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Since 2020, synchrotron radiation facilities in several Asia-Pacific countries have been collaborating in a major project called "SYNAPSE"(Synchrotrons for Neuroscience: an Asia-Pacific Scientific Enterprise). They use x-ray imaging to attack in a coordinated fashion one of the major issues in modern science: the structure of animal and human brains, including neurons and connections. The objective is to develop Google-like maps also including detailed structural and functional information for selected regions of interest. The sheer mass of data needed for the objective poses huge problems for the acquisition, processing, storage and use of images. In order to complete the task within a reasonable time, the key element of the SYNAPSE strategy is the parallel and coordinated work of several facilities on the same specimens. This article reviews different aspects of the enterprise, including the foundations of synchrotron radiation, coherence and of its role in advanced imaging, electron accelerators, x-ray optics and detectors. This will provide the foundation for an extensive presentation of the different components of SYNAPSE, with an overview of results already obtained within the consortium.

Mapping the large neural networks of animal and human brains is a fundamental but so far elusive task, because of the massive amount of data and the consequent prohibitively long image taking and processing times. We developed an effective strategy called “AXON” (Accelerated X-ray Observation of Neurons) to solve this problem. AXON can achieve comprehensive whole-brain mapping within a reasonable time by combining fast image taking and processing, plus two other critical performances: three-dimensional (3D) imaging with high and isotropic spatial resolution, and multi-scale resolution. We successfully tested this strategy with coordinated experiments at four synchrotron facilities in Japan, Taiwan, Singapore and Korea on two animal models, Drosophila and mouse. Its performances notably allowed full 3D mapping of the Drosophila brain in a few days. With reasonable improvements, AXON can deliver full mapping of large animal and human brains on a realistic time scale of a few years.

Determining the filtration function and biochemical status of kidney at the single glomerulus level remains hardly accessible, even from biopsies. Here, we provide evidence that IR spectro-microscopy is a suitable method to account for the filtration capacity of individual glomeruli along with related physio-pathological condition. A ∼4 μm voxel resolution 3D IR image reconstruction is built from consecutive tissue sections, thus, providing a 3D IR spectrum matrix of an individual glomerulus. The filtration capacity of glomeruli was quantitatively determined after BaSO4 perfusion, and additional chemical data could be used to determined oxidative stress effects and fibrosis, thus, combining functional and biochemical information from the same 3D IR spectrum matrix. This analytical approach was applied on mice with unilateral ureteral obstruction (UUO) inducing chronic kidney disease. Compared to the healthy condition, UUO induced a significant drop in glomeruli filtration capacity (−17 ± 8% at day 4 and −...

Here, we introduce a unique high-throughput infrared (IR) microscopy method that combines automated image correction and subsequent spectral data analysis for 3D-IR image reconstruction., Three-dimensional (3D) histology is the next frontier for modern anatomo-pathology. Characterizing abnormal parameters in a tissue is essential to understand the rationale of pathology development. However, there is no analytical technique, in vivo or histological, that is able to discover such abnormal features and provide a 3D distribution at microscopic resolution. Here, we introduce a unique high-throughput infrared (IR) microscopy method that combines automated image correction and subsequent spectral data analysis for 3D-IR image reconstruction. We performed spectral analysis of a complete organ for a small animal model, a mouse brain with an implanted glioma tumor. The 3D-IR image is reconstructed from 370 consecutive tissue sections and corrected using the X-ray tomogram of the organ for an accurate quantitative analysis of the chemical content. A 3D matrix of 89 × 106 IR spectra is generated, allowing us to separate the tumor mass from healthy brain tissues based on various anatomical, chemical, and metabolic parameters. We demonstrate that quantitative metabolic parameters can be extracted from the IR spectra for the characterization of the brain vs. tumor metabolism (assessing the Warburg effect in tumors). Our method can be further exploited by searching for the whole spectral profile, discriminating tumor vs. healthy tissue in a non-supervised manner, which we call ‘spectromics’.

