Biomimicking natural nanopatterned topology by 3D laser lithography

Natural nanopatterned surfaces (nNPS) present on insect wings have demonstrated bactericidal activity [1, 2]. Fabricated nanopatterned surfaces (fNPS) derived by characterization of these wings have also shown superior bactericidal activity [2]. However bactericidal NPS topologies vary in both geometry and chemical characteristics of the individual features in different insects and fabricated surfaces, rendering it difficult to ascertain the optimum geometrical parameters underling bactericidal activity. This situation calls for the adaptation of new and emerging techniques, which are capable of fabricating and characterising comparable structures to nNPS from biocompatible materials. In this research, CAD drawn nNPS representing an area of 10 μm x10 μm was fabricated on a fused silica glass by Nanoscribe photonic professional GT 3D laser lithography system using two photon polymerization lithography. The glass was cleaned with acetone and isopropyl alcohol thrice and a drop of IP-DIP photoresist from Nanoscribe GmbH was cast onto the glass slide prior to patterning. Photosensitive IP-DIP resist was polymerized with high precision to make the surface nanopatterns using a 780 nm wavelength laser. Both moving-beam fixedsample (MBFS) and fixed-beam moving-sample (FBMS) fabrication approaches were tested during the fabrication process to determine the best approach for the precise fabrication of the required nanotopological pattern. Laser power was also optimized to fabricate the required fNPS, where this was changed from 3mW to 10mW to determine the optimum laser power for the polymerization of the photoresist for fabricating FNPS..

Progress in resolving bio-nano interactions microscopically to understand the bactericidal activity of dragonfly wing nanopillars

Bactericidal mechanism of nanototography is a highly debated, yet unresolved phenomenon. Investigation of bacteria-nanotopography interactions therefore has become a significant research interest for the development of advanced bactericidal nano textured surfaces for the control of bacterial adhesion to prevent their survival on biomaterials. The unique nanototography of dragonfly wings with nanopillars surface are of great interest among researchers to develop antibacterial surfaces with mechanical approach. The mechanical bactericidal activity of dragonfly wing is a property of this unique nanopillars which are made of hydrocarbons and usually known as cuticle layer. Due to their size, composition and beam sensitive nature of these nanopillars, it is difficult to characterise the bacterial-nanopillars interactions using traditional electron microscopic methods. However, to understand mechanistic bactericidal activity, characterisation is needed at a further later step where bacteria-nanopillar interaction takes place. This natural bio-nano interface needs to be resolved in order to understand the bactericidal activity and develop efficient nano structures on to biomaterials. Several attempts have been taken to characterise this bio-nano interface on synthetic surfaces although characterisation of such interface on natural surface has shown very little success . As the fabricated surfaces are different in chemistry, architecture and bactericidal activity, it is vital to characterise the interface on a natural surface. Here, we reveal the bacteria-nanotopography interaction of dragonfly wing and grampositive Staphylococcus aureus bacterium and discuss empirical approach on characterising the natural interface and their limitations. We have used TEM and Helium ion microscopy (HIM) to resolve the interfaces of Escherichia coli and Staphylococcus aureus on dragonfly wing nanopillars. Bacterial cells were incubated for 30 minutes on dragonfly wing was used to reveal the bio-nano interaction of bacteria and the nanotopography of dragonfly wing. Then samples were fixed with 2% glutaraldehyde and subjected to membrane staining and ethanol dehydration prior to imaging. In TEM samples were resin embedded, and for HIM samples were used without coating, so that we present uninterrupted natural interactions between S.aureus and nanopillars with clarity and detail. Ne milling followed by He imaging shows the interface between the nanopillars of dragonfly wing and Escherichia coli (Figure 1) Staphylococcus aureus (Figure 2). Using HIM we have identified that bacteria anchor to nanopillars only at certain positions and membrane in between is intact. We also see that Escherichia coli extra cellular polymeric substances (EPS) has been secreted on the nanopillars during this interaction. This causes bacteria to adhere strongly and makes a stress response. In order to understand the interaction clearly, bacterium was milled at various positions and microgrraphed at various angles. We have observed that Staphylococcus aureus bacteria contact the nanopillars only at certain points anchoring bacterium to the surface. Even after bacterium is flattened out, we could not observe membrane has collapsed. In between these anchor points membrane remains intact. It can be clearly noted from the images that bacterial membrane is blended with the nanopillars and their borders cannot be distinguished. This remarks integration of nanopillars with lipid bilayer. As nanopillars and membrane are organic and hydrophobic in nature this kind of dissolving interaction would be chemically expected. The results further confirms the findings in our previous paper.1 However further biochemical assessments are necessary for a comprehensive explanation. These observations are different to some previous computational modelling predictions done without the use of microscopic evidence.2,3

