and understanding the therapeutic mechanism. Functional metal nanoclusters can be effective inhibitory agents against microorganisms according to various characterization methods, but quantitative three-dimensional (3D) spatial structural analysis of intact cells is lacking. Herein, using coherent X-ray diffraction imaging, we performed in situ 3D visualization of unstained Staphylococcus aureus cells treated with peptide-mineralized Au-cluster probes at a resolution of ∼47 nm. Subsequent 3D mass-density mapping and quantitative structural analyses of S. aureus in different degrees of destruction showed that the bacterial cell wall was damaged and cytoplasmic constituents were released from cells, confirming the significant antibacterial effects of the Au-cluster probe. This study provides a promising nondestructive approach for quantitative imaging and paves the way for further research into microbe-inhibitor drug interactions.

In vitro and in vivo characterization of insulin vesicles by electron microscopy.

Insulin is the main hypoglycemic hormone, promoting the absorption and storage of glucose and inhibiting its production. It is a hexamer composed of six insulin macromolecules and a Zn2+ and clustered in insulin vesicles of pancreatic ß cell. Most current research has focused on the in vivo imaging of whole cells while there are few detailed studies on structure of insulin vesicles. The precise content of Zn2+ in vesicles is not clear, and the aggregation state and location of insulin in insulin vesicles is not fully characterized, which hinders a thorough understanding of insulin secretion process and diseases caused by blood sugar regulation. Here, we performed electron microscopy (EM) studies on both whole cells (in vivo) and extracted isolated insulin vesicles by supercentrifugation (in vitro) to explore the location and distribution of insulin vesicles in pancreatic ß cells. Meanwhile, we analyzed the content of Zn2+ and Ca2+ through EM imaging and energy dispersive spectroscopy (EDS) mapping, and the content of Zn2+ was found to be proportional to the size of insulin vesicles. In addition, by taking advantage of TEM tomography, the three-dimensional structure of insulin vesicle was obtained by acquisition projections in different angles of insulin vesicle. This study provides a promising way to quantitative analysis of intracellular insulin, which may be of great significance to the study of diabetes and other blood sugar diseases.

Thermal effects of beam profiles on X-ray photon correlation spectroscopy at megahertz X-ray free-electron lasers.

X-ray free-electron lasers (XFELs) with megahertz repetition rates enable X-ray photon correlation spectroscopy (XPCS) studies of fast dynamics on microsecond and sub-microsecond time scales. Beam-induced sample heating is one of the central concerns in these studies, as the interval time is often insufficient for heat dissipation. Despite the great efforts devoted to this issue, few have evaluated the thermal effects of X-ray beam profiles. This work compares the effective dynamics of three common beam profiles using numerical methods. Results show that under the same fluence, the effective temperatures increase with the nonuniformity of the beam, such that the Gaussian beam profile yields a higher effective temperature than the donut-like and uniform profiles. Moreover, decreasing the beam sizes is found to reduce beam-induced thermal effects, in particular the effects of beam profiles.

Nanoscale Detection of Subcellular Nanoparticles by X-Ray Diffraction Imaging for Precise Quantitative Analysis of Whole Cancer Cells.

Nanoparticles show great potential for drug delivery systems in cancer treatment and diagnosis, which mainly rely on the interaction between nanoparticles and living cells. However, there is still a lack of accurate and large field-of-view imaging techniques to reveal the aggregation and distribution behavior of nanoparticles in whole cancer cells without being destroyed. Here, we demonstrated quantitative imaging of unstained and intact mouse breast cancer cells (4T1) containing 50 nm gold nanoparticles (Au@citrate NPs) using an X-ray scanning coherent diffraction imaging (ptychography) technique in a large field-of-view. A two-dimensional spatial resolution of 17 nm was achieved on the 4T1 cell. We combine X-ray ptychography and equally sloped tomography (EST) to perform three-dimensional structural mapping, distribution, and aggregation behavior of Au@citrate NPs in cancer cells. By taking full advantage of the large field-of-view, high-resolution, and quantitative imaging technique, the single intracellular Au@citrate NPs are observed and the amount of Au@citrate NPs in aggregations can be accurately quantified. In addition, the morphological changes of lysosomes containing Au@citrate NPs can be observed in the high-contrast mass density images. This study provides an approach for exploring quantitative analysis and physiological delivery of nanomaterials in intact cancer cells at nanoscale resolution, which may greatly benefit the interdisciplinary research of material science, nanomedicine, and nanotoxicology.

3D Imaging and Quantification of the Integrin at a Single-Cell Base on a Multisignal Nanoprobe and Synchrotron Radiation Soft X-ray Tomography Microscopy.