IR microscopy was first conceptualized in 1949 and the first commercial system was launched 1983. With the appearance of FPA detectors in the 90's, FTIR microscopy became a technique of choice for histology. Two decades later, the release of QCLs working in the mid-IR range refuels this promise by accelerating tremendously the acquisition of IR images for large tissue areas with high-quality spectra for chemical mapping of parameters of interest. The new QCL-IR imaging system allows a 150× faster spectral data acquisition at equivalent S/N level. The quality of spectral data is comparable while applying spectral curve-fitting treatments, thus showing that laser sources offer reliable signal over a large spectral range. If QCL-IR imaging system seem to offer the opportunity to develop routines for anatomo-pathology, several technological challenges stand in front of us to reach this goal to define the specs of an IR microscope dedicated to hospital.

The X-ray beamline TPS 02A is making use of the bending magnet source at the Taiwan Photon Source (TPS), which is dedicated to performing full-field hard X-ray projection imaging at micron size spatial resolutions in the photon energy range of 5 - 25 keV. It provides the photon flux >4.2 × 1011 photons·s−1 with energy resolution

Analytical techniques allowed describing the morphology of fossils, up the 3D level, and more recently, molecular remains are also characterized to understand evolutionary aspects of disappeared species. The preservation of original organic components in fossils is controversial, but elucidating evolutionary processes and phylogenetic relationships is invaluable. Infrared (IR) microscopy is one of the most interesting analytical technique, due to its non-destructive utilization and its sensitivity for extracting most of the inorganic and organic information in fossils. However, utilization of IR microscopy has rapidly raised controversies in paleontology, essentially because the interpretation of spectral data remains limited to experienced spectroscopists and also due to the nature of fossilized tissues, becoming mostly inorganic after the mineralization process. This review discusses the most important discoveries made by the use of IR microscopy for the analysis of fossils and provides a comprehensive IR bands database for interpreting fossil inorganic and organic contents.

Evidence has recently emerged that solid and diffuse tumors produce a specific extracellular matrix (ECM) for division and diffusion, also developing a specific interface with microvasculature. This ECM is mainly composed of collagens and their scaffolding appears to drive tumor growth. Although collagens are not easily analyzable by UV-fluorescence means, FTIR imaging has appeared as a valuable tool to characterize collagen contents in tissues, specially the brain, where ECM is normally devoid of collagen proteins. Here, we used FTIR imaging to characterize collagen content changes in growing glioma tumors. We could determine that C6-derived solid tumors presented high content of triple helix after 8–11 days of growth (typical of collagen fibrils formation; 8/8 tumor samples; 91 % of total variance), and further turned to larger α-helix (days 12–15; 9/10 of tumors; 94 % of variance) and β-turns (day 18–21; 7/8 tumors; 97 % of variance) contents, which suggest the incorporation of non-fibrillar collagen types in ECM, a sign of more and more organized collagen scaffold along tumor progression. The growth of tumors was also associated to the level of collagen produced (P

An original synthesis method based on X-ray irradiation produced gold nanoparticles (AuNPs) with two important properties for biomedical research: intense visible photoluminescence and very high accumulation in cancer cells. The nanoparticles, coated with MUA (11-mercaptoundecanoid acid), are very small (1.4 nm diameter); the above two properties are not present for even slightly larger sizes. The small MUA-AuNPs are non-cytotoxic (except for very high concentrations) and do not interfere with cancer cell proliferation. Multimodality imaging using visible light fluorescence and X-ray microscopy is demonstrated by tracing the nanoparticle-loaded tumor cells. (C) 2012 Elsevier Inc. All rights reserved.

