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.

Multimodal bioimaging using nanodiamond and gold hybrid nanoparticles.

Hybrid core-shell nanodiamond-gold nanoparticles were synthesized and characterized as a novel multifunctional material with tunable and tailored properties for multifunctional biomedical applications. The combination of nanostructured gold and nanodiamond properties afford new options for optical labeling, imaging, sensing, and drug delivery, as well as targeted treatment. ND@Au core-shell nanoparticles composed of nanodiamond (ND) core doped with Si vacancies (SiV) and Au shell were synthesized and characterized in terms of their biomedical applications. Several bioimaging modalities based on the combination of optical and spectroscopic properties of the hybrid nano-systems are demonstrated in cellular and developing zebrafish larvae models. The ND@Au nanoparticles exhibit isolated ND's Raman signal of sp3 bonded carbon, one-photon fluorescence of SiV with strong zero-phonon line at 740 nm, two-photon excited fluorescence of nanogold with short fluorescence lifetime and strong absorption of X-ray irradiation render them possible imaging agent for Raman mapping, Fluorescence imaging, two-photon Fluorescence Lifetime Imaging (TP-FLIM) and high-resolution hard-X-ray microscopy in biosystems. Potential combination of the imaging facilities with other theranostic functionalities is discussed.

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.

3D chemical imaging of the brain using quantitative IR spectro-microscopy.

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'.

3D Digital Pathology for a Chemical-Functional Analysis of Glomeruli in Health and Pathology.

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 -48 ± 14% at day 14) and volume (36 ± 10% at day 4 and 67 ± 13% at day 14), along a significant increase of oxidative stress (+61 ± 19% at day 4 and +84 ± 17% at day 14) and a change in the lipid-to-protein ratio (-8.2 ± 3.6% at day 4 and -18.1 ± 5.9% at day 14). Therefore, IR spectro-microscopy might be developed as a new 3D pathology resource for analyzing functional and biochemical parameters of glomeruli.

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.

Quantitative IR microscopy and spectromics open the way to 3D digital pathology.

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.

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.

Image alignment for tomography reconstruction from synchrotron X-ray microscopic images.

A synchrotron X-ray microscope is a powerful imaging apparatus for taking high-resolution and high-contrast X-ray images of nanoscale objects. A sufficient number of X-ray projection images from different angles is required for constructing 3D volume images of an object. Because a synchrotron light source is immobile, a rotational object holder is required for tomography. At a resolution of 10 nm per pixel, the vibration of the holder caused by rotating the object cannot be disregarded if tomographic images are to be reconstructed accurately. This paper presents a computer method to compensate for the vibration of the rotational holder by aligning neighboring X-ray images. This alignment process involves two steps. The first step is to match the "projected feature points" in the sequence of images. The matched projected feature points in the x-θ plane should form a set of sine-shaped loci. The second step is to fit the loci to a set of sine waves to compute the parameters required for alignment. The experimental results show that the proposed method outperforms two previously proposed methods, Xradia and SPIDER. The developed software system can be downloaded from the URL, http://www.cs.nctu.edu.tw/~chengchc/SCTA or http://goo.gl/s4AMx.

FTIR spectro-imaging of collagen scaffold formation during glioma tumor development.

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 ß-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 < 0.05). This study thus confirms that collagen scaffolding is a major event accompanying the angiogenic shift and faster tumor growth in solid glioma phenotypes.

The role of asbestos morphology on their cellular toxicity: an in vitro 3D Raman/Rayleigh imaging study.

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.

Environment acidity triggers release of recombinant adeno-associated virus serotype 2 from a tunable matrix.

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 (PEI(800)). 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.

Very small photoluminescent gold nanoparticles for multimodality biomedical imaging.

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.

X-ray imaging of tumor growth in live mice by detecting gold-nanoparticle-loaded cells.

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.

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.

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.

Imaging the cellular uptake of tiopronin-modified gold nanoparticles.

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.

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.

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.

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.