Poster Session 2 17:30 - 18:00 Tuesday, 6th July, 2021 Sessions Poster Session

17:30 - 17:31

9 Skin Image Analysis in Contact Capacitive Imaging and High Resolution Ultrasound Imaging

Mr Christos Bontozoglou1, Dr Xu Zhang2, Mrs Elena Chirikhina1, Dr Perry Xiao1 1London South Bank University, London, United Kingdom. 2Tongjing Zhejiang College, Jiaxing, China

Abstract Text

We present our latest research on skin image analysis in Contact Capacitive Imaging and High Resolution Ultrasound Imaging. Contact Capacitive Imaging is a novel imaging technique that can be used for in-vivo skin measurements [1-3]. With Contact Capacitive Imaging, we can analyze the skin water content, skin solvent penetrations, skin texture, and skin micro-relief analysis by mathematical algorithms and machine learning. High Resolution Ultrasound Imaging is the state of the art technology, and can produces high resolution images of the skin and superficial soft tissue to a vertical resolution of about 40 microns [4]. With High Resolution Ultrasound Imaging, we have studies the differences of different layers, such as stratum corneum, epidermis and dermis, around the different locations on the face and around different body parts. In this paper, we will first present the Contact Capacitive Imaging technology and High Resolution Ultrasound Imaging technique, then present the analyzed experimental results and discussions.

Keywords

Skin image analysis, capacitive imaging, high resolution ultrasound, machine learning, skin water content, skin solvent penetration, skin texture, skin thickness.

References

1. Ou, X., Pan, W., Xiao, P., In vivo skin capacitive imaging analysis by using grey level co-occurrence matrix (GLCM), International journal of pharmaceutics 460 (1-2), 28-32, 2014. 2. Bontozoglou, C. and Xiao, P. (2019). Applications of Capacitive Imaging in Human Skin Texture and Hair Analysis . MDPI Applied Sciences. 10 (1), p. 256. https://doi.org//10.3390/app10010256 3. Zhang, X., Pan, W., Bontozoglou, C., Chirikhina, E., Chen, D. and Xiao, P. (2019). Skin Capacitive Imaging Analysis Using Deep Learning GoogLeNet. Computing Conference 2020. London, UK 16 - 17 Jul 2019 Springer. 4. Chirikhina, E., Chirikhin, A., Xiao, P., Dewsbury-Ennis, S. and Bianconi, F. (2020). In Vivo Assessment of Water Content, Trans-Epidermial Water Loss and Thickness in Human Facial Skin. Applied Sciences. 10 (17), p. e6139. https://doi.org/10.3390/app10176139 17:31 - 17:32

15 Unscrambling mixed elements atom-by-atom by combining HAADF STEM and EDX

Dr. Annick De Backer1,2, Dr. Eva Bladt1,2, Dr. Zezhong Zhang1,2,3, Prof. Sara Bals1,2, Prof. Sandra Van Aert1,2 1EMAT, University of Antwerp, Antwerp, Belgium. 2NANOlab Center of Excellence, Antwerp, Belgium. 3Department of Materials, University of Oxford, Oxford, United Kingdom