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e., mapping of elemental, isotopic, and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro− to nano-meter spatial resolution. Our approach includes (i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, (ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and (iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (μ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance

Resolving Bio-Nano Interactions of E.coli Bacteria-Dragonfly Wing Interface with Helium Ion and 3D-Structured Illumination Microscopy to Understand Bacterial Death on Nanotopography

Obtaining a comprehensive understanding of the bactericidal mechanisms of natural nanotextured surfaces is crucial for the development of fabricated nanotextured surfaces with efficient bactericidal activity. However, the scale, nature, and speed of bacteria-nanotextured surface interactions make the characterization of the interaction a challenging task. There are currently several different opinions regarding the possible mechanisms by which bacterial membrane damage occurs upon interacting with nanotextured surfaces. Advanced imaging methods could clarify this by enabling visualization of the interaction. Charged particle microscopes can achieve the required nanoscale resolution but are limited to dry samples. In contrast, light-based methods enable the characterization of living (hydrated) samples but are limited by the resolution achievable. Here we utilized both helium ion microscopy (HIM) and 3D structured illumination microscopy (3D-SIM) techniques to understand the interaction of Gram-negative bacterial membranes with nanopillars such as those found on dragonfly wings. Helium ion microscopy enables cutting and imaging at nanoscale resolution while 3D-SIM is a super-resolution optical microscopy technique that allows visualization of live, unfixed bacteria at ~100 nm resolution. Upon bacteria-nanopillar interaction, the energy stored due to the bending of natural nanopillars was estimated and compared with fabricated vertically aligned carbon nanotubes. With the same deflection, shorter dragonfly wing nanopillars store slightly higher energy compared to carbon nanotubes. This indicates that fabricated surfaces may achieve similar bactericidal efficiency as dragonfly wings. This study reports in situ characterization of bacteria-nanopillar interactions in real-time close to its natural state. These microscopic approaches will help further understanding of bacterial membrane interactions with nanotextured surfaces and the bactericidal mechanisms of nanotopographies so that more efficient bactericidal nanotextured surfaces can be designed, fabricated, and their bacteria-nanotopography interactions can be assessed in situ.peerReviewe

Publication Data for Bandara et al 2021 - Front.Plant.Sci LRWhite Embedding Method

Data acquired in the framework of the SPP 2089, funded by the DFG. This data folder associated with the publication "Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in its Native Spatial Context" by Bandara et al. (2021), Frontiers in Plant Science Journal. DOI: 10.3389/fpls.2021.668929 The compressed folder contains ten subfolders corresponding to each technique as described below. Each folder named as Origin contains the original data, while processed data is included in the folder named as Export. In total there are 251 files. 1. CARD-FISH: This folder contains Epifluorescence micrographs presented in Figure 8d and 10a. Original data format is .czi from Carl Zeiss imaging. Exported images format is .tif. Folder contains two folders for individual samples. 2. Correlia_Registered_Datasets This folder contains two Correlia (https://www.ufz.de/correlia) projects used to register different microscopy images presented in Figure 8d and Figure9. 3. DarkField_Figure3_Map This folder contains stitched darkfield micrographs of CY0211 sample (Figure3). Text file contains the imaging information. 4. Epifluorescence This folder contains Epifluorescence images presented in Figure3, Figure 10 5. HIM This folder contains Helium ion micrographs presented in Figure 2 and Figure 6d 6. nanoSIMS This folder contains nanoSIMS data presented in Figure 6 and Figure 10 7. Raman This folder contains confocal Raman Microscopy data presented in Figure 7 8. Roughness Measurements This folder contains surface profile data presented in Figure 2b 9. SEM-EDX This folder contains SEM and EDX data presented in Figure 5 and Figure 8d 10.ToFSIMS This folder contains ToFSIMS data presented in Figure 4 Funding: This data was produced within the framework of the priority program SPP 2089, “Rhizosphere spatiotemporal organization-a key to rhizosphere functions” funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Projektnummer 403641683 (RI-903/7-1). Research work of Yalda Devoudpour was supported by Deutsche Forschungsgemeinschaft Integration of Refugee Scientists and Academics