This research discusses the influence of morphology of nanomagnetic materials (one-dimensional iron nanowires and zero-dimensional iron nanoparticles) on heating efficiency of the hyperthermia treatment. One-dimensional iron nanowires, synthesized by reducing method in external magnetic field, are explored in terms of their material properties, magnetic anisotropy, and cytotoxicity of EMT-6 cells. The magnetic anisotropy of an array of nanowires is examined in parallel and perpendicular magnetic fields by VSM. For the magnetic hyperthermia treatment tests, iron nanowires and nanoparticles with different concentrations are heated in alternating magnetic field to measure their actual heating efficiency and SLP heating properties. The shape effects of iron nanomaterials can be revealed from their heating properties. The cytotoxicity of nanowires with different concentrations is measured by its survival rate in EMT-6 with the cells cultivated for 6 and 24 hours.

Currently, only mass-spectrometry (MS) microscopy brings a quantitative analysis of chemical contents of tissue samples in 3D. Here, the reconstruction of a 3D quantitative chemical images of a biological tissue by FTIR spectro-microscopy is reported. An automated curve-fitting method is developed to extract all intense absorption bands constituting IR spectra. This innovation benefits from three critical features: (1) the correction of raw IR spectra to make them quantitatively comparable; (2) the automated and iterative data treatment allowing to transfer the IR-absorption spectrum into a IR-band spectrum; (3) the reconstruction of an 3D IR-band matrix (x, y, z for voxel position and a 4th dimension with all IR-band parameters). Spectromics, which is a new method for exploiting spectral data for tissue metadata reconstruction, is proposed to further translate the related chemical information in 3D, as biochemical and anatomical tissue parameters. An example is given with oxidative stress distribution and the reconstruction of blood vessels in tissues. The requirements of IR microscopy instrumentation to propose 3D digital histology as a clinical routine technology is briefly discussed.

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. Refereed/Peer-reviewed

Well-dispersed gold nanoparticles (NP) coated with tiopronin were synthesized by X-ray irradiation without reducing agents. High-resolution transmission electron microscopy shows that the average core diameters of the NPs can be systematically controlled by adjusting the tiopronin to Au mole ratio in the reaction. Three methods were used to study the NP uptake by cells: quantitative measurements by inductively coupled plasma mass spectrometry, direct imaging with high lateral resolution transmission electron microscopy and transmission X-ray microscopy. The results confirmed that the NP internalization mostly occurred via endocytosis and concerned the cytoplasm. The particles, in spite of their small sizes, were not found to arrive inside the cell nuclei. The synthesis without reducing agents and solvents increased the biocompatibility as required for potential applications in analysis and biomedicine in general Refereed/Peer-reviewed

The spatial distribution of X-ray synthesized, PEG coated Au nanoparticles in cancer-bearing mice and their time dependent accumulation were investigated with inductive coupled plasma – optical emission spectroscopy (ICP-OES), transmission electron microscopy (TEM) and histological imaging. The results conclusively demonstrate that the particles strongly accumulate in tumor regions; up to ∼25 times more than in normal muscle tissue. This accumulation increases with the time after injection for up to ∼12 h in tumor, spleen and liver tissues, whereas for most non-tumor regions it saturates or decreases (blood, lung, brain, heart, and kidney). The impact of this result is discussed with special emphasis on passive targeted drug delivery and could also be used for the delineation and early-stage imaging of small tumors. Refereed/Peer-reviewed

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. Electronic supplementary material The online version of this article (doi:10.1186/s12951-015-0140-2) contains supplementary material, which is available to authorized users.

Amphiboles caused cohorts of deaths in exposed workers, leading to some of the largest class actions in the industry. Once inhaled, these inorganic fibers are thought to be both chemically and morphologically toxic, and their biopersistence in the lungs over decades lead to progressive pathologies, mesothelioma, and asbestosis. However, this exceptionally long chronicity for human pathologies suggests that chemical toxicity is certainly low, suggesting that morphological parameters could be more relevant in the pathology. Here, we developed a 3D Raman/optical imaging methodology in vitro to characterize both morphological and chemical parameters of cell/fiber interactions. We determined that lung cells could vesiculate amphiboles with length below 5 μm or could embed those not exceeding 15 μm in their fibrous extracellular matrix. Lung cells can thus develop defense strategies for handling the biopersistence of inorganic species, which may thus have major impact for biosafety issues related to nanomaterials.

