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.

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.

Computerized dynamic occlusal analysis and its correlation with static characters in post-orthodontic patients using the T-Scan system and the ABO objective grading system.

OBJECTIVES: This study was conducted to detect the overall performance of both static and dynamic occlusion in post-orthodontic patients using quantified methods, and to ascertain the correlation between the two states of occlusion. MATERIALS AND METHODS: A total of 112 consecutive patients evaluated by ABO-OGS were included in this study. Based on the pre-treatment Angle's classification of the malocclusion, samples were divided into four groups. After removing orthodontic appliances, each patients underwent the American Board of Orthodontic objective grading system (ABO-OGS) and T-Scan evaluations. All the scores were compared within these groups. Statistical evaluation included reliability tests, multivariate ANOVA, and correlation analyses (p < 0.05 was considered significant). RESULTS: The mean ABO-OGS score was satisfactory and did not differ by Angle classifications. The indices making substantial contributions to ABO-OGS were occlusal contacts, occlusal relationships, overjet, and alignment. Disocclusion time in post-orthodontic patients was longer than normal. Occlusion time, disocclusion time, and force distribution during dynamic motions were considerably influenced by static ABO-OGS measurements, especially occlusal contacts, buccolingual inclination, and alignment. CONCLUSION: Post-orthodontic cases that passed the static evaluation of clinicians and ABO-OGS may nevertheless be left with dental casts interference in dynamic motions. Both static and dynamic occlusion should be extensively evaluated before ending orthodontic treatment. Further research is needed on dynamic occlusal guidelines and standards.

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.

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.

Comparison of Biological Characteristics of Different Paranasal Sinus Mucosa in Goats.

BACKGROUND/PURPOSE: Prosthetic implants are the primary treatment for patients with edentulism. This study aims to explore and compare the biological characteristics of mucosal thickness and tensile strength of the paranasal sinuses (maxillary and frontal sinuses) in goats, thereby providing a theoretical basis and guidance for mucosa-related problems involved in maxillary sinus lifting. MATERIALS AND METHODS: The paranasal sinus mucosa (maxillary sinus crest, maxillary sinus floor and frontal sinus mucosa) was obtained from the goats for use in maxillary sinus lifting. The mucosa was made into tissue section specimens and evaluated by a computer with built-in screenshot software and an optical microscope with a graduated eyepiece. A total of 3 readings were randomly selected and recorded. The mucosa was clamped with a laboratory-made clamp device. After connecting the push-pull meter, the mucosa exposed by the inner ring of the clamp device was pressed vertically and uniformly until it ruptured. The strength value was read and recorded. The left and right ends of the mucosa were connected with the clamp device; horizontal tension was applied evenly to the mucosa until the mucosa ruptured. The strength value was read and recorded. The normality test, analysis of variance, LSD pairwise comparison and linear regression were performed for each group of data. RESULTS: The thicknesses of the maxillary sinus crest mucosa, floor mucosa and frontal sinus mucosa in goats were 410.03 ± 65.97 um, 461.33 ± 91.37 um and 216.90 ± 46.47 um, respectively. There were significant differences between the maxillary sinus crest and frontal sinus and the maxillary sinus floor and frontal sinus (P < .05). The range of tensile strength of the maxillary sinus crest mucosa, floor mucosa and frontal sinus mucosa in goats was 0.48 ± 0.10 kg, 0.54 ± 0.11kg and 0.20 ± 0.05kg, respectively. There were significant differences between the maxillary sinus crest and frontal sinus and the maxillary sinus floor and frontal sinus (P < .05). Tensile strength was positively correlated with the thickness of the mucosa of the maxillary and frontal sinuses (P < .05). CONCLUSION: The mucosal thickness and tensile strength of the maxillary sinus crest and floor were greater than those of the frontal sinus mucosa. There was a positive correlation between the tensile strength and the thickness of the mucosa.

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.

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.

Inhibition of H3K4 demethylation induces autophagy in cancer cell lines.

Epigenetic factors and related small molecules have emerged to be strongly involved in autophagy process. Here we report that 2-PCPA and GSK-LSD1, two inhibitors of histone H3K4 demethylase KDM1A/LSD1, induce autophagy in multiple mammalian cell lines. The two small molecules induce accumulation of LC3II, formation of autophagosome and autolysosome, and SQSTM1/p62 degradation. 2-PCPA treatment inhibits cell proliferation through cell cycle arrest but does not inducing cell death. Exogenous expression of KDM1A/LSD1 impaired the autophagic phenotypes triggered by 2-PCPA. The autophagy induced by 2-PCPA requires LC3-II processing machinery. But depletion of BECN1 and ULK1 with siRNA did not affect the LC3-II accumulation triggered by 2-PCPA. 2-PCPA treatment induces the change of global gene expression program, including a series of autophagy-related genes, such as SQSTM1/p62. Taken together, our data indicate that KDM1A/LSD1 inhibitors induce autophagy through affecting the expression of autophagy-related genes and in a BECN1-independent manner.

