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: Quantitative annular dark field scanning transmission electron microscopy (ADF STEM) has become a powerful technique to characterise nano-particles on an atomic scale. Because of their limited size and beam sensitivity, the atomic structure of such particles may become extremely challenging to determine. Therefore keeping the incoming electron dose to a minimum is important. However, this may reduce the reliability of quantitative ADF STEM which will here be demonstrated for nano-particle atom-counting. Based on experimental ADF STEM images of a real industrial catalyst, we discuss the limits for counting the number of atoms in a projected atomic column with single atom sensitivity. We diagnose these limits by combining a thorough statistical method and detailed image simulations.

Abstract: We introduce a Bayesian genetic algorithm for reconstructing atomic models of monotype crystalline nanoparticles from a single projection using Z-contrast imaging. The number of atoms in a projected atomic column obtained from annular dark field scanning transmission electron microscopy images serves as an input for the initial three-dimensional model. The algorithm minimizes the energy of the structure while utilizing a priori information about the finite precision of the atom-counting results and neighbor-mass relations. The results show promising prospects for obtaining reliable reconstructions of beam-sensitive nanoparticles during dynamical processes from images acquired with sufficiently low incident electron doses.

Abstract: An efficient model-based estimation algorithm is introduced to quantify the atomic column positions and intensities from atomic resolution (scanning) transmission electron microscopy ((S)TEM) images. This algorithm uses the least squares estimator on image segments containing individual columns fully accounting for overlap between neighbouring columns, enabling the analysis of a large field of view. For this algorithm, the accuracy and precision with which measurements for the atomic column positions and scattering cross-sections from annular dark field (ADF) STEM images can be estimated, has been investigated. The highest attainable precision is reached even for low dose images. Furthermore, the advantages of the model-based approach taking into account overlap between neighbouring columns are highlighted. This is done for the estimation of the distance between two neighbouring columns as a function of their distance and for the estimation of the scattering cross-section which is compared to the integrated intensity from a Voronoi cell. To provide end-users this well-established quantification method, a user friendly program, StatSTEM, is developed which is freely available under a GNU public license.

Abstract: A new methodology is presented to count the number of atoms in multimetallic nanocrystals by combining energy dispersive X-ray spectroscopy (EDX) and high angle annular dark field scanning transmission electron microscopy (HAADF STEM). For this purpose, the existence of a linear relationship between the incoherent HAADF STEM and EDX images is exploited. Next to the number of atoms for each element in the atomic columns, the method also allows quantification of the error in the obtained number of atoms, which is of importance given the noisy nature of the acquired EDX signals. Using experimental images of an Au@Ag core–shell nanorod, it is demonstrated that 3D structural information can be extracted at the atomic scale. Furthermore, simulated data of an Au@Pt core–shell nanorod show the prospect to characterize heterogeneous nanostructures with adjacent atomic numbers.

Abstract: Melanoma arises from pigment-producing cells called melanocytes located in the basal layers of the epidermis of the skin. Cytoglobin (CYGB) is a ubiquitously expressed hexacoordinated globin that is highly enriched in me­lanocytes and frequently downregulated during melanomagenesis. Previously, we showed that non-thermal plasma (NTP)-produced reactive oxygen and nitrogen species (RONS) lead to the formation of an intra­ molecular disulfide bridge that would allow CYGB to function as a redox-sensitive protein. Here, we investigate the cytotoxic effect of indirect NTP treatment in two melanoma cell lines with divergent endogenous CYGB expression levels, and we explore the role of CYGB in determining treatment outcome. Our findings are consistent with previous studies supporting that NTP cytotoxicity is mediated through the production of RONS and leads to apoptotic cell death in melanoma cells. Furthermore, we show that NTP-treated solutions elicit an antioxidant response through the activation of nuclear factor erythroid 2–related factor 2 (NRF2). The knock­ down and overexpression of CYGB respectively sensitizes and protects melanoma cells from RONS-induced apoptotic cell death. The presence of CYGB enhances heme-oxygenase 1 (HO-1) and NRF2 protein expression levels, whereas the absence impairs their expression. Moreover, analysis of the CYGB-dependent transcriptome demonstrates the tumor suppressor long non-coding RNA maternally expressed 3 (MEG3) as a hitherto unde­ scribed link between CYGB and NRF2. Thus, the presence of CYGB, at least in melanoma cells, seems to play a central role in determining the therapeutic outcome of RONS-inducing anticancer therapies, like NTP-treated solutions, possessing both tumor-suppressive and oncogenic features. Hence, CYGB expression could be of in­ terest either as a biomarker or as a candidate for future targeted therapies in melanoma.

