Abstract: Melanoma remains a deadly cancer despite significant advances in immune checkpoint blockade and targeted therapies. The incidence of melanoma is also growing worldwide, which highlights the need for novel treatment options and strategic combination of therapies. Here, we investigate non-thermal plasma (NTP), an ionized gas, as a promising, therapeutic option. In a melanoma mouse model, direct treatment of tumors with NTP results in reduced tumor burden and prolonged survival. Physical characterization of NTP treatment in situ reveals the deposited NTP energy and temperature associated with therapy response, and whole transcriptome analysis of the tumor identified several modulated pathways. NTP treatment also enhances the cancer-immunity cycle, as immune cells in both the tumor and tumor-draining lymph nodes appear more stimulated to perform their anti-cancer functions. Thus, our data suggest that local NTP therapy stimulates systemic, anti-cancer immunity. We discuss, in detail, how these fundamental insights will help direct the translation of NTP technology into the clinic and inform rational combination strategies to address the challenges in melanoma therapy.

Abstract: Ru catalysts are part of a set of late transition metal nanocatalysts that have garnered much interest for catalytic applications such as ammonia synthesis and fuel cell production. Their performance varies greatly depending on their morphology and size, these catalysts are widely studied using electron microscopy. Using recent developments in Annular Dark Field (ADF) Scanning Transmission Electron Microscopy (STEM) quantification techniques, a rapid atom counting procedure was utilized to document the evolution of a heterogeneous Ru catalyst supported on carbon black. Areas of the catalyst were imaged for approximately 15 minutes using ADF STEM. When the Ru clusters were exposed to the electron beam, the clusters changed phase from amorphous to crystalline. To quantify the thickness of the crystalline clusters, two techniques were applied (simulation and statistical decomposition) and compared. These techniques show that stable face centredcubic crystal structures in the form of rafts, between 2 and 8 atoms thick, were formed after the initial wetting of the carbon support.

Abstract: High angle annular dark field scanning transmission electron microscopy (HAADF STEM) images provide sample information which is sensitive to the chemical composition. The image intensities indeed scale with the mean atomic number Z. To some extent, chemically different atomic column types can therefore be visually distinguished. However, in order to quantify the atomic column composition with high accuracy and precision, model-based methods are necessary. Therefore, an empirical incoherent parametric imaging model can be used of which the unknown parameters are determined using statistical parameter estimation theory (Van Aert et al., 2009, [1]). In this paper, it will be shown how this method can be combined with frozen lattice multislice simulations in order to evolve from a relative toward an absolute quantification of the composition of single atomic columns with mixed atom types. Furthermore, the validity of the model assumptions are explored and discussed.

Abstract: This chapter provides an overview of statistical and quantitative methodologies that have pushed (scanning) transmission electron microscopy ((S)TEM) toward accurate and precise measurements of unknown structure parameters for understanding the relation between the structure of a material and its properties. Hereby, statistical parameter estimation theory has extensively been used which enabled not only measuring atomic column positions, but also quantifying the number of atoms, and detecting atomic columns as accurately and precisely as possible from experimental images. As a general conclusion, it can be stated that advanced statistical techniques are ideal tools to perform quantitative electron microscopy at the atomic scale. In the future, statistical methods will continue to be developed and novel quantification procedures will open up new possibilities for studying material structures at the atomic scale.

Abstract: By combining statistical parameter estimation and model-order selection using a Bayesian framework, the maximum a posteriori (MAP) probability rule is proposed in this chapter as an objective and quantitative method to detect atom columns from high-resolution scanning transmission electron microscopy (HRSTEM) images. The validity and usefulness of this approach is demonstrated to both simulated and experimental annular dark-field (ADF) STEM images, but also to simultaneously acquired annular bright-field (ABF) and ADF STEM image data.

Abstract: In this chapter, a statistical model-based method to count the number of atoms of monotype crystalline nanostructures from high-resolution annular dark-field (ADF) scanning transmission electron microscopy (STEM) images is discussed in detail together with a thorough study on the possibilities and inherent limitations. We show that this method can be applied to nanocrystals of arbitrary shape, size, and atom type. The validity of the atom-counting results is confirmed by means of detailed image simulations and it is shown that the high sensitivity of our method enables us to count atoms with single atom sensitivity.