Abstract Text

A new methodology is presented to count the number of atoms in heterogenous nanocrystals by combining energy dispersive X-ray (EDX) and high angle annular dark field scanning transmission electron microscopy (HAADF STEM). Our new method is applied to determine the number of atoms of a Au@Ag core-shell nanorod and creates opportunities to investigate heterogeneous nanostructures with adjacent atomic numbers. Heterogeneous nanocrystals are of great scientific and technological interest because of their unique electronic, optical or catalytic properties. These properties are largely controlled by the exact arrangement of the atoms. Therefore, quantitative structure determination is essential for the development of new nanocrystals. HAADF STEM provides images with atomic resolution where intensities scale with number of atoms and the atomic number Z. For homogeneous nanocrystals, the number of atoms can therefore be counted from HAADF STEM images [1]. For mixed columns, all types of elements will contribute differently to the image intensities thus significantly complicating the quantitative interpretation of the image intensities. So far, the first steps towards unscrambling mixed elements at the atomic scale were made using HAADF STEM and by exploiting the so-called atomic lensing model [2]. This model describes the dynamical diffraction as a superposition of individual atoms focusing the incident electrons. However, this approach is limited to heterogeneous structures with elements having distinct atomic numbers. Here we propose combining HAADF STEM images and elemental maps acquired by EDX spectroscopy [3] enabling atom-counting for heterogeneous nanostructures, even when the difference in atomic number is only one. The so-called scattering cross-section, corresponding to the total intensity of electrons scattered by a single atomic column, has been shown to be a successful performance measure for atom-counting and composition determination in HAADF STEM [1,4-6]. Similarly, EDX STEM scattering cross-sections can be defined from the elemental maps. For a quantitative comparison with simulations, the experimental scattering cross-sections need to be calibrated in order to match them with simulated scattering cross-sections. For HAADF STEM, the calibrated values can be obtained from the images which are normalized with respect to the incident probe intensity on the detector. Since both HAADF STEM and EDX imaging are incoherent techniques, a linear relation between the EDX and HAADF STEM scattering cross-sections is assumed. Normalization constants for the EDX scattering cross-sections are then required for the different elements to determine the scaling between the scattering cross-sections from the HAADF STEM image and EDX elemental maps and enable a comparison with simulations for the EDX scattering cross-sections. In order to confirm the linear relation, experimental STEM images and EDX maps of a CeO2 nanoparticle have been acquired from which scattering cross- sections have been determined. The scattering cross-sections are represented in a scatter plot indeed suggesting a linear relationship (Figure 1). As a proof of concept, the combination of EDX and HAADF STEM images is used to count the number of atoms for a Ag-coated Au nanorod. The EDX mapping was done for 60 minutes, and the results have been stored every 5 minutes followed by the acquisition of a calibrated HAADF STEM image shown in Figure 2(a). Scattering cross-sections have been measured over time for both the STEM images and the Ag and Au EDX maps. Those scattering cross-sections have been averaged over time in order to get mean values together with a measure for their precision. Using an iterative weighted least squares minimization, the experimental scattering cross-sections have been matched to simulated values by estimating the normalization constants for the EDX scattering cross-sections, assuming a linear scaling between the STEM and EDX scattering cross-sections. The simulated scattering cross-sections are predicted using the atomic lensing model. In this manner, channeling effects on both the HAADF STEM and the EDX scattering cross- sections are included. The resulting number of Ag and Au atoms for each atomic column are shown in Figure 2(b). In conclusion, a new quantitative framework will be presented to count the number of atoms in heterogeneous nanocrystals by combining HAADF STEM and EDX signals. This methodology opens up new possibilities for the characterization of heterogeneous nanostructures with adjacent atomic numbers at the atomic scale [7]. Figure 1 (a) Experimental HAADF STEM image of a Ce nanoparticle. (b) Experimental atomic resolution EDX elemental map for the Ce L shell. (c) The EDX scattering cross-sections as a function of the HAADF STEM scattering cross-sections. The grey line highlights the linear relation between the scattering cross-sections of both techniques.

Figure 2 (a) Experimental HAADF STEM images and EDX elemental maps for Ag and Au. (b) The total number of atoms and the number of Au and Ag atoms. Keywords atom-counting bimetallic nanoparticles energy dispersive X-ray spectroscopy quantitative scanning transmission electron microscopy

References

[1] S. Van Aert et al., Physical Review B 87 (2013), p. 064107. [2] K.H.W. van den Bos et al., Physical Review Letters 116 (2016), p. 246101. [3] A.J. D’Alfonso et al., Physical Review B 81 (2010), p. 100101. [4] S. Van Aert et al., Ultramicroscopy 109 (2009), p. 1236. [5] H. E et al., Ultramicroscopy 133 (2013), p. 109. [6] G.T. Martinez et al., Ultramicroscopy 187 (2018), p. 84. [7] This work was supported by the European Research Council (Grant 770887 PICOMETRICS to SVA and Grant 815128 REALNANO to SB, Grant 823717 ESTEEM3). The authors acknowledge financial support from the Research Foundation Flanders (FWO, Belgium) through project fundings and postdoctoral grants to ADB and EB. 17:32 - 17:33

