Laboratory Liquid-Jet X-ray Microscopy and X-ray Fluorescence Imaging for Biomedical Applications.

Diffraction-limited resolution and low penetration depth are fundamental constraints in optical microscopy and in vivo imaging. Recently, liquid-jet X-ray technology has enabled the generation of X-rays with high-power intensities in laboratory settings. By allowing the observation of cellular processes in their natural state, liquid-jet soft X-ray microscopy (SXM) can provide morphological information on living cells without staining. Furthermore, X-ray fluorescence imaging (XFI) permits the tracking of contrast agents in vivo with high elemental specificity, going beyond attenuation contrast. In this study, we established a methodology to investigate nanoparticle (NP) interactions in vitro and in vivo, solely based on X-ray imaging. We employed soft (0.5 keV) and hard (24 keV) X-rays for cellular studies and preclinical evaluations, respectively. Our results demonstrated the possibility of localizing NPs in the intracellular environment via SXM and evaluating their biodistribution with in vivo multiplexed XFI. We envisage that laboratory liquid-jet X-ray technology will significantly contribute to advancing our understanding of biological systems in the field of nanomedical research.

Laboratory phase-contrast nanotomography of unstained Bombus terrestris compound eyes.

Imaging the visual systems of bumblebees and other pollinating insects may increase understanding of their dependence on specific habitats and how they will be affected by climate change. Current high-resolution imaging methods are either limited to two dimensions (light- and electron microscopy) or have limited access (synchrotron radiation x-ray tomography). For x-ray imaging, heavy metal stains are often used to increase contrast. Here, we present micron-resolution imaging of compound eyes of buff-tailed bumblebees (Bombus terrestris) using a table-top x-ray nanotomography (nano-CT) system. By propagation-based phase-contrast imaging, the use of stains was avoided and the microanatomy could more accurately be reconstructed than in samples stained with phosphotungstic acid or osmium tetroxide. The findings in the nano-CT images of the compound eye were confirmed by comparisons with light- and transmission electron microscopy of the same sample and finally, comparisons to synchrotron radiation tomography as well as to a commercial micro-CT system were done.

Soft-Tissue Imaging in a Human Mummy: Propagation-based Phase-Contrast CT.

Purpose To evaluate phase-contrast CT as a noninvasive alternative to histology in the study of ancient soft tissue. Materials and Methods The imaging was performed between May 8 and June 13, 2017. A mummified human hand from ancient Egypt was imaged in a laboratory phase-contrast CT arrangement with propagation-based imaging. The experimental arrangement for propagation-based imaging included a microfocus x-ray source, a rotation stage for the sample, and an x-ray detector. The mummified hand was imaged in two different modes. First, a CT scan of the whole hand was performed in an overview arrangement. Then, a detailed scan of the tip of the middle finger was performed. With imaging distances tailored for a large magnification and to maximize the phase-contrast signal, the estimated resolution in the final images was 6-9 µm. Results The overview CT allowed identification of the tendons of the hand, as well as identification of arteries and nerves in the dehydrated soft tissue. In the detailed phase-contrast setting, virtual histology of the soft tissues of the fingertip could be performed. Blood vessels in the nail bed and the microanatomy of the bone marrow and hypodermis were imaged, and the layers of the skin could be distinguished. Round structures in the adipose tissue were identified as the remains of adipocytes. Conclusion Laboratory phase-contrast CT enables imaging of the anatomy and microanatomy of mummified soft tissue with sub-10-µm resolution and may serve as a complement or alternative to the classic invasive histologic methods used in soft-tissue paleopathology. © RSNA, 2018 Online supplemental material is available for this article.

Focused anti-scatter grid for background reduction in x-ray fluorescence tomography.

X-ray fluorescence (XRF) tomography is an emerging imaging technology with the potential for high spatial resolution molecular imaging. One of the key limitations is the background noise due to Compton scattering since it degrades the signal and limits the sensitivity. In this Letter, we present a linear focused anti-scatter grid that reduces the Compton scattering background. An anti-scatter grid was manufactured and evaluated both experimentally and theoretically with Monte Carlo simulations. The measurements showed a 31% increase in signal-to-background ratio, and simulations of an improved grid showed that this can easily be extended up to >75%. Simulated tomographies using the improved grid show a large improvement in reconstruction quality. The anti-scatter grid will be important for in vivo XRF tomography since the background reduction allows for faster scan times, lower doses, and lower nanoparticle concentrations.

