The new X-ray/visible microscopy MAXWELL technique for fast three-dimensional nanoimaging with isotropic resolution.

Microscopy by Achromatic X-rays With Emission of Laminar Light (MAXWELL) is a new X-ray/visible technique with attractive characteristics including isotropic resolution in all directions, large-volume imaging and high throughput. An ultrathin, laminar X-ray beam produced by a Wolter type I mirror irradiates the sample stimulating the emission of visible light by scintillating nanoparticles, captured by an optical system. Three-dimensional (3D) images are obtained by scanning the specimen with respect to the laminar beam. We implemented and tested the technique with a high-brightness undulator at SPring-8, demonstrating its validity for a variety of specimens. This work was performed under the Synchrotrons for Neuroscience-an Asia-Pacific Strategic Enterprise (SYNAPSE) collaboration.

High-resolution fast-tomography brain-imaging beamline at the Taiwan Photon Source.

The new Brain Imaging Beamline (BIB) of the Taiwan Photon Source (TPS) has been commissioned and opened to users. The BIB and in particular its endstation are designed to take advantage of bright unmonochromatized synchrotron X-rays and target fast 3D imaging, ∼1 ms exposure time plus very high ∼0.3 µm spatial resolution. A critical step in achieving the planned performances was the solution to the X-ray induced damaging problems of the detection system. High-energy photons were identified as their principal cause and were solved by combining tailored filters/attenuators and a high-energy cut-off mirror. This enabled the tomography acquisition throughput to reach >1 mm3 min-1, a critical performance for large-animal brain mapping and a vital mission of the beamline.

Imaging with Coherent X-rays: From the Early Synchrotron Tests to SYNAPSE.

The high longitudinal and lateral coherence of synchrotron X-rays sources radically transformed radiography. Before them, the image contrast was almost only based on absorption. Coherent synchrotron sources transformed radiography into a multi-faceted tool that can extract information also from "phase" effects. Here, we report a very simple description of the new techniques, presenting them to potential new users without requiring a sophisticated background in advanced physics. We then illustrate the impact of such techniques with a number of examples. Finally, we present the international collaboration SYNAPSE (Synchrotrons for Neuroscience-an Asia-Pacific Strategic Enterprise), which targets the use of phase-contrast radiography to map one full human brain in a few years.

Synchrotron radiation and X-ray free-electron lasers (X-FELs) explained to all users, active and potential.

Synchrotron radiation evolved over one-half century into a gigantic worldwide enterprise involving tens of thousands of researchers. Initially, almost all users were physicists. But now they belong to a variety of disciplines: chemistry, materials science, the life sciences, medical research, ecology, cultural heritage and others. This poses a challenge: explaining synchrotron sources without requiring a sophisticated background in theoretical physics. Here this challenge is met with an innovative approach that only involves elementary notions, commonly possessed by scientists of all domains.

Gold nano-mesh synthesis by continuous-flow X-ray irradiation.

X-ray irradiation has been extensively used in recent years as a fabrication step for nanoparticles and nanoparticle systems. A variant of this technique, continuous-flow X-ray irradiation, has recently been developed, and offers three important advantages: precise control of the irradiation dose, elimination of convection effects in the precursor solution, and suitability for large-scale production. Here, the use of this method to fabricate Au nano-meshes of interest as transparent and flexible electrodes for optoelectronics is reported. The study includes extensive characterization of the synthesis parameters and of the product properties, with rather encouraging results.

An enlightening procedure to explain the extreme power of synchrotron radiation.

A simple approach exploits quantum properties to justify the dependence on γ4 of the total synchrotron emitted power. It also clarifies some apparent puzzles and brings to light the underlying, multiple relativistic phenomena.

The simple physics of the bending magnet spectrum.

A new elementary model of the bending magnet synchrotron radiation is presented, with minimal mathematical formalism. The model explains features not justified by other simplified approaches; in particular, it brings to light the key role of the directional Doppler effect.

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.

Q&A: Why use synchrotron x-ray tomography for multi-scale connectome mapping?

To understand how information flows and is used in the human brain, we must map neural structures at all levels, providing visualizations similar to those of Google Earth for continents, countries, cities, and streets. Unfortunately, the imaging and processing techniques currently used in connectomics projects cannot achieve complete mapping for the brains of large animals within the timespan of a typical research career. However, feasible improvements in x-ray imaging would change this situation. This Q&A discusses synchrotron x-ray tomography, an exciting new approach for in situ mapping of whole-brain wiring diagrams at multiple levels of spatial resolution.

The relativistic foundations of synchrotron radiation.