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.

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.

Feasibility of Shape-Memory Ni/Ti Alloy Wire Containing Tube Elevators for Transcrestal Detaching Maxillary Sinus Mucosa: Ex Vivo Study.

BACKGROUND: Osteotome sinus floor elevation is a less invasive approach to augment an insufficient alveolar bone at the posterior maxilla for dental implantation. However, this approach has some limitations due to the lack of sinus lift tools available for clinical use and the small transcrestal access to the maxillary sinus floor. We recently invented shape-memory Ni/Ti alloy wire containing tube elevators for transcrestal detaching maxillary sinus mucosa, and developed goat ex vivo models for direct visualizing the effectiveness of detaching sinus mucosa in real time during transcrestal maxillary sinus floor elevation. METHODS: We evaluated our invented elevators, namely elevator 012 and elevator 014, for their effectiveness for transcrestal detaching maxillary sinus mucosa using the goat ex vivo models. We measured the length of sinus mucosa detached in mesial and distal directions or buccal and palatal directions, and the space volume created by detaching maxillary sinus mucosa in mesial, distal, buccal and palatal directions using the invented elevators. RESULTS: Elevator 012 had a shape-memory Ni/Ti alloy wire with a diameter of 0.012 inch, while elevator 014 had its shape-memory Ni/Ti alloy wire with a diameter of 0.014 inch. Elevator 012 could detach the goat maxillary sinus mucosa in the mesial or distal direction for 12.1±4.3 mm, while in the buccal or palatal direction for 12.5±6.7 mm. The elevator 014 could detach the goat maxillary sinus mucosa for 23.0±4.9 mm in the mesial or distal direction, and for 19.0±8.1 mm in the buccal or palatal direction. An average space volume of 1.7936±0.2079 ml was created after detaching the goat maxillay sinus mucosa in both mesial/distal direction and buccal/palatal direction using elevator 012; while the average space volume created using elevator 014 was 1.8764±0.2366 ml. CONCLUSION: Both two newly invented tube elevators could effectively detach the maxillary sinus mucosa on the goat ex vivo sinus models. Moreover, elevator 014 has advantages over the elevator 012 for the capability to detach sinus mucosa.

Histone H3K4 methyltransferase Mll1 regulates protein glycosylation and tunicamycin-induced apoptosis through transcriptional regulation.

Disrupting protein glycosylation induces ER (endoplasmic reticulum) stress, resulting in the activation of UPR (unfolded protein response) pathways. A key function of the UPR is to restore ER homeostasis, but prolonged or unsolved ER stress can lead to apoptosis. MLL1 (Mixed Lineage Leukemia 1, also named ALL-1 or HRX), a histone H3K4 methyltransferase in mammals, plays important roles in leukemogenesis, transcriptional regulation, cell cycle and development. Here, we find that Mll1 deficiency enhances UPR and apoptosis induced by the glycosylation inhibitor TM (tunicamycin). The abnormal regulation of the UPR appears to be caused by a defect in protein glycosylation. Furthermore, Mll1 directly binds to the promoters of H6pd, Galnt12 and Ugp2, which regulates H3K4 trimethylation and the subsequent expression of these genes. The knockdown of H6pd, Galnt12 or Ugp2 enhances TM-induced apoptosis in Mll1(+/+)MEF cells, whereas the ectopic expression of these proteins inhibits TM-induced apoptosis in Mll1(-/-) MEF cells. Together, our data suggest that the maturation of glycoproteins in the ER is subject to regulation at the epigenetic level by a histone methyltransferase whose abnormality can lead to cancer and developmental defects.

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.

Strain Heterogeneity and Extended Defects in Halide Perovskite Devices.

Strain is an important property in halide perovskite semiconductors used for optoelectronic applications because of its ability to influence device efficiency and stability. However, descriptions of strain in these materials are generally limited to bulk averages of bare films, which miss important property-determining heterogeneities that occur on the nanoscale and at interfaces in multilayer device stacks. Here, we present three-dimensional nanoscale strain mapping using Bragg coherent diffraction imaging of individual grains in Cs0.1FA0.9Pb(I0.95Br0.05)3 and Cs0.15FA0.85SnI3 (FA = formamidinium) halide perovskite absorbers buried in full solar cell devices. We discover large local strains and striking intragrain and grain-to-grain strain heterogeneity, identifying distinct islands of tensile and compressive strain inside grains. Additionally, we directly image dislocations with surprising regularity in Cs0.15FA0.85SnI3 grains and find evidence for dislocation-induced antiphase boundary formation. Our results shine a rare light on the nanoscale strains in these materials in their technologically relevant device setting.

Three-dimensional coherent x-ray diffraction imaging of molten iron in mantle olivine at nanoscale resolution.