The development of three-dimensional (3D) single-cell imaging and protein quantitative methods can provide more comprehensive information for diagnoses. We report the design and synthesis of a multisignal nanoprobe (AuGdNC@BSA-CV) for single-cell 3D imaging and quantifying the integrin αIIbß3 using correlated synchrotron radiation soft X-ray tomography microscopy and an iterative tomographic algorithm termed equally sloped tomography for the first time. Moreover, on the basis of the Au or Gd content of our nanoprobe, the number of integrin αIIbß3 on a single cell also can be accurately quantified (1.5 × 107 per cell) via inductively coupled plasma mass spectrometry.

Is GSH Chelated Pt Molecule Inactive in Anti-Cancer Treatment? A Case Study of Pt6 GS4.

Platinum (Pt) drugs are widely used in anti-cancer treatment although many reports advocated that tumor cells could inactivate Pt drugs via glutathione-Pt (GSH-Pt) adducts formation. To date, GSH chelated Pt molecules have not been assessed in cancer treatment because GSH-Pt adducts are not capable of killing cancer cells, which is widely accepted and well followed. In this report, endogenous biothiol is utilized to precisely synthesize a GSH chelated Pt molecule (Pt6 GS4 ). This Pt6 GS4 molecule can be well taken up by aggressive triple negative breast cancer (TNBC) cells. Subsequently, its metabolites could enter nuclei to interact with DNA, finally the DNA-Pt complex triggers TNBC cell apoptosis via the p53 pathway. Impressively, high efficacy for anti-cancer treatment is achieved by Pt6 GS4 both in vitro and in vivo when compared with traditional first-line carboplatin in the same dosage. Compared with carboplatin, Pt6 GS4 keeps tumor bearing mice alive for a longer time and is non-toxic for the liver and kidneys. This work opens a route to explore polynuclear Pt compound with accurate architecture for enhancing therapeutic effects and reducing systemic toxicity.

Characterizing the intrinsic properties of individual XFEL pulses via single-particle diffraction.

With each single X-ray pulse having its own characteristics, understanding the individual property of each X-ray free-electron laser (XFEL) pulse is essential for its applications in probing and manipulating specimens as well as in diagnosing the lasing performance. Intensive research using XFEL radiation over the last several years has introduced techniques to characterize the femtosecond XFEL pulses, but a simple characterization scheme, while not requiring ad hoc assumptions, to address multiple aspects of XFEL radiation via a single data collection process is scant. Here, it is shown that single-particle diffraction patterns collected using single XFEL pulses can provide information about the incident photon flux and coherence property simultaneously, and the X-ray beam profile is inferred. The proposed scheme is highly adaptable to most experimental configurations, and will become an essential approach to understanding single X-ray pulses.

Potential of Attosecond Coherent Diffractive Imaging.

Attosecond science has been transforming our understanding of electron dynamics in atoms, molecules, and solids. However, to date almost all of the attoscience experiments have been based on spectroscopic measurements because attosecond pulses have intrinsically very broad spectra due to the uncertainty principle and are incompatible with conventional imaging systems. Here we report an important advance towards achieving attosecond coherent diffractive imaging. Using simulated attosecond pulses, we simultaneously reconstruct the spectrum, 17 probes, and 17 spectral images of extended objects from a set of ptychographic diffraction patterns. We further confirm the principle and feasibility of this method by successfully performing a ptychographic coherent diffractive imaging experiment using a light-emitting diode with a broad spectrum. We believe this work clears the way to an unexplored domain of attosecond imaging science, which could have a far-reaching impact across different disciplines.

Interaction Principle Between Coagulation Factor X and Fullerene Derivatives with Different Hydrophilic-Hydrophobic Properties for Anticoagulation.

Recent experiments have found that fullerenols can inhibit coagulation factor X (FXa) activity and have the effects on anticoagulation. But the interactions between fullerene derivatives and FXa are still lacking which are crucial for the new inhibitors designs and applications. In this study, we investigated the interaction principle between FXa and fullerenol molecules (C60(OH)24)/carboxyfullerene molecules (C60(C(COOH)2)2) with different hydrophilic-hydrophobic properties via AutoDock Vina. We performed molecular docking to obtain the binding mode conformations of C60(OH)24/C60(C(COOH)2)2 to FXa and investigated multibody adsorption behaviors of C60(OH)24/C60(C(COOH)2)2 to FXa. Then we analyzed the interactions between FXa and C60(OH)24/C60(C(COOH)2)2 to obtain the absorption driving mechanism. We found C60(C(COOH)2)2 was more stable to bind to the active site of FXa compared with C60(OH)24 with lower binding energy during the competitive absorptions. The adsorption behaviors of fullerene derivatives C60(OH)24 and C60(C(COOH)2)2 were different as well during their multibody absorptions. The absorption of C60(OH)24 was driven by hydrophilic interactions while that of C60(C(COOH)2)2 was driven by hydrophobic interactions. These results can be used to guide the design and optimization of the fullerene derivative anticoagulant through inhibiting the activity of FXa.