Special relativity (SR) determines the properties of synchrotron radiation, but the corresponding mechanisms are frequently misunderstood. Time dilation is often invoked among the causes, whereas its role would violate the principles of SR. Here it is shown that the correct explanation of the synchrotron radiation properties is provided by a combination of the Doppler shift, not dependent on time dilation effects, contrary to a common belief, and of the Lorentz transformation into the particle reference frame of the electromagnetic field of the emission-inducing device, also with no contribution from time dilation. Concluding, the reader is reminded that much, if not all, of our argument has been available since the inception of SR, a research discipline of its own standing.

A compact synchrotron-based transmission X-ray microscope.

A compact transmission X-ray microscope has been designed and implemented based on a cylindrical symmetry around the optical axis that sharply limits the instabilities due to thermal mechanical drift. Identical compact multi-axis closed-loop actuation modules drive different optical components. The design is modular and simplifies the change of individual parts, e.g. the use of different magnification and focusing devices. This compact instrument can be easily transported between laboratory and synchrotron facilities and quickly put into operation. An automated alignment mechanism simplifies the assembly of different modules after transportation. After describing the design details, the results of the first tests are presented.

Cu(In(1-x)Ga(x))S2 nanocrystals and films: low-temperature synthesis with size and composition control.

We demonstrate a single-step X-ray irradiation process that yields high-quality Cu(In1-xGax)S2 nanocrystals in colloidal solutions, with complete control of size and composition. Thin films produced by drop-casting exhibit high-quality photoresponse, confirming that our process is suitable for microelectronics applications.

Gold nanoparticles as high-resolution X-ray imaging contrast agents for the analysis of tumor-related micro-vasculature.

BACKGROUND: Angiogenesis is widely investigated in conjunction with cancer development, in particular because of the possibility of early stage detection and of new therapeutic strategies. However, such studies are negatively affected by the limitations of imaging techniques in the detection of microscopic blood vessels (diameter 3-5 µm) grown under angiogenic stress. We report that synchrotron-based X-ray imaging techniques with very high spatial resolution can overcome this obstacle, provided that suitable contrast agents are used. RESULTS: We tested different contrast agents based on gold nanoparticles (AuNPs) for the detection of cancer-related angiogenesis by synchrotron microradiology, microtomography and high resolution X-ray microscopy. Among them only bare-AuNPs in conjunction with heparin injection provided sufficient contrast to allow in vivo detection of small capillary species (the smallest measured lumen diameters were 3-5 µm). The detected vessel density was 3-7 times higher than with other nanoparticles. We also found that bare-AuNPs with heparin allows detecting symptoms of local extravascular nanoparticle diffusion in tumor areas where capillary leakage appeared. CONCLUSIONS: Although high-Z AuNPs are natural candidates as radiology contrast agents, their success is not guaranteed, in particular when targeting very small blood vessels in tumor-related angiography. We found that AuNPs injected with heparin produced the contrast level needed to reveal--for the first time by X-ray imaging--tumor microvessels with 3-5 µm diameter as well as extravascular diffusion due to basal membrane defenestration. These results open the interesting possibility of functional imaging of the tumor microvasculature, of its development and organization, as well as of the effects of anti-angiogenic drugs.

Fabrication of single crystal CuGaS2 nanorods by X-ray irradiation.

CuGaS(2) nanorods were synthesized by irradiating the precursor solution with intense X-rays. The products are single crystal nanorods with preferential [220] growth and a uniform size distribution. We also report on the photoresponse of drop-cast films of these nanorods.

Quantitative analysis of nanoparticle internalization in mammalian cells by high resolution X-ray microscopy.

BACKGROUND: Quantitative analysis of nanoparticle uptake at the cellular level is critical to nanomedicine procedures. In particular, it is required for a realistic evaluation of their effects. Unfortunately, quantitative measurements of nanoparticle uptake still pose a formidable technical challenge. We present here a method to tackle this problem and analyze the number of metal nanoparticles present in different types of cells. The method relies on high-lateral-resolution (better than 30 nm) transmission x-ray microimages with both absorption contrast and phase contrast -- including two-dimensional (2D) projection images and three-dimensional (3D) tomographic reconstructions that directly show the nanoparticles. RESULTS: Practical tests were successfully conducted on bare and polyethylene glycol (PEG) coated gold nanoparticles obtained by x-ray irradiation. Using two different cell lines, EMT and HeLa, we obtained the number of nanoparticle clusters uptaken by each cell and the cluster size. Furthermore, the analysis revealed interesting differences between 2D and 3D cultured cells as well as between 2D and 3D data for the same 3D specimen. CONCLUSIONS: We demonstrated the feasibility and effectiveness of our method, proving that it is accurate enough to measure the nanoparticle uptake differences between cells as well as the sizes of the formed nanoparticle clusters. The differences between 2D and 3D cultures and 2D and 3D images stress the importance of the 3D analysis which is made possible by our approach.