Abstract: Many current anti-cancer therapies rely on increasing the intracellular reactive oxygen and nitrogen species (RONS) contents with the aim to induce irreparable damage, which subsequently results in tumor cell death. A novel tool in cancer therapy is the use of cold atmospheric plasma (CAP), which has been found to be very effective in the treatment of many different cancer cell types in vitro as well as in vivo, mainly through the vast generation of RONS. One of the key determinants of the cell's fate will be the interaction of RONS, generated by CAP, with important proteins, i.e. redox-regulatory proteins. One such protein is cytoglobin (CYGB), a recently discovered globin proposed to be involved in the protection of the cell against oxidative stress. In this study, the effect of plasma-produced RONS on CYGB was investigated through the treatment of CYGB with CAP for different treatment times. Spectroscopic analysis of CYGB showed that although chemical modifications occur, its secondary structure remains intact. Mass spectrometry experiments identified these modifications as oxidations of mainly sulfur-containing and aromatic amino acids. With longer treatment time, the treatment was also found to induce nitration of the heme. Furthermore, the two surface-exposed cysteine residues of CYGB were oxidized upon treatment, leading to the formation of intermolecular disulfide bridges, and potentially also intramolecular disulfide bridges. In addition, molecular dynamics and docking simulations confirmed, and further show, that the formation of an intramolecular disulfide bond, due to oxidative conditions, affects the CYGB 3D structure, thereby opening the access to the heme group, through gate functioning of His117. Altogether, the results obtained in this study (1) show that plasma-produced RONS can extensively oxidize proteins and (2) that the oxidation status of two redox-active cysteines lead to different conformations of CYGB.

Abstract: Background: Previous studies have demonstrated the potential beneficial effect of N-acetylcysteine (NAC) in chronic obstructive pulmonary disease (COPD). However, the required dose and responder phenotype remain unclear. The current study investigated the effect of high-dose NAC on airway geometry, inflammation, and oxidative stress in COPD patients. Novel functional respiratory imaging methods combining multislice computed tomography images and computer-based flow simulations were used with high sensitivity for detecting changes induced by the therapy. Methods: Twelve patients with Global Initiative for Chronic Obstructive Lung Disease stage II COPD were randomized to receive NAC 1800 mg or placebo daily for 3 months and were then crossed over to the alternative treatment for a further 3 months. Results: Significant correlations were found between image-based resistance values and glutathione levels after treatment with NAC (P = 0.011) and glutathione peroxidase at baseline (P = 0.036). Image-based resistance values appeared to be a good predictor for glutathione peroxidase levels after NAC (P = 0.02), changes in glutathione peroxidase levels (P = 0.035), and reduction in lobar functional residual capacity levels (P = 0.00084). In the limited set of responders to NAC therapy, the changes in airway resistance were in the same order as changes induced by budesonide/formoterol. Conclusion: A combination of glutathione, glutathione peroxidase, and imaging parameters could potentially be used to phenotype COPD patients who would benefit from addition of NAC to their current therapy. The findings of this small pilot study need to be confirmed in a larger pivotal trial.

Abstract: Animal models are being used extensively in pre-clinical and safety assessment studies to assess the effectiveness and safety of new chemical entities and delivery systems. Although never entirely replacing the need for animal testing, the use of computer simulations could eventually reduce the amount of animals needed for research purposes and refine the data acquired from the animal studies. Computational fluid dynamics is a powerful tool that makes it possible to simulate flow and particle behavior in animal or patient-specific respiratory models, for purposes of inhaled delivery. This tool requires an accurate representation of the respiratory system, respiration and dose delivery attributes. The aim of this study is to develop a representative airway model of the Sprague-Dawley rat using static and dynamic micro-CT scans. The entire respiratory tract was modeled, from the snout and nares down to the central airways at the point where no distinction could be made between intraluminal air and the surrounding tissue. For the selection of the representative model, variables such as upper airway movement, segmentation length, airway volume and size are taken into account. Dynamic scans of the nostril region were used to illustrate the characteristic morphology of this region in anaesthetized animals. It could be concluded from this study that it was possible to construct a highly detailed representative model of a Sprague-Dawley rat based on imaging modalities such as micro-CT scans

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: Patients with sleep related breathing disorders (SRBD) can be treated with nasal continuous positive airway pressure (nCPAP) or with several upper airway (UA) interventions. While nCPAP is almost always effective when clinically well tolerated, the therapeutic outcome of UA surgery and UA devices is difficult to predict. The improvement of our capability to perform the latter predictions more accurately is of great clinical importance since it will prevent patients to be treated with ineffective, sometimes irreversible, techniques. In this review we describe the importance of new imaging methods in this respect. We first refer to the most important pathophysiological mechanisms known so far to play a role in SRBD, indicating that functional imaging could elucidate these mechanisms in a patient specific manner. Then we describe the new technologies that make UA imaging more functional referring to the data as they are presently available. Finally we summarise the studies that do indicate that the use of functional imaging of the UA in SRBD patients may significantly help to predict the clinical outcome after UA interventions.