Publication Data for Bandara et al 2021 - Front.Plant.Sci LRWhite Embedding Method

Data acquired in the framework of the SPP 2089, funded by the DFG. This data folder associated with the publication "Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in its Native Spatial Context" by Bandara et al. (2021), Frontiers in Plant Science Journal. DOI: 10.3389/fpls.2021.668929 The compressed folder contains ten subfolders corresponding to each technique as described below. Each folder named as Origin contains the original data, while processed data is included in the folder named as Export. In total there are 251 files. 1. CARD-FISH: This folder contains Epifluorescence micrographs presented in Figure 8d and 10a. Original data format is .czi from Carl Zeiss imaging. Exported images format is .tif. Folder contains two folders for individual samples. 2. Correlia_Registered_Datasets This folder contains two Correlia (https://www.ufz.de/correlia) projects used to register different microscopy images presented in Figure 8d and Figure9. 3. DarkField_Figure3_Map This folder contains stitched darkfield micrographs of CY0211 sample (Figure3). Text file contains the imaging information. 4. Epifluorescence This folder contains Epifluorescence images presented in Figure3, Figure 10 5. HIM This folder contains Helium ion micrographs presented in Figure 2 and Figure 6d 6. nanoSIMS This folder contains nanoSIMS data presented in Figure 6 and Figure 10 7. Raman This folder contains confocal Raman Microscopy data presented in Figure 7 8. Roughness Measurements This folder contains surface profile data presented in Figure 2b 9. SEM-EDX This folder contains SEM and EDX data presented in Figure 5 and Figure 8d 10.ToFSIMS This folder contains ToFSIMS data presented in Figure 4 Funding: This data was produced within the framework of the priority program SPP 2089, “Rhizosphere spatiotemporal organization-a key to rhizosphere functions” funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Projektnummer 403641683 (RI-903/7-1). Research work of Yalda Devoudpour was supported by Deutsche Forschungsgemeinschaft Integration of Refugee Scientists and Academics

Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on <i>Escherichia coli</i>

Nanotextured surfaces (NTSs) are critical to organisms as self-adaptation and survival tools. These NTSs have been actively mimicked in the process of developing bactericidal surfaces for diverse biomedical and hygiene applications. To design and fabricate bactericidal topographies effectively for various applications, understanding the bactericidal mechanism of NTS in nature is essential. The current mechanistic explanations on natural bactericidal activity of nanopillars have not utilized recent advances in microscopy to study the natural interaction. This research reveals the natural bactericidal interaction between <i>E. coli</i> and a dragonfly wing’s (<i>Orthetrum villosovittatum</i>) NTS using advanced microscopy techniques and proposes a model. Contrary to the existing mechanistic models, this experimental approach demonstrated that the NTS of <i>Orthetrum villosovittatum</i> dragonfly wings has two prominent nanopillar populations and the resolved interface shows membrane damage occurred without direct contact of the bacterial cell membrane with the nanopillars. We propose that the bacterial membrane damage is initiated by a combination of strong adhesion between nanopillars and bacterium EPS layer as well as shear force when immobilized bacterium attempts to move on the NTS. These findings could help guide the design of novel biomimetic nanomaterials by maximizing the synergies between biochemical and mechanical bactericidal effects