42 Imaging across biological length scales with laser free confocal microscopy

Dr Phillipa Timmins1, Dr Mika Ruonala2, Dr Rimas Jus̆kaitis1, Dr Mark Neil3, Dr Martin Booth1,4, Dr Tony Wilson4, Dr Michael Shaw5,6, Dr Kirti Prakash5 1Aurox Ltd, Culham Science Centre, Abingdon, United Kingdom. 2Image Computing and Information Technologies, Frankfurt, Germany. 3Photonics Group, Department of Physics, Imperial College London, London, United Kingdom. 4Department of Engineering Science, University of Oxford, Oxford, United Kingdom. 5National Physical Laboratory, Teddington, United Kingdom. 6Department of Computer Science, Faculty of Engineering Sciences, University College London, London, United Kingdom

Abstract Text

Aperture correlation microscopy, also known as differential spinning disk or laser free confocal (LFC) microscopy, combines the light efficiency of structured illumination with the acquisition speed (up to 100 FPS) of a spinning disk confocal system. By using an incoherent light source, such as an LED, LFC presents an attractive alternative to laser-based instruments for optical sectioning, allowing fast 3D imaging with enhanced spectral flexibility, less photobleaching and a relatively low instrument price. In addition, the Aurox LFC system offers a modular and flexible hardware configuration in terms of the imaging camera, light source, filters and the objective lens. In this paper, we image a variety of biological samples ranging from nm to mm length scales highlighting the versatility of the Aurox LFC system for cell biology, neuroscience, virology and cancer research. We present a comprehensive characterization of the instrument in terms of spatial resolution, axial response, sample irradiance and compare the performance with state-of-the-art laser scanning confocal and structured illumination microscopes.

Keywords spinning disk confocal, laser free confocal, structured illumination, spatial resolution, axial response, sample irradiance, modular imaging 17:33 - 17:34

44 Characterizations of Responsive Photonic Liquid Crystal Network Coatings on Flexible Plastic Substrates by Atomic Force Microscopy

Dr. Lanti Yang1, Dr. Nadia Grossiord2, Dr. Ellen van Heeswijk3, Prof.Dr. Albertus Schenning3 11. Technology & Innovation, SABIC, Bergen op Zoom, Netherlands. 2Technology & Innovation, SABIC, Bergen op Zoom, Netherlands. 32. Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven, Netherlands

Abstract Text

Summary This abstract highlights a novel Atomic Force Microscopy (AFM) approach to gain valuable insights into various liquid crystal networks (LCN) on plastic substrates. Studies using AFM-quantitative nanomechanical mapping (AFM-QNM) for the LCN alignment, adhesion to plastic substrates and the nano- mechanical properties of the LCN coating characterizations are described. The results accelerate the design and development of various responsive LCN on flexible plastic substrates for a variety of applications.

Modifications to add stimulus-triggered functionality to surfaces have enabled many new advances such as self-healing, thermal regulation, energy scavenging, sensing and anti-(bio)fouling. In particular, coatings based on cholesteric liquid crystal networks (LCN) are versatile materials to create stimuli-responsive coatings onto plastic substrates, which lead to lightweight and flexible applications, notably in healthcare [1, 2]. During the design and application of stimuli-triggered LCN coatings on plastic substrates, the main challenges are 1) coating adhesion to the substrate; 2) cholesteric liquid crystal alignment, which governs the (optical) properties of the functional coating; 3) combination of both responsiveness and mechanical wear resistance and 4) response specificity and sensitivity towards a stimulus. Therefore, to assist the design and development of such a coating system on plastics, advanced characterizations for both a thorough understanding of the LCN coating morphology and quantifying the coating mechanical properties are crucial. Here, we present a new method based on AFM-QNM in combination with a neat sample preparation procedure for multiple LCN coatings on plastic substrates characterizations.

