The work described in the doctoral thesis aims to enhance the functionality of a scanning electron microscope (SEM) by integrating miniaturized versions of cleanroom tools used in microfabrication. The thesis presents the integration of substrate heating, in-situ thermal atomic layer deposition (ALD), sputtering, and thermal evaporation within the SEM. These techniques are expected to enable fast and efficient fabrication of proof-of-concept devices with minimal resources. The thesis also discusses the challenges, limitations, and future work of the integrated cleanroom processes in SEM.@en

To design electron lens systems, applying a fully automated optimization routine has not yet been feasible, especially for the case where the optimization has many free variables of the lens system, such as all parameters that define the geometry of the lens electrodes and the voltage of each electrode. Hence, the study of the implementation of different optimization procedures has not yet been possible either. In one of our previous studies, we have proposed to use the so-called Second Order Electrode Method (SOEM) which performs the electrostatic field calculations in a very short time by the approximations of the field near the optical axis. There, using SOEM in field calculation, a Genetic Algorithm (GA) was successfully implemented to optimize the electron lens systems. One of the questions that has not been studied and answered in the literature yet, is whether the GA is the most suitable option among different optimization techniques for the design/optimization of electron lens systems. In this paper, by implementing the SOEM technique as the field calculation method, different optimization procedures are implemented and their performances are compared. For this study, a typical six electrode lens system is employed. The implemented optimization techniques include calculus-based local optimization (‘Fmin’) and metaheuristic methods such as GA, Particle Swarm Optimization (PSO), and Simulated Annealing (SA). The results demonstrate that the population-based global optimization techniques like GA and PSO significantly outperform single-based local optimization methods such as ‘Fmin’ and SA. Additionally, PSO shows slightly better performance than GA, although it cannot be concluded that PSO will always outperform GA for every electron lens design problem. Furthermore, in the comparison between the two single-based optimization techniques, the metaheuristic approach (SA) outperforms the calculus-based one (‘Fmin’). Hence, we recommend implementing metaheuristic, global, population-based optimization techniques like GA and PSO for the optimization electron lens systems.@en

Monte Carlo simulations are frequently used to describe electron–matter interaction in the 0–50 keV energy range. It often takes hours to simulate electron microscope images using first-principle physical models. In an attempt to maintain a reasonable speed, empirical models are sometimes used. We present an open-source software package with first-principle physical models, which can run on GPUs for fast results. Typical electron microscope images can be obtained in minutes instead of hours on consumer-grade hardware, without any loss of accuracy.@en

Development of synthetic bactericidal surfaces is a drug-free route to the prevention of implant-associated infections. Surface nanotopographies with specific dimensions have been shown to kill various types of bacterial strains through a mechanical mechanism, while regulating stem cell differentiation and tissue regeneration. The effective ranges of dimensions required to simultaneously achieve both aims are in the <200 nm range. Here, a nanoscale additive manufacturing (=3D printing) technique called electron beam induced deposition (EBID) is used to fabricate nanopillars with reproducible and precisely controlled dimensions and arrangements that are within those effective ranges (i.e. a height of 190 nm, a diameter of 80 nm, and an interspacing of 170 nm). When compared to the flat surface, the nanopatterned surfaces show a significant bactericidal activity against both Escherichia coli and Staphylococcus aureus (with respective killing efficiencies of 97 ± 1% and 36 ± 5%). Direct penetration of nanopatterns into the bacterial cell wall leads to the disruption of the cell wall and cell death. The more rigid cell wall of S. aureus is consistent with the decreased killing efficiency. These findings support the development of nanopatterns with precisely controlled dimensions that are capable of killing both Gram-negative and Gram-positive bacteria.@en

There is a growing interest for patterning on curved or tilted surfaces using electron beam lithography. Computational proximity correction techniques are well established for flat surfaces and perpendicular exposure, but for curved and tilted surfaces adjustments are needed as the dose distribution is no longer cylindrically symmetric with respect to the surface normal. A graphical processing unit -accelerated 3D Monte Carlo simulation, based on first-principle scattering models, is used to simulate the asymmetric dose distribution. Based on that, an approximate adjustment is made to an existing high-performance proximity effect correction (PEC) algorithm aimed at the correct exposure of a pattern of nanowires on a 17° tilted surface. It was experimentally verified that using the adjusted PEC indeed leads to a more uniform exposure on tilted surfaces.