Abstract: In this chapter, the principles of statistical parameter estimation theory for a quantitative analysis of atomic-resolution electron microscopy images are introduced. Within this framework, electron microscopy images are described by a parametric statistical model. Here, parametric models are introduced for different types of electron microscopy images: reconstructed exit waves, annular dark-field (ADF) scanning transmission electron microscopy (STEM) images, and simultaneously acquired ADF and annular bright-field (ABF) STEM images. Furthermore, the Cramér-Rao lower bound (CRLB) is introduced, i.e. a theoretical lower bound on the variance of any unbiased estimator. This CRLB is used to quantify the precision of the structure parameters of interest, such as the atomic column positions and the integrated atomic column intensities.

Abstract: In two subjects with a unilateral diaphragmatic paralysis and complaints of dyspnea, a completely different treatment approach was chosen despite similar anatomical and physiological abnormalities. These decisions were supported by the results generated by Functional Respiratory Imaging (FRI). FRI was able to generate functional information with respect to lobar ventilation and local drug deposition. In one subject, it was found that some lobes were poorly ventilated and drug deposition simulation showed that some regions were undertreated. This subject underwent a diaphragm plication to restore the ventilation. In the other subject, it was found that all lobes were still ventilated. A conservative approach with regular follow-up was chosen to wait for spontaneous recovery of the diaphragmatic function. Both subjects improved subjectively and objectively. These cases demonstrate how novel medical imaging techniques such as FRI can be used to personalize respiratory treatment in subjects with unilateral diaphragmatic paralysis.

Abstract: The Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification of chronic obstructive pulmonary disease (COPD) does not always match with other clinical disease descriptors such as exacerbation frequency and quality of life, indicating that forced expiratory volume in 1 s (FEV1) is not a perfect descriptor of the disease. The aim of this study was to find out whether changes in airway geometry after inhalation of the most commonly used inhalation therapy in severe COPD can more adequately be described with an image-based approach than with spirometry. 10 COPD GOLD stage III patients were assessed in a double-blind crossover study. Airway volumes were analysed using segmentation of multi-slice computed tomography (MSCT) images; airway resistance was determined using computational fluid dynamics (CFD). Distal airway volume significantly increased (p=0.011) in patients 4 h after receiving a budesonide/formoterol combination from 9.6+/-4.67 cm(3) to 10.14+/-4.81 cm(3). Also CFD-determined airway resistance significantly decreased (p=0.047) from 0.051+/-0.021 kPa.s.L-1 to 0.043+/- 0.019 kPa.s.L-1. None of the lung function parameters showed a significant change. Only functional residual capacity (FRC) showed a trend to decline (p=0.056). Only the image-based parameters were able to predict the visit at which the combination product was administered. This study showed that imaging is a sensitive, complementary tool to describe changes in airway structure.

Abstract: Background: Inhaled formulations using extrafine particles of long-acting beta(2)-agonists and corticosteroids were developed to optimize asthma treatment. Findings that these combinations reach and treat smaller airways more effectively are predominantly based on general non-specific outcomes with little information on regional characteristics. Objectives: This study aims to assess long-term effects of extrafine beclomethasone/formoterol on small airways of asthmatic patients using novel functional imaging methods. Methods: Twenty-four stable asthma patients were subdivided into three groups (steroid naive, n = 7; partially controlled, n = 6; well controlled, n = 11). Current treatment was switched to a fixed combination of extrafine beclomethasone/formoterol (Foster (R); Chiesi Pharmaceuticals, Parma, Italy). Patients underwent lung function evaluation and thorax high-resolution computerized tomography (HRCT) scan. Local airway resistance was obtained from computational fluid dynamics (CFD). Results: After 6 months, the entire population showed improvement in pre-bronchodilation imaging parameters, including small airway volume (p = 0.0007), resistance (p = 0.011), and asthma control score (p = 0.016). Changes in small airway volume correlated with changes in asthma control score (p = 0.004). Forced expiratory volume in 1 s (p = 0.044) and exhaled nitric oxide (p = 0.040) also improved. Functional imaging provided more detail and clinical relevance compared to lung function tests, especially in the well-controlled group where only functional imaging parameters showed significant improvement, while the correlation with asthma control score remained. Conclusions: Extrafine beclomethasone/formoterol results in a significant reduction of small airway obstruction, detectable by functional imaging (HRCT/CFD). Changes in imaging parameters correlated significantly with clinically relevant improvements. This indicates that functional imaging is a useful tool for sensitive assessment of changes in the respiratory system after asthma treatment. Copyright (C) 2013 S. Karger AG, Basel

