Design and performance of an X-ray scanning microscope at the Hard X-ray Nanoprobe beamline of NSLS-II.

A hard X-ray scanning microscope installed at the Hard X-ray Nanoprobe beamline of the National Synchrotron Light Source II has been designed, constructed and commissioned. The microscope relies on a compact, high stiffness, low heat dissipation approach and utilizes two types of nanofocusing optics. It is capable of imaging with ∼15 nm × 15 nm spatial resolution using multilayer Laue lenses and 25 nm × 26 nm resolution using zone plates. Fluorescence, diffraction, absorption, differential phase contrast, ptychography and tomography are available as experimental techniques. The microscope is also equipped with a temperature regulation system which allows the temperature of a sample to be varied in the range between 90 K and 1000 K. The constructed instrument is open for general users and offers its capabilities to the material science, battery research and bioscience communities.

Ancient administrative handwritten documents: X-ray analysis and imaging.

Handwritten characters in administrative antique documents from three centuries have been detected using different synchrotron X-ray imaging techniques. Heavy elements in ancient inks, present even for everyday administrative manuscripts as shown by X-ray fluorescence spectra, produce attenuation contrast. In most cases the image quality is good enough for tomography reconstruction in view of future applications to virtual page-by-page `reading'. When attenuation is too low, differential phase contrast imaging can reveal the characters from refractive index effects. The results are potentially important for new information harvesting strategies, for example from the huge Archivio di Stato collection, objective of the Venice Time Machine project.

X-ray irradiation synthesis of PEG-coated Au-Pd nanoparticles.

We demonstrate that the combination of x-ray irradiation and capping by polyethylene glycol (PEG) produces excellent flexibility in controlling the structure of Au-Pd nanoparticles while preserving their catalytic performance. We specifically adopted two different fabrication methods: co-reduction and seed-assisted reduction. In both cases, precursor composition plays an important role in controlling the phases and size of the bimetallic nanoparticles. The optimal catalytic performance is obtained with the highest Pd concentration and when the nanoparticles consist of a Au core and a Pd shell.

Gold nanoparticles as multimodality imaging agents for brain gliomas.

BACKGROUND: Nanoparticles can be used for targeted drug delivery, in particular for brain cancer therapy. However, this requires a detailed analysis of nanoparticles from the associated microvasculature to the tumor, not easy because of the required high spatial resolution. The objective of this study is to demonstrate an experimental solution of this problem, based in vivo and post-mortem whole organ imaging plus nanoscale 3-dimensional (3D) X-ray microscopy. RESULTS: The use of gold nanoparticles (AuNPs) as contrast agents paved the way to a detailed high-resolution three dimensional (3D) X-ray and fluorescence imaging analysis of the relation between xenografted glioma cells and the tumor-induced angiogenic microvasculature. The images of the angiogenic microvessels revealed nanoparticle leakage. Complementary tests showed that after endocytotic internalization fluorescent AuNPs allow the visible-light detection of cells. CONCLUSIONS: AuNP-loading of cells could be extended from the case presented here to other imaging techniques. In our study, they enabled us to (1) identify primary glioma cells at inoculation sites in mice brains; (2) follow the subsequent development of gliomas. (3) Detect the full details of the tumor-related microvasculature; (4) Finding leakage of AuNPs from the tumor-related vasculature, in contrast to no leakage from normal vasculature.

Microstructure of atmospheric particles revealed by TXM and a new mode of influenza virus transmission.

For control of influenza, firstly it is important to find the real virus transmission media. Atmospheric aerosol particles are presumably one of the media. In this study, three typical atmospheric inhaled particles in Shanghai were studied by the synchrotron based transmission X-ray microscopes (TXM). Three dimensional microstructure of the particles reveals that there are many pores contained in, particularly the coal combustion fly particles which may be possible virus carrier. The particles can transport over long distance and cause long-range infections due to its light weight. We suggest a mode which is droplet combining with aerosol mode. By this mode the transmission of global and pandemic influenzas and infection between inland avian far from population and poultry or human living in cities along coast may be explained.

Firefly light flashing: oxygen supply mechanism.