Abstract: Introduction: Noninvasive ventilation (NIV) is a well-established treatment for acute-on-chronic respiratory failure in hypercapnic COPD patients. Less is known about the effects of a long-term treatment with NIV in hypercapnic COPD patients and about the factors that may predict response in terms of improved oxygenation and lowered CO2 retention.Methods: In this study, we randomized 15 patients to a routine pharmacological treatment (n = 5, age 66 [standard deviation ± 6] years, FEV1 30.5 [±5.1] %pred, PaO2 65 [±6] mmHg, PaCO2 52.4 [±6.0] mmHg) or to a routine treatment and NIV (using the Synchrony BiPAP device [Respironics, Inc, Murrsville, PA]) (n = 10, age 65 [±7] years, FEV1 29.5 [±9.0] %pred, PaO2 59 [±13] mmHg, PaCO2 55.4 [±7.7] mmHg) for 6 months. We looked at arterial blood gasses, lung function parameters and performed a low-dose computed tomography of the thorax, which was later used for segmentation (providing lobe and airway volumes, iVlobe and iVaw) and post-processing with computer methods (providing airway resistance, iRaw) giving overall a functional image of the separate airways and lobes.Results: In both groups there was a nonsignificant change in FEV1 (NIV group 29.5 [9.0] to 38.5 [14.6] %pred, control group 30.5 [5.1] to 36.8 [8.7] mmHg). PaCO2 dropped significantly only in the NIV group (NIV: 55.4 [7.7] → 44.5 [4.70], P = 0.0076; control: 52.4 [6.0] → 47.6 [8.2], NS). Patients actively treated with NIV developed a more inhomogeneous redistribution of mass flow than control patients. Subsequent analysis indicated that in NIV-treated patients that improve their blood gases, mass flow was also redistributed towards areas with higher vessel density and less emphysema, indicating that flow was redistributed towards areas with better perfusion. There was a highly significant correlation between the % increase in mass flow towards lobes with a blood vessel density of >9% and the increase in PaO2. Improved ventilation–perfusion match and recruitment of previously occluded small airways can explain the improvement in blood gases.Conclusion: We can conclude that in hypercapnic COPD patients treated with long-term NIV over 6 months, a mass flow redistribution occurs, providing a better ventilation–perfusion match and hence better blood gases and lung function. Control patients improve homogeneously in iVaw and iRaw, without improvement in gas exchange since there is no improved ventilation/perfusion ratio or increased alveolar ventilation. These differences in response can be detected through functional imaging, which gives a more detailed report on regional lung volumes and resistances than classical lung function tests do. Possibly only patients with localized small airway disease are good candidates for long-term NIV treatment. To confirm this and to see if better arterial blood gases also lead to better health related quality of life and longer survival, we have to study a larger population.

Abstract: Background: Salbutamol and ipratropium bromide improve lung function in patients with chronic obstructive pulmonary disease (COPD). However, their bronchodilating effect has not yet been compared in the central and distal airways. Functional imaging using computational fluid dynamics offers the possibility of making such a comparison. The objective of this study was to assess the effects of salbutamol and ipratropium bromide on the geometry and computational fluid dynamics-based resistance of the central and distal airways.Methods: Five patients with Global Initiative for Chronic Obstructive Lung Disease Stage III COPD were randomized to a single dose of salbutamol or ipratropium bromide in a crossover manner with a 1-week interval between treatments. Patients underwent lung function testing and a multislice computed tomography scan of the thorax that was used for functional imaging. Two hours after dosing, the patients again underwent lung function tests and repeat computed tomography.Results: Lung function parameters, including forced expiratory volume in 1 second, vital capacity, overall airway resistance, and specific airway resistance, changed significantly after administration of each product. On functional imaging, the bronchodilating effect was greater in the distal airways, with a corresponding drop in airway resistance, compared with the central airways. Salbutamol and ipratropium bromide were equally effective at first glance when looking at lung function tests, but when viewed in more detail with functional imaging, hyporesponsiveness could be shown for salbutamol in one patient. Salbutamol was more effective in the other patients.Conclusion: This pilot study gives an innovative insight into the modes of action of salbutamol and ipratropium bromide in patients with COPD, using the new techniques of functional imaging and computational fluid dynamics.

Abstract: The feasibility of algae harvesting with submerged flat panel membranes was investigated as pre-concentration step prior to centrifugation. Polishing of the supernatant coming from the centrifuge was evaluated as well. The effect of membrane polymer (polyvinyl chloride [PVC], polyethersulfone polyvinyl-pyrollidone [PES-PVP], poly vinylidene fluoride [PVDF]), pore size (microfiltration [MF], ultrafiltration [UF]), algae cell concentrations and species were investigated at lab-scale. In addition, backwashing as fouling control was compared to standard relaxation. PVDF was the superior polymer, and UF showed better fouling resistance. Backwashing outperformed relaxation in fouling control. The backwashable membranes allowed up to 300% higher fluxes compared to commercial flat panel benchmark (PVC) membranes. Estimations on energy consumption for membrane filtration followed by centrifugation revealed relatively low values of 0.169 kW h/kg of dry weight of algae compared to 0.5 kW h/kg for algae harvesting via classical centrifuge alone. (C) 2012 Elsevier Ltd. All rights reserved.