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BACKGROUND.Implant-associated infection (IAI) is a rising complication in bone-related medical treatments using metal implants. Preventive measures, such as implant surfaces with integrated antibacterial properties, are hence required. The emergence of antibiotic-resistant pathogens has led to a growing interest in inorganic nanoparticles as antibacterial agents. In addition, implants should simultaneously stimulate bone tissue regeneration and integration to improve implant longevity. Additive manufacturing (AM) of orthopedic implants has attracted interest as improved bone tissue regeneration and integration was demonstrated by AM porous implants. AM porous implants, however, are at enhanced risks of IAIs; these porous implants require integrated antibacterial properties. This study focused on a systematic comparison of the antibacterial properties of AM porous Ti6Al4V implant surfaces with silver nanoparticles (Ag NPs), copper nanoparticles (Cu NPs) and/or zinc nanoparticles (Zn NPs) against methicillin resistant Staphylococcus aureus (MRSA). METHODS.Medical grade porous Ti6Al4V implants were additively manufactured using selective laser melting (SLM). Subsequently, the SLM Ti6Al4V implants were biofunctionalized by plasma electrolytic oxidation (PEO) using an electrolyte that consisted of Ca/P-based species and ratios of Ag NPs, Cu NPs and/or Zn NPs. Implants with 0, 25, 75 and 100% of Ag NPs, Cu NPs and/or Zn NPs were synthesized. After biofunctionalization, the surface morphology, structure and chemistry of the implants were investigated using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The antibacterial properties of the implants against MRSA were studied in vitro as well as ex vivo. The leachable antibacterial activity was studied in an agar diffusion assay, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Ag+, Cu2+ and Zn2+ ions was determined by a micro-dilution assay and the bactericidal activity of the implants was quantified by a colony-forming unit (CFU) count in vitro against surface-adherent and non-adherent MRSA. Furthermore, ex vivo antibacterial properties were determined using a femoral murine infection model mimicking the in vivo environment. RESULTS.Biofunctionalization of the porous SLM Ti6Al4V implants by PEO resulted in a uniform and homogeneous micro-/nano-porous oxide layer that covered the entire surface of the implants. SEM and EDS analysis demonstrated the presence of Ag NPs, Cu NPs and Zn NPs in the TiO2 surface layer. The agar diffusion assay demonstrated a strong leachable antibacterial activity for the implants with Ag NPs and Cu NPs or Zn NPs, while no leachable antibacterial activity was observed for implants without Ag NPs. Antibacterial testing showed that Ag+, Cu2+ and Zn2+ ions, as well the combinations of the ions, were growth inhibitory and bactericidal to MRSA. In addition, implants with ≥ 50% of Ag NPs and combinations of ≥ 75% of Ag NPs and Cu NPs or Zn NPs, fully eradicated surface-adherent and non-adherent MRSA within 24 h and prevented biofilm formation up to 24 h. Moreover, strong ex vivo bactericidal properties against MRSA were demonstrated for these implants within 24 h. CONCLUSION. Biofunctionalized surfaces with Ag NPs and Cu NPs or Zn NPs on porous SLM Ti6Al4V implants showed strong in vitro and ex vivo antibacterial properties against MRSA. The implants with 50% of Ag NPs and combinations of 75% of Ag NPs and 25% of Cu NPs or Zn NPs are promising for further development, to prevent IAIs and improve the longevity of the implants in clinical applications.

Due to competition of host and bacterial cells to adhere and grow on implant surfaces, implants are frequently associated with a high risk of peri-implant infections. The microorganisms that are abundantly present during peri-implant infections are Staphylococcus bacteria and a variety of other less abundant bacteria as for example Escherichia coli. For this reason, some studies have been focusing on creating nanopatterns that might reduce bacterial colonization when used as implant surface topography. However, most of this research showed that the specified nanopatterns were only exceedingly bactericidal to either Gram-positive or Gram-negative bacteria. The aim of this novel study is to investigate the bactericidal effects of nanopatterns with pillar diameter of ~ 80 nm, a height of ~ 190 nm and an interpillar distance of ~ 170 nm on Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The patterns were incubated with S. aureus and E. coli for 18 hours at 37 °C in their specific growth medium. The bactericidal effects were examined with scanning electron microscopy (SEM) by assessing the bacterial cell morphology and the amount of damaged bacterial cells found on the patterns. The patterns were able to damage approximately 96.9 ± 1.2% of E. coli and 83.9 ± 22.8% of S. aureus cells. The severity of bacterial cell damage has led to believe that the percentage of dead bacterial cells was a sufficient measure for the bactericidal efficacy of the pattern. Based on these results there is a convincing assumption that these specific pillar parameters can be used as a bactericidal surface topography on bone implants. Nevertheless, follow up experiments should be done in combination with live imaging of the cells to establish possible long-term bacterial- and host cell effects on topography and gain more insights into the proposed bactericidal mechanism(s).