We report quantitative 3D coherent x-ray diffraction imaging of a molten Fe-rich alloy and crystalline olivine sample, synthesized at 6 GPa and 1800 °C, with nanoscale resolution. The 3D mass density map is determined and the 3D distribution of the Fe-rich and Fe-S phases in the olivine-Fe-S sample is observed. Our results indicate that the Fe-rich melt exhibits varied 3D shapes and sizes in the olivine matrix. This work has potential for not only improving our understanding of the complex interactions between Fe-rich core-forming melts and mantle silicate phases but also paves the way for quantitative 3D imaging of materials at nanoscale resolution under extreme pressures and temperatures.

Effect of local application of bone morphogenetic protein -2 on experimental tooth movement and biological remodeling in rats.

Background: This study attempts to detect the potential effects of local bone morphogenetic protein -2 (BMP-2) on orthodontic tooth movement and periodontal tissue remodeling. Methods: Forty adult SD rats were randomly divided into four groups: blank control group, unilateral injection of BMP-2 on the pressure side or tension side of orthodontic teeth and bilateral injection of BMP-2. Their maxillary first molar was moved by a 30 g constant force closed coil spring. 60 µL of BMP-2 with a concentration of 0.5 µg/mL was injected into each part at a time. In addition, three rats were selected as healthy control rats without any intervention. Fluorescent labeled BMP-2 was used to observe the distribution of exogenous BMP-2 in tissues. Micro-CT was used to measure the microscopic parameters of tooth displacement, trabecular bone and root absorption volume. Three different histological methods were used to observe the changes of tissue remodeling, and then the number of osteoclasts and the content of collagen fibers were calculated. Results: Compared with the blank control group, BMP-2 injection reduced the movement distance and increased the collagen fiber content and bone mass (p < 0.01). There was no significant difference in tooth movement distance, BV/TV ratio and BMD between injection sites in unilateral injection group (p > 0.05). In the case of bilateral injection of BMP-2, the osteogenesis is enhanced. Unilateral injection of BMP-2 did not promote root resorption, but double injection showed root resorption (p < 0.01). Conclusion: Our study does show that the osteogenesis of BMP-2 is dose-dependent rather than site-dependent when a certain amount of BMP-2 is applied around orthodontic teeth. Local application of BMP-2 around orthodontic teeth in an appropriate way can enhance bone mass and tooth anchorage without increasing the risk of root absorption volume. However, high levels of BMP-2 may cause aggressive root resorption. These findings are of great significance, that is, BMP-2 is an effective target for regulating orthodontic tooth movement.

A drug-loaded composite coating to improve osteogenic and antibacterial properties of Zn-1Mg porous scaffolds as biodegradable bone implants.

Zinc (Zn) alloy porous scaffolds produced by additive manufacturing own customizable structures and biodegradable functions, having a great application potential for repairing bone defect. In this work, a hydroxyapatite (HA)/polydopamine (PDA) composite coating was constructed on the surface of Zn-1Mg porous scaffolds fabricated by laser powder bed fusion, and was loaded with a bioactive factor BMP2 and an antibacterial drug vancomycin. The microstructure, degradation behavior, biocompatibility, antibacterial performance and osteogenic activities were systematically investigated. Compared with as-built Zn-1Mg scaffolds, the rapid increase of Zn2+, which resulted to the deteriorated cell viability and osteogenic differentiation, was inhibited due to the physical barrier of the composite coating. In vitro cellular and bacterial assay indicated that the loaded BMP2 and vancomycin considerably enhanced the cytocompatibility and antibacterial performance. Significantly improved osteogenic and antibacterial functions were also observed according to in vivo implantation in the lateral femoral condyle of rats. The design, influence and mechanism of the composite coating were discussed accordingly. It was concluded that the additively manufactured Zn-1Mg porous scaffolds together with the composite coating could modulate biodegradable performance and contribute to effective promotion of bone recovery and antibacterial function.

Quantitative analysis of the effect of radiation on mitochondria structure using coherent diffraction imaging with a clustering algorithm.

Radiation damage and a low signal-to-noise ratio are the primary factors that limit spatial resolution in coherent diffraction imaging (CDI) of biomaterials using X-ray sources. Introduced here is a clustering algorithm named ConvRe based on deep learning, and it is applied to obtain accurate and consistent image reconstruction from noisy diffraction patterns of weakly scattering biomaterials. To investigate the impact of X-ray radiation on soft biomaterials, CDI experiments were performed on mitochondria from human embryonic kidney cells using synchrotron radiation. Benefiting from the new algorithm, structural changes in the mitochondria induced by X-ray radiation damage were quantitatively characterized and analysed at the nanoscale with different radiation doses. This work also provides a promising approach for improving the imaging quality of biomaterials with XFEL-based plane-wave CDI.