Enhancement of phase retrieval capability in ptychography by using strongly scattering property of the probe-generating device.

In common ptychographic coherent diffractive imaging (PCDI) systems, the probe-generating devices typically exhibit strong scattering, which is not fully used. Here, we report the reasonableness of using the diffraction pattern of the probe-generating device as the frequency-domain information of the scanning probe located in the sample plane, and we propose a method introducing this frequency-domain information into an iterative process to improve the imaging quality of PCDI. The new method was demonstrated using both a visible laser source and a synchrotron radiation X-ray source; the proposed method significantly improved the imaging quality in both demonstrations.

Yellow Emission from Low Coordination Site of Sr2 SiO4 :Eu2+ , Ce3+ : Influence of Lanthanide Dopants on the Electron Density and Crystallinity in Crystal Site Engineering Approach.

Lanthanide doping through a crystal site engineering approach tunes the emission wavelength suitable for LED applications, but weak emission from low coordination sites remains a huge challenge. Herein the use of a sensitizer is reported to enhance the emission strength and unravel the crystallinity and phase, as this approach demands a large amount of dopants. Doping of Eu2+ ions at SrO10 and SrO9 sites of Sr2 SiO4 (S2 S), respectively, tunes the emission from green to yellow and controlled doping of a Ce3+ sensitizer quadruples the quantum efficiency of yellow emission. Remarkably, doping of Eu2+ at the SrO9 site produces polycrystals, whereas co-doping of Ce3+ and Eu2+ at the same site produces single crystals. DFT calculations further delineate the underlying changes wherein strong interaction of dopant with its neighbours determines the electron density, and thus the crystallinity and phase, rather than usual explanation of aliovalent conditions, which is further substantiated by TEM results. Irrespective of dopant valence, use of large amounts of dopants and their interaction with the host is responsible for the crystallinity and phase change (α'-S2 S to ß-S2 S). The XPS valence band spectra experimentally evidences the changes in bonding nature of O 2p and O 2s orbitals of silicate and its electron density, due to doping at the two sites. In short, the outcomes resulting from this work could be extended for the development of other two-coordination site lanthanide-doped materials and crystallization of inorganic materials.

Role of Synthesis Method on Luminescence Properties of Europium(II, III) Ions in ß-Ca2SiO4: Probing Local Site and Structure.

The europium ion probes the symmetry disorder in the crystal structure, although the distortion due to charge compensation in the case of aliovalent dopant remains interesting, especially preparation involves low and high temperatures. This work studies the preparation of the ß-Ca2SiO4 (from here on C2S) particle from Pechini (C2SP) and hydrothermal (C2SH) methods, and its luminescence variance upon doping with Eu2+ and Eu3+ ions. The blue shift of the charge-transfer band (CTB) in the excitation spectra indicates a larger Eu3+-O2- distance in Eu3+ doped C2SH. The changes in vibrational frequencies due to stretching and bending vibrations in the FTIR and the Raman spectra and binding energy shift in the XPS analysis confirmed the distorted SiO44- tetrahedra in C2SH. The high hydrothermal temperature and pressure produce distortion, which leads to symmetry lowering although doping of aliovalent ion may slightly change the position of the Ca atoms. The increasing asymmetry ratio value from C2SP to C2SH clearly indicates that the europium ion stabilized in a more distorted geometry. It is also supported by Judd-Ofelt analysis. The concentration quenching and site-occupancy of Eu3+ ions in two nonequivalent sites of C2S were discussed. The charge state and concentration of europium ions in C2SP and C2SH were determined using X-ray photoelectron spectroscopy measurements. The C2S particles were studied by X-ray powder diffraction, FTIR, Raman, BET surface area, TGA/DTA, electron microscopy, XPS, and luminescence spectroscopy. The impact of citrate ion on the morphology and particle size of C2SH has been hypothesized on the basis of the microscopy images. This study provides insights that are needed for further understanding the structure of C2S and thereby improves the applications in optical and biomedical areas and cement hydration.

Synchrotron X-ray Microtomography with Improved Image Quality by Ring Artifacts Correction for Structural Analysis of Insects.

