Soft X-ray microscopy (SXM) is a powerful technique for high-resolution biomedical imaging, enabling the observation of bio-nano interactions in near-native conditions without the need for heavy metal staining and fluorescence labeling. A laboratory soft X-ray microscope (LSXM) was developed to bridge the resolution gap between light microscopy and electron microscopy in cellular imaging. However, LSXMs employ a lower-brightness X-ray source in comparison to those operated in synchrotron facilities, which can negatively affect the contrast of X-ray micrographs. Therefore, proper sample preparation is essential to achieve optimal imaging results. This paper details an LSXM sample preparation protocol for investigating cellular nanoparticle uptake. Samples are prepared using optimized parameters for both manual plunge-freezing and automated vitrification, ensuring the rapid transition of biological material into a solid state with controllable thickness in the 5-10 mu m range, preserving cellular structures and enabling optimal X-ray transmission for cellular imaging. We demonstrate the effectiveness of this protocol in facilitating the observation of nanoparticle uptake in two different biological samples: murine macrophages and acanthamoeba. Controlling ice thickness improves X-ray transmission through the specimen, enhancing the contrast and image quality of SXM.

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.

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.

Lipid-based nanoparticles are organic nanostructures constituted of phospholipids and cholesterol, displaying high in vivo biocompatibility. They have been demonstrated as effective nanocarriers for drug delivery and targeting. Mapping liposome distribution is crucial as it enables a precise understanding of delivery kinetics, tissue targeting efficiency, and potential off-target effects. Recently, ruthenium-encapsulated liposomes have shown potential for targeted drug delivery, photodynamic therapy, and optical fluorescence imaging. In the present work, we design Ru(bpy)3-encapsulated liposomes (Ru-Lipo) empowering optical and X-ray fluorescence (XRF) properties for dual mode imaging and demonstrate the passivation role of liposomes over the free Ru(bpy)3 compound. We employ whole-body XRF imaging to map the in vivo biodistribution of Ru-Lipo in mice, enabling tumor detection and longitudinal studies with elemental specificity and resolution down to the sub-millimeter scale. Quantitative XRF computed tomography on extracted organs permits targeting efficiency evaluations. These findings highlight the promising role of XRF imaging in pharmacokinetic studies and theranostic applications for the rapid optimization of drug delivery and assessment of targeting efficiency.

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.

Respiratory X-ray imaging enhanced by phase contrast has shown improved airway visualization in animal models. Limitations in current X-ray technology have nevertheless hindered clinical translation, leaving the potential clinical impact an open question. Here, we explore phase-contrast chest radiography in a realistic in silico framework. Specifically, we use preprocessed virtual patients to generate in silico chest radiographs by Fresnel-diffraction simulations of X-ray wave propagation. Following a reader study conducted with clinical radiologists, we predict that phase-contrast edge enhancement will have a negligible impact on improving solitary pulmonary nodule detection (6 to 20 mm). However, edge enhancement of bronchial walls visualizes small airways (<2 mm), which are invisible in conventional radiography. Our results show that phase-contrast chest radiography could play a future role in observing small-airway obstruction (e.g., relevant for asthma or early-stage chronic obstructive pulmonary disease), which cannot be directly visualized using current clinical methods, thereby motivating the experimental development needed for clinical translation. Finally, we discuss quantitative requirements on distances and X-ray source/detector specifications for clinical implementation of phase-contrast chest radiography.

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 (similar to 10 mu 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.

In recent years, the design and synthesis of bio-compatible coatings leading to hybrid nanoparticles (NPs) as the contrast agents have gained substantial relevance. Furthermore, the addition of several functionalities for bio-imaging applications represents a key step for non-invasive bio-diagnostics. In this context, we design and utilize hybrid nanostructures for X-ray fluorescence computed tomography (XFCT). The combination of a ceramic or metallic core–based on MoO2, Rh or Ru–with a protective shell allows the generation of bio-compatible nanohybrids for dual mode bio-imaging, where the core NPs constitute the X-ray fluorescence (XRF) contrast agents [1]–[3]. Core NPs are synthesized via polyol, hydrothermal or microwave-assisted hydrothermal methods, yielding uniform shape and high dispersibility in aqueous media. Different approaches have been pursued for the fabrication of a bio-compatible shell coating. A modified sol-gel based silica coating process, doped with a commercial fluorophore (Cy5.5), was developed and shown to be applicable to both ceramic and metallic NPs [4], forming core-shell NPs with both optical and X-ray fluorescence properties. Alternatively, carbon quantum dots (CQDs) were synthesized via citrate pyrolysis using microwave-assisted hydrothermal method, exhibiting uniform size distribution (1.6±0.4 nm) and excitation-independent emission (440 nm). Conjugation of these CQDs, via cross-linking, with Rh NPs led to excitation-independent hybrid NPs, with a red-shifted emission wavelength (520 nm), attributed to the reduction of pyrrolic nitrogen on CQDs [5]. These hybrid NPs exhibit improved in vitro biocompatibility in comparison with bare XRF contrast agents. Furthermore, the optical fluorescence–provided by Cy5.5 or CQDs–allows the localization of the NPs in the intracellular environment while the XRF signal from the core NPs is utilized for XFCT, in small animals, leading to both a microscopic and macroscopic bio-imaging contrast agent.

Multimodal contrast agents in biomedical imaging enable the collection of more comprehensive diagnostic information. In the present work, we design hybrid ruthenium-decorated superparamagnetic iron oxide nanoparticles (NPs) as the contrast agents for both magnetic resonance imaging (MRI) and X-ray fluorescence computed tomography (XFCT). The NPs are synthesized via a one-pot polyol hot injection route, in diethylene glycol. In vivo preclinical studies demonstrate the possibility of correlative bioimaging with these contrast agents. The complementarity allows accurate localization, provided by the high contrast of the soft tissues in MRI combined with the elemental selectivity of XFCT, leading to NP detection with high specificity and resolution. We envision that this multimodal imaging could find future applications for early tumor diagnosis, improved long-term treatment monitoring, and enhanced radiotherapy planning.

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 intra-operatively. In the present paper we investigate a laboratory propagation-based phase-contrast x-ray computed tomography (CT) system that provides the resolution, contrast, and, potentially, the speed for this purpose. The system relies on a liquid-metal jet micro-focus source and a scintillator-coated CMOS detector. The study is performed on paraffin-embedded non-stained samples of human pancreatic neuroendocrine tumors, liver intrahepatic cholangiocarcinoma, and pancreatic serous cystic neoplasm (benign). We observe tumors with distinct and sharp edges having cellular resolution (similar to 10 mu m) as well as many assisting histological landmarks, allowing for resection margin assessment. All x-ray data is compared with classical histology. The agreement is excellent, and we conclude that the method has potential for intra-operative three-dimensional virtual histology.