Firefly luminescence is an intriguing phenomenon with potential technological applications, whose biochemistry background was only recently established. The physics side of this phenomenon, however, was still unclear, specifically as far as the oxygen supply mechanism for light flashing is concerned. This uncertainty is due to the complex microscopic structure of the tracheal system: without fully knowing its geometry, one cannot reliably test the proposed mechanisms. We solved this problem using synchrotron phase contrast microtomography and transmission x-ray microscopy, finding that the oxygen consumption corresponding to mitochondria functions exceeds the maximum rate of oxygen diffusion from the tracheal system to the photocytes. Furthermore, the flashing mechanism uses a large portion of this maximum rate. Thus, the flashing control requires passivation of the mitochondria functions, e.g., by nitric oxide, and switching of the oxygen supply from them to photoluminescence.

X-ray microscopy and tomography detect the accumulation of bare and PEG-coated gold nanoparticles in normal and tumor mouse tissues.

We demonstrate that, with appropriate staining, high-resolution X-ray microscopy can image complicated tissue structures--cerebellum and liver--and resolve large or small amounts of Au nanoparticles in these tissues. Specifically, images of tumor tissue reveal high concentrations of accumulated Au nanoparticles. PEG (poly(ethylene glycol)) coating is quite effective in enhancing this accumulation and significantly modifies the mechanism of uptake by reticuloendothelial system (RES) organs.

Full-field microimaging with 8 keV X-rays achieves a spatial resolutions better than 20 nm.

Fresnel zone plates (450 nm thick Au, 25 nm outermost zone width) used as objective lenses in a full field transmission reached a spatial resolution better than 20 nm and 1.5% efficiency with 8 keV photons. Zernike phase contrast was also realized without compromising the resolution. These are very significant achievements in the rapid progress of high-aspect-ratio zone plate fabrication by combined electron beam lithography and electrodeposition.

Hard x-ray Zernike microscopy reaches 30 nm resolution.

Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.

One-pot tuning of Au nucleation and growth: from nanoclusters to nanoparticles.

We describe a simple and effective method to obtain colloidal surface-functionalized Au nanoparticles. The method is primarily based on irradiation of a gold solution with high-flux X-rays from a synchrotron source in the presence of 11-mercaptoundecanoic acid (MUA). Extensive tests of the products demonstrated high colloidal density as well as excellent stability, shelf life, and biocompatibility. Specific tests with X-ray diffraction, UV-visible spectrometry, visible microscopy, Fourier transform infrared spectroscopy, dark-field visible-light scattering microscopy, and transmission electron microscopy demonstrated that MUA, being an effective surfactant, not only allows tunable size control of the nanoparticles, but also facilitates functionalization. The nanoparticle sizes were 6.45 ± 1.58, 1.83 ± 1.21, 1.52 ± 0.37 and 1.18 ± 0.26 nm with no MUA and with MUA-to-Au ratios of 1:2, 1:1, and 3:1. The MUA additionally enabled functionalization with l-glycine. We thus demonstrated flexibility in controlling the nanoparticle size over a large range with narrow size distribution.

One-pot synthesis of AuPt alloyed nanoparticles by intense x-ray irradiation.

We synthesized AuPt alloyed nanoparticles in colloidal solution by a one-pot procedure based on synchrotron x-ray irradiation in the presence of PEG (polyethylene glycol). The exclusive presence of alloyed nanoparticles with fcc structure was confirmed by several different experiments including UV-vis spectroscopy, x-ray diffraction (XRD) and transmission electron microscopy (TEM). The composition of the AuPt alloyed nanoparticles can be varied in a continuous fashion by simply varying the feed ratios of Au and Pt precursors. The nanoparticles exhibited colloidal stability and biocompatibility, important for potential applications.

Detecting small lung tumors in mouse models by refractive-index microradiology.

Refractive-index (phase-contrast) radiology was able to detect lung tumors less than 1 mm in live mice. Significant micromorphology differences were observed in the microradiographs between normal, inflamed, and lung cancer tissues. This was made possible by the high phase contrast and by the fast image taking that reduces the motion blur. The detection of cancer and inflammation areas by phase contrast microradiology and microtomography was validated by bioluminescence and histopathological analysis. The smallest tumor detected is less than 1 mm(3) with accuracy better than 1 × 10(-3) mm(3). This level of performance is currently suitable for animal studies, while further developments are required for clinical application.