Abstract: Computational fluid dynamics (CFD) is a technique that is used increasingly in the biomedical field. Solving the flow equations numerically provides a convenient way to assess the efficiency of therapies and devices, ranging from cardiovascular stents and heart valves to hemodialysis workflows. Also in the respiratory field CFD has gained increasing interest, especially through the combination of three dimensional image reconstruction which results in highend patient-specific models. This paper provides an overview of clinical applications of CFD through image based modeling, resulting from recent studies performed in our center. We focused on two applications: assessment of the efficiency of inhalation medication and analysis of endobronchial valve placement. In the first application we assessed the mode of action of a novel bronchodilator in 10 treated patients and 4 controls. We assessed the local volume increase and resistance change based on the combination of imaging and CFD. We found a good correlation between the changes in volume and resistance coming from the CFD results and the clinical tests. In the second application we assessed the placement and effect of one way endobronchial valves on respiratory function in 6 patients. We found a strong patientspecific result of the therapy where in some patients the therapy resulted in complete atelectasis of the target lobe while in others the lobe remained inflated. We concluded from these applications that CFD can provide a better insight into clinically relevant therapies.

Abstract: Urban atmospheres in modern cities carry characteristic mixtures of particulate pollution which are potentially aggravating for chronic respiratory patients (CRP). Although air quality surveys can be detailed, the obtained information is not always useful to evaluate human health effects. This paper presents a novel approach to estimate particle deposition rates in airways of CRP, based on real air pollution data. By combining computational fluid dynamics with physical-chemical characteristics of particulate pollution, deposition rates are estimated for particles of different toxicological relevance, that is, minerals, iron oxides, sea salts, ammonium salts, and carbonaceous particles. Also, it enables some qualitative evaluation of the spatial, temporal, and patient specific effects on the particle dose upon exposure to the urban atmosphere. Results show how heavy traffic conditions increases the deposition of anthropogenic particles in the trachea and lungs of respiratory patients (here, +0.28 and +1.5 μg·h1, respectively). In addition, local and synoptic meteorological conditions were found to have a strong effect on the overall dose. However, the pathology and age of the patient was found to be more crucial, with highest deposition rates for toxic particles in adults with a mild anomaly, followed by mild asthmatic children and adults with severe respiratory dysfunctions (7, 5, and 3 μg·h1, respectively).

Abstract: Energy filtered TEM, EFTEM, provides three-dimensional data, two spatial and one spectral dimension. We propose to acquire these data by measuring a series of images with a defocused energy filter. It will be shown that each image is a projection of the data on the detector and that reconstruction of the data out of a sufficient number of such projections using a tomographic reconstruction algorithm is possible. This technique uses only a fraction of the electron dose an energy filtered series (EFS) needs for the same spectral and spatial resolution and the same mean signal-to-noise ratio. (c) 2005 Elsevier B.V. All rights reserved.

Abstract: Objective: We aim to investigate if anatomical and functional properties of the upper airway using computerized 3D models derived from computed tomography (CT) scans better predict obstructive sleep apnea (OSA) severity than standard clinical markers. Methods: Consecutive children with suspected OSA underwent polysomnography, clinical assessment of upper airway patency, and a CT scan while awake. A three-dimensional (3D) reconstruction of the pharyngeal airway was built from these images, and computational fluid dynamics modeling of low inspiratory flow was performed using open-source software. Results: Thirty-three children were included (23 boys; mean age, was 6.0 +/- 3.2 y). OSA was diagnosed in 23 patients. Children with OSA had a significantly lower volume of the overlap region between tonsils and the adenoids (median volume, 1408 mm compared to 2173 mm; p = 0.04), a lower mean cross-sectional area at this location (median volume, 69.3 mm(2) compared to 114.3 mm(2); p = 0.04), and a lower minimal cross-sectional area (median volume, 17.9 mm(2) compared to 25.9 mm(2); p = 0.05). Various significant correlations were found between several imaging parameters and the severity of OSA, most pronounced for upper airway conductance (r = -0.46) (p < 0.01) for correlation between upper airway conductance and the apnea-hypopnea index. No differences or significant correlations were observed with clinical parameters of upper airway patency. Preliminary data after treatment showed that none of the patients with residual OSA had their smallest cross-sectional area located in segment 3, and this frequency was significantly lower than in their peers whose sleep study normalized (64%; p = 0.05). Conclusion: Functional imaging parameters are highly correlated with OSA severity and are a more powerful correlate than clinical scores of upper airway patency. Preliminary data also showed that we could identify differences in the upper airway of those subjects who did not benefit from a local upper airway treatment. (c) 2013 Elsevier B.V. All rights reserved.