X-ray microtomography using correlation of near-field speckles for material characterization.

Nondestructive microscale investigation of objects is an invaluable tool in life and materials sciences. Currently, such investigation is mainly performed with X-ray laboratory systems, which are based on absorption-contrast imaging and cannot access the information carried by the phase of the X-ray waves. The phase signal is, nevertheless, of great value in X-ray imaging as it is complementary to the absorption information and in general more sensitive to visualize features with small density differences. Synchrotron facilities, which deliver a beam of high brilliance and high coherence, provide the ideal condition to develop such advanced phase-sensitive methods, but their access is limited. Here we show how a small modification of a laboratory setup yields simultaneously quantitative and 3D absorption and phase images of the object. This single-shot method is based on correlation of X-ray near-field speckles and represents a significant broadening of the capabilities of laboratory-based X-ray tomography.

Removal of ring artifacts in microtomography by characterization of scintillator variations.

Ring artifacts reduce image quality in tomography, and arise from faulty detector calibration. In microtomography, we have identified that ring artifacts can arise due to high-spatial frequency variations in the scintillator thickness. Such variations are normally removed by a flat-field correction. However, as the spectrum changes, e.g. due to beam hardening, the detector response varies non-uniformly introducing ring artifacts that persist after flat-field correction. In this paper, we present a method to correct for ring artifacts from variations in scintillator thickness by using a simple method to characterize the local scintillator response. The method addresses the actual physical cause of the ring artifacts, in contrary to many other ring artifact removal methods which rely only on image post-processing. By applying the technique to an experimental phantom tomography, we show that ring artifacts are strongly reduced compared to only making a flat-field correction.

Noise analysis of speckle-based x-ray phase-contrast imaging.

Speckle-based x-ray phase-contrast imaging has drawn increasing interest in recent years as a simple, multimodal, cost-efficient, and laboratory-source adaptable method. We investigate its noise properties to help further optimization on the method and further comparison with other phase-contrast methods. An analytical model for assessing noise in a differential phase signal is adapted from studies on the digital image correlation technique in experimental mechanics and is supported by simulations and experiments. The model indicates that the noise of the differential phase signal from speckle-based imaging has a behavior similar to that of the grating-based method.

Tomographic reconstruction in soft x-ray microscopy using focus-stack back-projection.

Tomographic reconstruction in soft x-ray microscopy is a powerful technique for obtaining high-resolution 3D images of biological samples. However, the depth of focus of such zone-plate-based microscopes is typically shorter than the thickness of many relevant biological objects, challenging the validity of the projection assumption used in conventional reconstruction algorithms. In order to make full use of the soft x-ray microscopes' high resolution, the tomographic reconstruction needs to take the depth of focus into account. Here we present a method to achieve high resolution in the full sample when the depth of focus is short compared to the sample thickness. The method relies on the back-projection of focus-stacked image data from x-ray microscopy. We demonstrate the method on theoretical and experimental data.

Speckle-based x-ray phase-contrast imaging with a laboratory source and the scanning technique.

The speckle-based scanning method for x-ray phase-contrast imaging is implemented with a liquid-metal-jet source. Using the two-dimensional scanning technique, the phase shift introduced by the object is retrieved in both transverse orientations, and the limitations on spatial resolution inherent to the speckle-tracking technique are avoided. This method opens up possibilities of new high-resolution multimodal applications for lab-based phase-contrast x-ray imaging.

3D simulation of the image formation in soft x-ray microscopes.

In water-window soft x-ray microscopy the studied object is typically larger than the depth of focus and the sample illumination is often partially coherent. This blurs out-of-focus features and may introduce considerable fringing. Understanding the influence of these phenomena on the image formation is therefore important when interpreting experimental data. Here we present a wave-propagation model operating in 3D for simulating the image formation of thick objects in partially coherent soft x-ray microscopes. The model is compared with present simulation methods as well as with experiments. The results show that our model predicts the image formation of transmission soft x-ray microscopes more accurately than previous models.

Laboratory x-ray fluorescence tomography for high-resolution nanoparticle bio-imaging.