Several films with LCN coatings on flexible plastic substrates were prepared using a protocol described in Ref. 3. For the film cross-section characterizations, the films were first cut to a suitable size and subsequently carefully sandwiched between two supporting plastic sheets. The whole was then cryo-microtomed at -120°C with a LEICA EM UC7 microtome to obtain a nano-scale flat cross-sectional surface. All AFM measurements were performed on a Dimension FastScan AFM system (Bruker) AFM using high-accuracy quantitative nanomechanical mapping mode (AFM-HA-QNM) with a frequency of 0.7 Hz. The AFM tip had a spring constant of 40 N m−1 (Bruker, RTESPA-300-30). To understand the nanomechanical properties with LC coatings with different crosslinking degrees, the elastic modulus of the sample was fitted from the force curve using the Derjaguin–Muller–Toropov (DMT) model referencing a polycarbonate calibration sample with a known modulus of 2.6 GPa.

When (helix-shaped) cholesteric LCPs are oriented perpendicularly to a substrate, periodic changes of the refractive index due to the LC orientation change result in the creation of parallel planes of which the properties are described by Bragg’s law. Consequently, the pitch (p) of the helix changes due to intercalation of molecules or thermal expansion, which results in a shift of the reflection band and therefore a change in terms of optical properties. Since controlled LCN alignment is directly correlated to the optical properties of the coating, it is critical to be able to assess the alignment at different locations of the coating. Imaging the alignment of LCN can be a challenge for electron microscopy imaging due to the low material contrast difference. However, AFM-QNM high resolution imaging was able to clearly identify LCN alignment in our samples (Fig.1). Clear parallel lines in the LCN coating layer are well visualized in the images. The images also show a constant parallel alignment with the same pitch size at different locations of the LCN coating. Furthermore, the modulus and adhesion mapping images provide information on the viscoelastic properties of the LCN coating, the plastic substrates and an applied adhesion layer. The results also help assess the interface between different layers. More details of the interface characterizations on different design systems will be shown in the presentation. In short, the study demonstrates that using an adhesion layer between LCN and plastic substrates as shown in Fig. 1 can strengthen interlayer adhesion.

In the coating system shown in Figure 2, the mechanical properties such as flexibility and swelling behavior of the network play vital roles on the network’s responsive behavior. The responsiveness and color of LCNs are controlled by changing the cross-linking density and molecular weight. The designed system opens a new route to tune the functional (optical) and responsive properties with specificity and sensitivity. Here, AFM-QNM characterizations played a crucial role to indisputably prove these points, by enabling direct correlation and quantification of the relationship between coating color, cross-linking density and elastic modulus [3].

In conclusion, a novel AFM approach was developed to characterize various LCN coatings on plastic substrates. The characterizations provided valuable information on LCN alignments, adhesion to plastic substrate, and mechanical properties. Furthermore, our approach contributed to the in-depth understanding of the factors governing the stimulus-triggered response of the LCN coatings. This research therefore helped to solve the main challenges of designing stimuli-triggered, light weight and flexible LCN coatings on plastic substrates.

Figure 1 AFM-QNM images to demonstrate the LCN coating on Plastic substrate using an adhesive layer. The images are from different signal channels with (a) height image, (b) modulus mapping image, (c) adhesion mapping image. A high magnification AFM height image in the LCN coating area is shown in (d) to demonstrate the pitch size with a schematic drawing of LCN alignment. Figure 2 A) Photo of the patterned coating. The color boxes indicate the high-crosslinking (blue) and low- crosslinking (red) areas of the LCN coating areas used for AFM measurements. (b) Schematic drawing to demonstrate the pitch size changes in the reflection color shift. (C) the modulus distribution of high- crosslinking area with an inserted modulus mapping image (D) the modulus distribution of low-crosslinking area with an inserted modulus mapping image.

