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

Patients with orthopaedic implants often experience implant-associated infections (IAI), one of the most prominent causes for implant failure. IAIs are caused by pathogens adhering to the implant surface. One method to prevent IAIs is through the use of surface topography modifications. Cellular behaviour has been shown to be influenced by substrate surface topographies. Ideally, a topography that can simultaneously kill bacteria and promote osseointegration is desirable. In previous research, proved-to-be-bactericidal nanopillars of ~180 nm in height and ~80 nm in diameter were fabricated at 170 nm interspace, resulting in a bactericidal efficiency of 36 ± 5% for Staphylococcus aureus (S. aureus). The current research aimed to enhance this bactericidal efficiency by producing nanopatterns with different interspace distances and introducing disorder, while keeping the same nanopillar dimensions. The patterns have been produced by using electron beam induced deposition (EBID) at 100, 170, 300 and 500 nm interspacing. A disordered version of the 170, 300 and 500 nm samples was included with 45, 110 and 210 nm disorder distance, respectively. In order to keep the nanopillar dimensions consistent, EBID parameters were systematically optimised. The nanopatterns were interfaced with S. aureus cells and the bactericidal efficiencies were determined by counting the number of healthy, deformed and dead bacterial cells. For the ordered 100, 170, 300 and 500 nm interspace nanopatterns, bactericidal efficiencies of 62.3 ± 23.1%, 45.0 ± 31.4%, 8.6 ± 4.2% and 3.7 ± 2.3% were found, respectively. For the disordered 170, 300 and 500 nm interspace nanopatterns, bactericidal efficiencies of 45.9 ± 29.2%, 14.7 ± 7.2% and 12.7 ± 9.8% were found, respectively. These results showed a significantly higher bactericidal effect on lower-interspace nanopatterns. Disordered nanopatterns showed only slightly higher bactericidal efficiencies when compared to the ordered nanopatterns. This data indicated that bactericidal efficiencies could be increased relative to the previous nanopattern design. The findings of this research aid in developing antibacterial nanopatterns that are capable of killing gram-positive bacteria and support the path to prevention of IAIs.

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).

The presence of implants in the body is frequently associated with a high risk of peri-implant infections. The host and the bacterial cells compete to adhere and grow on the implant surface. Therefore, producing an implant that can promote the stem cells function (e.g., osteogenic differentiation) while reducing bacterial colonization is vital. By applying controlled topography on the surface, the behavior of stem cells and bacteria can be influenced. In this study, early interactions between bacteria and osteogenic nanopatterns were investigated. Five different nanopatterns were produced by electron beam induced deposition. The nanopatterns were then incubated with E. coli K12 strain for 18 hours. Thereafter, the samples were investigated by scanning electron microscope to assess the morphology and the adhesion of bacteria. Based on the experimental results, most of the nanopatterns exhibited a positive effect in reducing bacterial cells adhesion. Moreover, two nanopatterns with heights of 130 nm and 60 nm exhibited bactericidal properties. Possible bactericidal mechanisms have been proposed based on closer examination of the bacteria-nanopattern interface and computational analysis of the interfacial forces. This study investigated the effects of osteogenic nanopatterns on bacterial cells response for the first time. The findings suggested that nanopatterns with both osteogenic and antibacterial properties may be developed for bone implant applications.

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.

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.