We demonstrate that nanoparticle x-ray fluorescence computed tomography in mouse-sized objects can be performed with very high spatial resolution at acceptable dose and exposure times with a compact laboratory system. The method relies on the combination of the 24 keV line-emission from a high-brightness liquid-metal-jet x-ray source, pencil-beam-forming x-ray optics, photon-counting energy-dispersive detection, and carefully matched (Mo) nanoparticles. Phantom experiments and simulations show that the arrangement significantly reduces Compton background and allows 100 µm detail imaging at dose and exposure times compatible with small-animal experiments. The method provides a possible path to in vivo molecular x-ray imaging at sub-100 µm resolution in mice.

Iterative nanoparticle bioengineering enabled by x-ray fluorescence imaging.

Nanoparticles (NPs) are currently developed for drug delivery and molecular imaging. However, they often get intercepted before reaching their target, leading to low targeting efficacy and signal-to-noise ratio. They tend to accumulate in organs like lungs, liver, kidneys, and spleen. The remedy is to iteratively engineer NP surface properties and administration strategies, presently a time-consuming process that includes organ dissection at different time points. To improve this, we propose a rapid iterative approach using whole-animal x-ray fluorescence (XRF) imaging to systematically evaluate NP distribution in vivo. We applied this method to molybdenum-based NPs and clodronate liposomes for tumor targeting with transient macrophage depletion, leading to reduced accumulations in lungs and liver and eventual tumor detection. XRF computed tomography (XFCT) provided 3D insight into NP distribution within the tumor. We validated the results using a multiscale imaging approach with dye-doped NPs and gene expression analysis for nanotoxicological profiling. XRF imaging holds potential for advancing therapeutics and diagnostics in preclinical pharmacokinetic studies.

Damage investigation on tungsten and diamond diffractive optics at a hard x-ray free-electron laser.

Focusing hard x-ray free-electron laser radiation with extremely high fluence sets stringent demands on the x-ray optics. Any material placed in an intense x-ray beam is at risk of being damaged. Therefore, it is crucial to find the damage thresholds for focusing optics. In this paper we report experimental results of exposing tungsten and diamond diffractive optics to a prefocused 8.2 keV free-electron laser beam in order to find damage threshold fluence levels. Tungsten nanostructures were damaged at fluence levels above 500 mJ/cm(2). The damage was of mechanical character, caused by thermal stress variations. Diamond nanostructures were affected at a fluence of 59 000 mJ/cm(2). For fluence levels above this, a significant graphitization process was initiated. Scanning Electron Microscopy (SEM) and µ-Raman analysis were used to analyze exposed nanostructures.

Organ uptake, toxicity and skin clearance of ruthenium contrast agents monitored in vivo by x-ray fluorescence.

Aims: To investigate the distribution and toxicity of ruthenium nanoparticles (Ru NPs) injected intravenously in mice. Methods: We synthesized Ru NPs, followed their biodistribution by x-ray fluorescence (XRF) imaging and evaluated organ toxicity by histopathology and gene expression. Results: Ru NPs accumulated, mainly in liver and spleen, where they were phagocyted by tissue macrophages, giving a transient inflammation and oxidative stress response that declined after 2 weeks. Ru NPs gradually accumulated in the skin, which was confirmed by microscopic examination of skin biopsies. Conclusion: Ru NP toxicity in recipient organs is transient. Particles are at least partially excreted by the skin, supporting a role for the skin as a nanoparticle clearing organ.

Ronchi test for characterization of nanofocusing optics at a hard x-ray free-electron laser.

We demonstrate the use of the classical Ronchi test to characterize aberrations in focusing optics at a hard x-ray free-electron laser. A grating is placed close to the focus and the interference between the different orders after the grating is observed in the far field. Any aberrations in the beam or the optics will distort the interference fringes. The method is simple to implement and can provide single-shot information about the focusing quality. We used the Ronchi test to measure the aberrations in a nanofocusing Fresnel zone plate at the Linac Coherent Light Source at 8.194 keV.

Can laboratory x-ray virtual histology provide intraoperative 3D tumor resection margin assessment?