Keywords

AFM-QNM, Responsive Photonic Liquid Crystal Network, Flexible Plastic Substrates, modulus mapping

References

References: [1] T. J. White, D. J. Broer, Programmable and adaptive mechanics with liquid crystal polymer networks and elastomers Nat. Mater., 2015, 14, 1087. [2] E. P. A. van Heeswijk, A. J. J. Kragt, N. Grossiord, and A. P. H. J. Schenning, Environmentally-responsive photonic polymers Chem. Commun., 2019, 55, 2880. [3] E. P. A. van Heeswijk, L. Yang, N. Grossiord, A. P. H. J. Schenning, Tunable Photonic Materials via Monitoring Step-Growth Polymerization Kinetics by Structural Colors, Adv. Funct. Mater., 2020, 30, 1906833 17:34 - 17:35

59 METEOR: an integrated top down cryo-CLEM imaging system

Marit Smeets1, Anna Bieber2, Cristina Capitanio2, Oda Schioetz2, Dr. Philipp Erdmann2,3, Prof. Juergen Plitzko2 1Delmic B.V., Delft, Netherlands. 2Max Planck Institute of Biochemistry, Martinsried, Germany. 3Fondazione Human Technopole, Milan, Italy

Abstract Text

Here we present METEOR, a top down fluorescence light microscope (FLM) that enables integrated cryo correlative light and electron microscopy (cryo-CLEM) and enhances lamella sample yield for cryo electron tomography (cryo-ET). Cryo-ET is emerging as a powerful technique to acquire high-resolution 3D structures such as intracellular organelles and protein complexes in their near-native cellular environment. However, the current sample preparation method can be challenging as it often requires cryo-focussed ion beam (FIB) milling to create an electron transparent lamella and correlative light microscopy to ensure the region of interest (ROI) is present in the lamella. METEOR reduces the number of transfer steps between microscopes, protecting the fragile sample from unnecessary contamination and damage. It also allows confirmation of the presence of the ROI in the lamella during and after FIB milling. We demonstrated the abilities of METEOR by preparing targeted cryo-lamellae of Saccharomyces cerevisiae overexpressing Ede1-tagged with eGFP. Ede1 is a selective autophagy receptor and involved in the recruitment of proteins that form large condensates at the plasma membrane and within autophagic bodies1. We show that the fluorescence data from METEOR can be used to target these condensates and confirm the presence of the condensates during and after milling. By employing cryo-ET on FIB milled lamellae the Ede1 containing condensates were visualized with a high resolution by cryo-TEM (see Figure 1). METEOR will increase the sample yield of specimens that need correlative microscopy to target protein structures and make correlative cryo-ET a more accessible and high-throughput technique.

Figure 1: Correlative cryo-FIB milling and cryo-ET workflow using METEOR, demonstrated on a sample of S. cerevisiae expressing eGFP-Ede1 A-D: Maximum intensity projections (MIPs) of fluorescence Z-stacks (A,C) and ion beam views (B,D) of a lamella before milling (A,B) and after fine milling (C,D). Fluorescence Z-stacks were acquired with excitation at 484 nm, an emission filter 525/30 nm and a z step size of 400 nm. E: Overlay of the final lamella MIP on the TEM lamella overview. Arrows in A, C and E indicate the targeted fluorescent punctum where the tomogram in (F) was taken. F: A 2D slice of a tomogram acquired on the indicated position on the lamella. The target structure, a phase separated END surrounded by fenestrated ER, can be clearly distinguished from the surrounding cytosol containing ribosomes. LD: lipid droplet

Keywords cryo-CLEM, FIB/SEM, cryo-FIB/SEM, cryo-ET, cryo-lamella References

1. Wilfling, F. et al. A Selective Autophagy Pathway for Phase-Separated Endocytic Protein Deposits. Molecular Cell 80, 1–15 (2020). 17:35 - 17:36

65 Rapid (FLASH-FLIM) imaging of protoporphyrin IX in a tumour mimic in real time using a CMOS based widefield fluorescence lifetime imaging camera