We present first experiments on electron beam induced deposition of silacyclohexane (SCH) and dichlorosilacyclohexane (DCSCH) under a focused high-energy electron beam (FEBID). We compare the deposition dynamics observed when growing pillars of high aspect ratio from these compounds and we compare the proximity effect observed for these compounds. The two precursors show similar behaviour with regards to fragmentation through dissociative ionization in the gas phase under single-collision conditions. However, while DCSCH shows appreciable cross sections with regards to dissociative electron attachment, SCH is inert with respect to this process. We discuss our deposition experiments in context of the efficiency of these different electron-induced fragmentation processes. With regards to the deposition dynamics, we observe a substantially faster growth from DCSCH and a higher saturation diameter when growing pillars with high aspect ratio. However, both compounds show similar behaviour with regards to the proximity effect. With regards to the composition of the deposits, we observe that the C/Si ratio is similar for both compounds and in both cases close to the initial molecular stoichiometry. The oxygen content in the DCSCH deposits is about double that of the SCH deposits. Only marginal chlorine is observed in the deposits of from DCSCH. We discuss these observations in context of potential approaches for Si deposition.@en

By placing, in vacuum, a stack of transmission dynodes (tynodes) on top of a CMOS pixel chip, a single free electron detector could be made with outstanding performance in terms of spatial and time resolution. The essential object is the tynode: an ultra thin membrane, which emits, at the impact of an energetic electron on one side, a multiple of electrons at the other side. The electron yields of tynodes have been calculated by means of GEANT-4 Monte Carlo simulations, applying special low-energy extensions. The results are in line with another simulation based on a continuous charge-diffusion model. By means of Micro Electro Mechanical System (MEMS) technology, tynodes and test samples have been realized. The secondary electron yield of several samples has been measured in three different setups. Finally, several possibilities to improve the yield are presented.

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In this paper two ways of modeling the secondary electron yields of an insula-
tor under electron irradiation, are discussed and coupled. The drift-diusion-
reaction model is used to investigate the secondary yields and charging eects.

With increasing prevalence of neurodegenerative diseases the need for an effective treatment increases. To date pharmacological treatment is the golden standard. However, the medicines used have many adverse effects and, only work for a short time period. The best solution would be to cure the disease and thus stop the degradation of neurons and replace the already lost neurons by healthy ones. With the development of regenerative medicine, strategies to replace the lost neurons are emerging. One of the approaches includes the use of neuronal progenitor cells to differentiate and grow into functional neurons. To achieve this, a suitable in vitro environment is needed that includes an optimal use of culture medium and substrate. Laminin has been shown to be an effective substrate for neuronal cell culture purposes as it is part of the native extracellular matrix in the nervous system. However, laminin represents a family of 20 isoforms as known so far and there is still a lot unknown about the functions of each isoform on neuronal cultures. In this study the effects of eight available human recombinant laminin isoforms (i.e., -111, -121, -211, -221, -411, -421, -511 and -521) on cell attachment, differentiation, neurite outgrowth and connectivity of cerebellar progenitor cells have been assessed by means of immunofluorescence techniques. A general laminin substrate stemming from mice sarcoma (L2020) was used as a control. The results show that Laminin- 211, -221 and -411 do not possess any neurite outgrowth and differentiation abilities and thus were not included in the remaining experiments. Between the remaining substrates used no difference was found on cell attachment and neuronal differentiation. Laminin-511 and -521 increased neurite outgrowth the most and laminin-421 the least. Laminin-111 took third place. Laminin-111, -511, -521 and L2020 were included in the connectivity experiments. Laminin-521 stimulated synaptogenesis to the largest extend followed by laminin-511 and then -111. Overall, laminin-511 and -521 showed the best results in this study and performed better than a general laminin substrate that studies mostly use.