Purpose: Surgery is an essential part of the curative plan for most patients affected with solid tumors. The outcome of such surgery, e.g., recurrence rates and ultimately patient survival, depends on several factors where the resection margin is of key importance. Presently, the resection margin is assessed by classical histology, which is time-consuming (several days), destructive, and basically only gives two-dimensional information. Clearly, it would be advantageous if immediate feedback on tumor extension in all three dimensions were available to the surgeon intraoperatively. Approach: We investigate a laboratory propagation-based phase-contrast x-ray computed tomography system that provides the resolution, the contrast, and, potentially, the speed for this purpose. The system relies on a liquid-metal jet microfocus source and a scintillator-coated CMOS detector. Our study is performed on paraffin-embedded non-stained samples of human pancreatic neuroendocrine tumors, liver intrahepatic cholangiocarcinoma, and pancreatic serous cystic neoplasm (benign). Results: We observe tumors with distinct and sharp edges having cellular resolution ( ∼ 10 µ m ) as well as many assisting histological landmarks, allowing for resection margin assessment. All x-ray data are compared with classical histology. The agreement is excellent. Conclusion: We conclude that the method has potential for intraoperative three-dimensional virtual histology.

Numerical model for tomographic image formation in transmission x-ray microscopy.

We present a numerical image-formation model for investigating the influence of partial coherence, sample thickness and depth-of-focus on the accuracy of tomographic reconstructions in transmission x-ray microscopes. The model combines wave propagation through the object by finite difference techniques with Fourier methods. We include a ray-tracing model to analyse the origin of detrimental stray light in zone plate-based x-ray microscopes. These models allow optimization of x-ray microscopy systems for quantitative tomographic imaging of thick objects. Results show that both the depth-of-focus and the reconstructed local absorption coefficient are highly dependent on the degree of coherence of the optical system.

Phase retrieval in X-ray phase-contrast imaging suitable for tomography.

In-line phase-contrast X-ray imaging provides images where both absorption and refraction contribute. For quantitative analysis of these images, the phase needs to be retrieved numerically. There are many phase-retrieval methods available. Those suitable for phase-contrast tomography, i.e., non-iterative phase-retrieval methods that use only one image at each projection angle, all follow the same pattern though derived in different ways. We outline this pattern and use it to compare the methods to each other, considering only phase-retrieval performance and not the additional effects of tomographic reconstruction. We also outline derivations, approximations and assumptions, and show which methods are similar or identical and how they relate to each other. A simple scheme for choosing reconstruction method is presented, and numerical phase-retrieval performed for all methods.

Laboratory soft-x-ray microscope for cryotomography of biological specimens.

Soft-x-ray cryotomography allows quantitative and high-resolution three-dimensional imaging of intact unstained cells. To date, the method relies on synchrotron-radiation sources, which limits accessibility for researchers. Here we present a laboratory water-window microscope for cryotomography. It is based on a λ=2.48 nm liquid-jet laser-plasma source, a normal-incidence multilayer condenser, a 30 nm zone-plate objective, and a cryotilt sample holder. We demonstrate high-resolution imaging, as well as quantitative tomographic imaging, of frozen intact cells. The reconstructed tomogram of the intracellular local absorption coefficient shows details down to ∼100 nm.

In Silico Phase-Contrast X-Ray Imaging of Anthropomorphic Voxel-Based Phantoms.

Propagation-based phase-contrast X-ray imaging is an emerging technique that can improve dose efficiency in clinical imaging. In silico tools are key to understanding the fundamental imaging mechanisms and develop new applications. Here, due to the coherent nature of the phase-contrast effects, tools based on wave propagation (WP) are preferred over Monte Carlo (MC) based methods. WP simulations require very high wave-front sampling which typically limits simulations to small idealized objects. Virtual anthropomorphic voxel-based phantoms are typically provided with a resolution lower than imposed sampling requirements and, thus, cannot be directly translated for use in WP simulations. In the present paper we propose a general strategy to enable the use of these phantoms for WP simulations. The strategy is based on upsampling in the 3D domain followed by projection resulting in high-resolution maps of the projected thickness for each phantom material. These maps can then be efficiently used for simulations of Fresnel diffraction to generate in silico phase-contrast X-ray images. We demonstrate the strategy on an anthropomorphic breast phantom to simulate propagation-based phase-contrast mammography using a laboratory micro-focus X-ray source.