Graham Hungerford1, Nathan Cumberbatch2, Adam Holland2, Kulwinder Sagoo1 1Horiba, Glasgow, United Kingdom. 2Horiba, Northampton, United Kingdom

Abstract Text

A widefield fluorescence lifetime camera making use of time-correlated single-photon counting (TCSPC) was employed to image a tissue phantom in real time. The tissue phantom consisted of gellan gum and Intralipid with an inclusion doped with Protophyrin IX (PpIX) to provide a tumour mimic. The aim was to explore the use of fluorescence lifetimes as a potential tool to demarcate tumour boundaries in fluorescence guided surgery (FGS). The camera was CMOS based, with each pixel containing a single-photon avalanche diode (SPAD) and TCSPC timing electronics. This enabled simultaneous fluorescence lifetime acquisition in each of the pixels and the ability to present a fluorescence lifetime image in real time. The identification of cancerous tissue can be subjective. The use of fluorescence, where a fluorophore can target a specific tissue and provide contrast with surrounding tissues has evolved, providing fluorescence guidance for surgical resection. Most instrumentation used in FGS relies on the intensity of the fluorescence emitted by the contrast agent, thus techniques are needed to quantify (ie standardise) the emission intensity. The fact that the fluorescence lifetime is an absolute measure, independent of fluorophore concentration, is advantageous as it can negate any calibration process. The most sensitive method to measure the fluorescence lifetime is that of TCSPC and recent developments in CMOS based technology, principally SPADs and photon timing electronics, have enabled sensors consisting of detector arrays with associated timing electronics to be fabricated [1]. A widefield approach minimises the number of moving parts (no scan module) and means that a parallel approach to data collection can be employed, with the ability to rapidly acquire data. The fluorescence from protoporphyrin IX (PpIX) has been employed to characterise cellular activity and assist in the visualisation of tumour cells. Its formation can be induced by 5-aminolevulonic acid (5-ALA) which is metabolised by tumour cells to form PpIX, where it is localised within the cells. Here PpIX was included in a construct consisting of lipid mixture, Intralipid [2] (employed to simulate fat content and optical scattering), in a gellan gum matrix and PpIX incorporated in intralipid in aqueous solution. The samples are imaged using commercial widefield TCSPC camera (Horiba Scientific FlimeraTM) based on a sensor chip with 192 x 128 pixels [3]. Each pixel contains both detection and photon timing enabling the Fluorescence Lifetime Acquisition by Simultaneous Histogramming (FLASH). This “FLASH-FLIM” approach enables widefield fluorescence lifetime images in real time to be acquired [4]. The emission of PpIX can be affected by the presence of photoproducts and aggregates [5] which can influence the monitored lifetime of the emission, especially if viewed using a longpass filter. Figure 1, measured using a Horiba Scientific DeltaFlex system, shows decay associated spectra (DAS) for PpIX in Intralipid. The presence of two emitting species was determined and means that the average lifetime will vary with emission wavelength; dependent on the relative quantities of the two emissions. Also the observed lifetime is therefore expected to be shorter than that of the monomeric form (16.5ns). Considering the whole wavelength range shown the average lifetime is close to 11 ± 3ns. Figure 1. Decay associated spectra and the average lifetime for PpIX in Intralipid.

Examining the margin of part of a PpIX labelled inclusion (region A, insert in figure 2) using the widefield FLIM camera showed that it was possible to acquire an image in a short period of time (1 second), with sufficient photons to obtain a lifetime image. As little as 200 photons events can yield lifetime data [6], however the accuracy of the lifetime determination will deteriorate with few counts and the lifetime obtained is not as large as expected. However if the purpose is to use it as a tool by which to gain contrast then its absolute value may not be of critical importance.

Figure 2. Intensity and average lifetime lifetime images of part of region A (inset) of a PpIX labelled inclusion within a gellan / Intralipid construct.