Ring artifacts are undesirable and complicate the analysis and interpretation of microstructures in synchrotron X-ray microtomography. Here, we propose a new method to improve the image quality of an object by removing the ring artifacts and investigate the efficiency of this process with tomographic images of a dried Tenebrio molitor. In this method, before the tomographic reconstruction, ring artifacts were identified and located in the sinograms as line artifacts. Then, the identified line artifacts were corrected as single point noise via image processing of the original projections. Eventually, the corresponding line artifacts were removed, resulting in reduced ring artifacts in the reconstructed tomographic images. Simulations verified the efficiency of the proposed method. This method was successfully applied for the structural analysis of the insect T. molitor, showing superior performance in reducing ring artifacts in the tomographic image without noticeable loss of structural information.

Three-dimensional structure determination from a single view.

The ability to determine the structure of matter in three dimensions has profoundly advanced our understanding of nature. Traditionally, the most widely used schemes for three-dimensional (3D) structure determination of an object are implemented by acquiring multiple measurements over various sample orientations, as in the case of crystallography and tomography, or by scanning a series of thin sections through the sample, as in confocal microscopy. Here we present a 3D imaging modality, termed ankylography (derived from the Greek words ankylos meaning 'curved' and graphein meaning 'writing'), which under certain circumstances enables complete 3D structure determination from a single exposure using a monochromatic incident beam. We demonstrate that when the diffraction pattern of a finite object is sampled at a sufficiently fine scale on the Ewald sphere, the 3D structure of the object is in principle determined by the 2D spherical pattern. We confirm the theoretical analysis by performing 3D numerical reconstructions of a sodium silicate glass structure at 2 A resolution, and a single poliovirus at 2-3 nm resolution, from 2D spherical diffraction patterns alone. Using diffraction data from a soft X-ray laser, we also provide a preliminary demonstration that ankylography is experimentally feasible by obtaining a 3D image of a test object from a single 2D diffraction pattern. With further development, this approach of obtaining complete 3D structure information from a single view could find broad applications in the physical and life sciences.

Enhanced ferroelectric-nanocrystal-based hybrid photocatalysis by ultrasonic-wave-generated piezophototronic effect.

An electric field built inside a crystal was proposed to enhance photoinduced carrier separation for improving photocatalytic property of semiconductor photocatalysts. However, a static built-in electric field can easily be saturated by the free carriers due to electrostatic screening, and the enhancement of photocatalysis, thus, is halted. To overcome this problem, here, we propose sonophotocatalysis based on a new hybrid photocatalyst, which combines ferroelectric nanocrystals (BaTiO3) and semiconductor nanoparticles (Ag2O) to form an Ag2O-BaTiO3 hybrid photocatalyst. Under periodic ultrasonic excitation, a spontaneous polarization potential of BaTiO3 nanocrystals in responding to ultrasonic wave can act as alternating built-in electric field to separate photoinduced carriers incessantly, which can significantly enhance the photocatalytic activity and cyclic performance of the Ag2O-BaTiO3 hybrid structure. The piezoelectric effect combined with photoelectric conversion realizes an ultrasonic-wave-driven piezophototronic process in the hybrid photocatalyst, which is the fundamental of sonophotocatalysis.

Quantitative Imaging of Single Unstained Magnetotactic Bacteria by Coherent X-ray Diffraction Microscopy.

Novel coherent diffraction microscopy provides a powerful lensless imaging method to obtain a better understanding of the microorganism at the nanoscale. Here we demonstrated quantitative imaging of intact unstained magnetotactic bacteria using coherent X-ray diffraction microscopy combined with an iterative phase retrieval algorithm. Although the signal-to-noise ratio of the X-ray diffraction pattern from single magnetotactic bacterium is weak due to low-scattering ability of biomaterials, an 18.6 nm half-period resolution of reconstructed image was achieved by using a hybrid input-output phase retrieval algorithm. On the basis of the quantitative reconstructed images, the morphology and some intracellular structures, such as nucleoid, polyß-hydroxybutyrate granules, and magnetosomes, were identified, which were also confirmed by scanning electron microscopy and energy dispersive spectroscopy. With the benefit from the quantifiability of coherent diffraction imaging, for the first time to our knowledge, an average density of magnetotactic bacteria was calculated to be ∼1.19 g/cm(3). This technique has a wide range of applications, especially in quantitative imaging of low-scattering biomaterials and multicomponent materials at nanoscale resolution. Combined with the cryogenic technique or X-ray free electron lasers, the method could image cells in a hydrated condition, which helps to maintain their natural structure.