Biocompatibility of Fe(3)O(4) nanoparticles evaluated by in vitro cytotoxicity assays using normal, glia and breast cancer cells.

In order to reveal the biocompatibility of Fe(3)O(4) nanoparticles and bipolar surfactant tetramethylammonium 11-aminoundecanoate cytotoxicity tests were performed as a function of concentration from low (0.1 microg ml(-1)) to higher concentration (100 microg ml(-1)) using various human glia, human breast cancer and normal cell lines. Cytotoxicity tests for human glia (D54MG, G9T, SF126, U87, U251, U373), human breast cancer (MB157, SKBR3, T47D) and normal (H184B5F5/M10, WI-38, SVGp12) cell lines exhibited almost nontoxicity and reveal biocompatibility of Fe(3)O(4) nanoparticles in the concentration range of 0.1-10 microg ml(-1), while accountable cytotoxicity can be seen at 100 microg ml(-1). The results of our studies suggest that Fe(3)O(4) nanoparticles coated with bipolar surfactant tetramethylammonium 11-aminoundecanoate are biocompatible and promising for bio-applications such as drug delivery, magnetic resonance imaging and magnetic hyperthermia.

Tailored Au nanorods: optimizing functionality, controlling the aspect ratio and increasing biocompatibility.

Monodisperse gold nanorods with high aspect ratio were synthesized by x-ray irradiation. Irradiation was first used to stimulate the creation of seeds. Afterward, nanorod growth was stimulated either by chemical reduction or again by x-ray irradiation. In the last case, the entire process took place without reducing agents. The shape of the final products could be controlled by modulating the intensity of the x-ray irradiation during the seed synthesis. In turn, the nanorod aspect ratio determines the absorption wavelength of the nanorods that can thus be optimized for different applications. Likewise, the aspect ratio influences the uptake of the nanorods by HeLa cells.

Synchrotron microangiography studies of angiogenesis in mice with microemulsions and gold nanoparticles.

We present an effective solution for the problem of contrast enhancement in phase-contrast microangiography, with the specific objective of visualising small (<8 microm) vessels in tumor-related microangiogenesis. Different hydrophilic and hydrophobic contrast agents were explored in this context. We found that an emulsified version of the hydrophobic contrast agents Lipiodol provides the best contrast and minimal distortion of the circulation and vessel structure. Such emulsions are reasonably biocompatible and, with sizes of 0 +/- 0.8 microm, sufficient to diffuse to the smallest vessel and still provide reasonable contrast. We also explored the use of Au nanoparticle colloids that could be used not only to enhance contrast but also for interesting applications in nanomedicine. Both the Lipiodol microemulsions and Au nanoparticle colloids can be conjugated with medicines or cell specific labeling agents and their small size can allow the study of the diffusion of contrast agents through the vessel leakage. This enables direct imaging of drug delivery which is important for cancer treatment.

Simple dose rate measurements for a very high synchrotron X-ray flux.

Dose measurements based on methylene blue (MB) bleaching, widely used for ultraviolet light, can also be applied to X-rays including very high flux levels. This method has been tested by using both MB bleaching and Fricke dosimetry for a conventional monochromatic X-ray source and then for 'white-beam' synchrotron radiation. The results show that MB bleaching dosimetry can easily measure X-ray doses up to at least 10(5) Gy s(-1), as long as the MB concentration is sufficiently high. This condition can be verified from the deviations from linearity of the bleaching versus exposure time.

Coupled tomography and distinct-element-method approach to exploring the granular media microstructure in a jamming hourglass.