Altered branching patterns of Purkinje cells in mouse model for cortical development disorder.

Disrupted cortical cytoarchitecture in cerebellum is a typical pathology in reeler. Particularly interesting are structural problems at the cellular level: dendritic morphology has important functional implication in signal processing. Here we describe a combinatorial imaging method of synchrotron X-ray microtomography with Golgi staining, which can deliver 3-dimensional(3-D) micro-architectures of Purkinje cell(PC) dendrites, and give access to quantitative information in 3-D geometry. In reeler, we visualized in 3-D geometry the shape alterations of planar PC dendrites (i.e., abnormal 3-D arborization). Despite these alterations, the 3-D quantitative analysis of the branching patterns showed no significant changes of the 77 ± 8° branch angle, whereas the branch segment length strongly increased with large fluctuations, comparing to control. The 3-D fractal dimension of the PCs decreased from 1.723 to 1.254, indicating a significant reduction of dendritic complexity. This study provides insights into etiologies and further potential treatment options for lissencephaly and various neurodevelopmental disorders.

X-ray-induced changes in wettability.

We observed that hard X-ray irradiation modifies the wettability of a variety of inorganic materials. The smooth surfaces of all tested inorganic materials (ZnO, p-Si, Al2O3, SrTiO3, TiN, ZnS, CuO, Ag2O, and Cr2O3) change during irradiation to a state of superhydrophilic wettability, and such changes are explained by the accumulation of positive surface charges by photoelectron emission. The initial wettability state is re-established within several minutes of storage in deionized water.

The serpin Spn5 is essential for wing expansion in Drosophila melanogaster.

Serpins, a superfamily of protease inhibitors, control proteolytic cascades in many physiological processes. Genomic studies have revealed the presence of a high number of serpin-encoding genes in Drosophila melanogaster, but their functions remain largely unknown. In a biochemical screen designed to detect protease inhibitors that may be implicated in early Drosophila development, we identified in embryos a ligand that forms a 67 kDa SDS-stable complex with the broad spectrum protease trypsin. Characterization of this ligand revealed it to be the recently described serpin, Spn5. Expression analysis by in situ and Northern blot hybridization indicated maternal transmission of the transcript as well as zygotic expression in many larval, pupal and adult tissues. Targeted repression by RNA interference did not alter early embryogenesis but resulted in a complete defect in the unfolding and expansion of the wings of freshly eclosed mutant flies, without other detectable effects on development.

Nondestructive Characterization by Advanced Synchrotron Light Techniques: Spectromicroscopy and Coherent Radiology.

The advanced characteristics of synchrotron light has led in recent years to the development of a series of new experimental techniques to investigate chemical and physical properties on a microscopic scale. Although originally developed for materials science and biomedical research, such techniques find increasing applications in other domains - and could be quite useful for the study and conservation of cultural heritage. Specifically, they can nondestructively provide detailed chemical composition information that can be useful for the identification of specimens, for the discovery of historical links based on the sources of chemical raw materials and on chemical processes, for the analysis of damage, their causes and remedies and for many other issues. Likewise, morphological and structural information on a microscopic scale is useful for the identification, study and preservation of many different cultural and historical specimens. We concentrate here on two classes of techniques: in the first case, photoemission spectromicroscopy. This is the result of the advanced evolution of photoemission techniques like ESCA (Electron Microscopy for Chemical Analysis). By combining high lateral resolution to spectroscopy, photoemission spectromicroscopy can deliver fine chemical information on a microscopic scale in a nondestructive fashion. The second class of techniques exploits the high lateral coherence of modern synchrotron sources, a byproduct of the quest for high brightness or brilliance. We will see that such techniques now push radiology into the submicron scale and the submillisecond time domain. Furthermore, they can be implemented in a tomographic mode, increasing the information and becoming potentially quite useful for the analysis of cultural heritage specimens.

Full-field hard x-ray microscopy below 30 nm: a challenging nanofabrication achievement.

The fabrication of devices to focus hard x-rays is one of the most difficult-and important-challenges in nanotechnology. Here we show that Fresnel zone plates combining 30 nm external zones and a high aspect ratio finally bring hard x-ray microscopy beyond the 30 nm Rayleigh spatial resolution level and measurable spatial frequencies down to 20-23 nm feature size. After presenting the overall nanofabrication process and the characterization test results, we discuss the potential research impact of these resolution levels.