Successful design of a pH responsive polyelectrolyte-based virus delivery matrix with extracellular release triggered by tumor acidosis has been achieved. Recombinant adeno-associated virus serotype 2 (AAV2) is loaded in the polyelectrolyte-based matrix (AAV2-matrix), which is formed by a biodegradable copolymer of poly(polyethylene glycol-1-(3-aminopropyl)imidazole-DL-aspartic acid) with tuned pH response based on inclusion of polyethyleneimine (PEI800). Physico-chemical properties of AAV2-matrix are optimized to minimize cellular interactions until a tumor acidosis-like environment protonates the matrix, reverses ζ-potential and causes particles to swell, releasing the AAV2 virus. The pH-dependent release is highly controllable and potentially useful to optimize site specific viral delivery. Refereed/Peer-reviewed

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.

We show that sufficient concentrations of gold nanoparticles produced by an original synthesis method in EMT-6 and CT-26 cancer cells make it possible to detect the presence, necrosis and proliferation of such cells after inoculation in live mice. We first demonstrated that the nanoparticles do not interfere with the proliferation process. Then, we observed significant differences in the tumor evolution and the angiogenesis process after shallow and deep inoculation. A direct comparison with pathology optical images illustrates the effectiveness of this approach Refereed/Peer-reviewed

Biocompatible Au nanoparticles with surfaces modified by PEG (polyethylene glycol) were developed in view of possible applications for the enhancement of radiotherapy. Such nanoparticles exhibit preferential deposition at tumor sites due to the enhanced permeation and retention (EPR) effect. Here, we systematically studied their effects on EMT-6 and CT26 cell survival rates during irradiation for a dose up to 10 Gy with a commercial biological irradiator (E(average) = 73 keV), a Cu-Kalpha(1) x-ray source (8.048 keV), a monochromatized synchrotron source (6.5 keV), a radio-oncology linear accelerator (6 MeV) and a proton source (3 MeV). The percentage of surviving cells after irradiation was found to decrease by approximately 2-45% in the presence of PEG-Au nanoparticles ([Au] = 400, 500 or 1000 microM). The cell survival rates decreased as a function of the dose for all sources and nanoparticle concentrations. These results could open the way to more effective cancer irradiation therapies by using nanoparticles with optimized surface treatment. Difficulties in applying MTT assays were also brought to light, showing that this approach is not suitable for radiobiology.

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. Refereed/Peer-reviewed

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. Refereed/Peer-reviewed

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.

We investigated iron oxide nanoparticles with two different surface modifications, dextran coating and cross-linked dextran coating, showing that their different internalization affects their capability to enhance radiation damage to cancer cells. The internalization was monitored with an ultrahigh resolution transmission x-ray microscope (TXM), indicating that the differences in the particle surface charge play an essential role and dominate the particle-cell interaction. We found that dextran-coated iron oxide nanoparticles cannot be internalized by HeLa and EMT-6 cells without being functionalized with amino groups (the cross-linked dextran coating) that modify the surface potential from -18 mV to 13.4 mV. The amount of cross-linked dextran-coated iron oxide nanoparticles uptaken by cancer cells reached its maximum, 1.33 x 10(9) per HeLa cell, when the co-culture concentration was 40 microg Fe mL(-1) or more. Standard tests indicated that these internalized nanoparticles increased the damaging effects of x-ray irradiation, whereas they are by themselves biocompatible. These results could lead to interesting therapy applications; furthermore, iron oxide also produces high contrast for magnetic resonance imaging (MRI) in the diagnosis and therapy stages.

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.

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.