We describe an approach for exploring microscopic properties of granular media that couples x-ray microtomography and distinct-element-method (DEM) simulations through image analysis. We illustrate it via the study of the intriguing phenomenon of instant arching in an hourglass (in our case a cylinder filled with a polydisperse mixture of glass beads that has a small circular shutter in the bottom). X-ray tomography provides three-dimensional snapshots of the microscopic conditions of the system both prior to opening the shutter, and thereafter, once jamming is completed. The process time in between is bridged using DEM simulation, which settles to positions in remarkably good agreement with the x-ray images. Specifically designed image analysis procedures accurately extract the geometrical information, i.e., the positions and sizes of the beads, from the raw x-ray tomographs, and compress the data representation from initially 5 gigabytes to a few tens of kilobytes per tomograph. The scope of the approach is explored through a sensitivity analysis to input data perturbations in both bead sizes and positions. We establish that accuracy of size--much more than position--estimates is critical, thus explaining the difficulty in considering a mixture of beads of different sizes. We further point to limits in the replication ability of granular flows away from equilibrium; i.e., the difficulty of numerically reproducing chaotic motion.

Toxic Effects of Size-tunable Gold Nanoparticles on Caenorhabditis elegans Development and Gene Regulation.

We utilized size-tunable gold nanoparticles (Au NPs) to investigate the toxicogenomic responses of the model organism Caenorhabditis elegans. We demonstrated that the nematode C. elegans can uptake Au NPs coated with or without 11-mercaptoundecanoic acid (MUA), and Au NPs are detectable in worm intestines using X-ray microscopy and confocal optical microscopy. After Au NP exposure, C. elegans neurons grew shorter axons, which may have been related to the impeded worm locomotion behavior detected. Furthermore, we determined that MUA to Au ratios of 0.5, 1 and 3 reduced the worm population by more than 50% within 72 hours. In addition, these MUA to Au ratios reduced the worm body size, thrashing frequency (worm mobility) and brood size. MTT assays were employed to analyze the viability of cultured C. elegans primary neurons exposed to MUA-Au NPs. Increasing the MUA to Au ratios increasingly reduced neuronal survival. To understand how developmental changes (after MUA-Au NP treatment) are related to changes in gene expression, we employed DNA microarray assays and identified changes in gene expression (e.g., clec-174 (involved in cellular defense), cut-3 and fil-1 (both involved in body morphogenesis), dpy-14 (expressed in embryonic neurons), and mtl-1 (functions in metal detoxification and homeostasis)).

Free-electron-laser coherent diffraction images of individual drug-carrying liposome particles in solution.

Using the excellent performances of a SACLA (RIKEN/HARIMA, Japan) X-ray free electron laser (X-FEL), coherent diffraction imaging (CDI) was used to detect individual liposome particles in water, with or without inserted doxorubicin nanorods. This was possible because of the electron density differences between the carrier, the liposome, and the drug. The result is important since liposome nanocarriers at present dominate drug delivery systems. In spite of the low cross-section of the original ingredients, the diffracted intensity of drug-free liposomes was sufficient for spatial reconstruction yielding quantitative structural information. For particles containing doxorubicin, the structural parameters of the nanorods could be extracted from CDI. Furthermore, the measurement of the electron density of the solution enclosed in each liposome provides direct evidence of the incorporation of ammonium sulphate into the nanorods. Overall, ours is an important test for extending the X-FEL analysis of individual nanoparticles to low cross-sectional systems in solution, and also for its potential use to optimize the manufacturing of drug nanocarriers.

Ultra-short metformin pretreatment for clomiphene citrate-resistant polycystic ovary syndrome.

OBJECTIVE: To evaluate the effect of ultra-short (12 days) metformin pretreatment in clomiphene-citrate (CC) resistant polycystic ovary syndrome (PCOS). METHOD: Eighty women with CC-resistant PCOS were randomly allocated to metformin pretreatment or usual treatment. Forty women received 1500 mg metformin daily for 12 days, followed by clomiphene 150 mg daily for 5 days along with metformin. Forty women (control group) received the same dose of clomiphene but no metformin pretreatment. RESULTS: In the metformin group, 17 (42.5%) women ovulated, and 6 (15%) conceived. In the control group, 5 (12.5%) women ovulated but none conceived. Compared with the control group, the metformin group had significantly higher ovulation (P = 0.03) and pregnancy rates (P = 0.026). CONCLUSION: Twelve days of metformin pretreatment improves ovulation and pregnancy rates in women with CC-resistant PCOS.