If the use of the fluorescence lifetime parameter is to be used as a tool in FGS then the ability to visualise, at a rate that would enable real time feedback to a surgeon is of importance. To assess this the sample was moved on the stage under the FLIM camera for a period of 20 seconds and the data acquired as the inclusion boundaries were traversed. A montage of the images at a rate of 6 frames per second. This rate would give sufficient real time feedback and enable sufficient photons for the lifetime parameter to be used as a tool in this instance. The outcome is shown in figure 3. Figure 3. Montage, left, (6 fps, images left to right) of a 20 second measurement moving the tissue construct under the FLIM camera so that the boundaries of the PpIX inclusion are traversed. Right comparison of change in intensity and average lifetime from each frame.

The analysis of the frames, in terms of average lifetime and change in intensity shows that a greater change towards the boundaries of the inclusion are observed using the fluorescence lifetime parameter as compared to the intensity measure (figure 3). Thus showing the potential of its usage in FGS applications. The acquisition speed using the parallel FLASH-FLIM approach shows the potential of using the fluorescence lifetime as a parameter that can distinguish the tumour margin, while giving real time feedback, in the area of fluorescence guided surgery.

Keywords

FLIM, Fluorescence guided surgery, TCSPC, Tumour phantom

References

[1] C. Bruschini et al., 2019. Light Sci. Appl. 8, 87. [2] M.Lepore and I. Del, 2019. Open Biol. J., 13, 163-172. [3] R.K. Henderson et al., 2019. IEEE J. Solid-State Circuits. 54, 1907-1916. [4] K. Sagoo et al., 2021. Methods Appl. Fluoresc. 9, 015002. [5] L. Alston et al., 2018. J. Biomed. Opt., 23, 097002. [6] D. McLoskey et al., 2011. Meas. Sci. Technol. 22, 067001. 17:36 - 17:37

96 Beam-dose controlled atomic resolution EELS mapping of beam sensitive catalyst supports.

Dr. Trung Tran, Dr. Dogan Ozkaya Johnson Matthey Technology Centre, Reading, United Kingdom

Abstract Text

Probe-corrected STEM lead to not only incoherent and high-resolution imaging of individual atomic columns but also to spectroscopic imaging based on STEM-EELS at atomic level [1]. Also, through developments made in instrumentation [2, 3], extracting signal components from spectrum images using Multivariate Statistic Analysis (MSA) [3] and formulating the physics of delocalisation and radiation damage associated with electron-electron inelastic scattering [4], spectroscopic imaging has become part of a more routine analysis. However, many of the practical atomic resolution EELS mapping experiments has been successfully demonstrated on beam-stable (less sensitive to knock-on damage) crystalline structures (usually perovskite- like). Although being established as a popular and fascinating technique, EELS atomic resolution mapping is still challenging for a range of various crystalline samples including nanoscale ones, mainly due to the stability issue. Tackling this depends on optimisation of detection and balancing between beam dose and signal/noise ratio. Here we will demonstrate that combination of experimental design and data processing (including MSA-based denoising) is necessary based on a study of signal collection efficiency and sample stability. The advantage of multiple-frame acquisition will also be demonstrated. Some of the developments such as dual-EELS [5] has made a significant difference in EELS technology and has produced maps far superior to EDX in most of modern atomic-resolution systems, not only for spatial & energy resolution but also for signal/noise even at high energy loss (above ~2000 eV) excitations. Figure 1 shows EELS mapping of Al (K-edge), O (K-edge) and Co (L-edge) in the [101] zone axis of a cobalt blue (CoAl2O4) [6] crystal. Figure 1. EELS maps of Al (green) , O (red) and Co (blue) for [101] CoAl2O4

Keywords

EELS, beam sensitive, atomic resolution

References

1. Pennycook, S. J., et al., 2009, J. Electron Microscopy, 58(3), 87. 2. Krivanek, O. L., 1992, Microsc. Microanal. Microstruct, 2, 315. 3. Watanabe, M., Kanno, M., and Okunishi, E., 2010, JEOL News, 45, 1. 4. Egerton, R. F, 2017, Ultramicroscopy, 180, 115. 5. Scott, J. et. al., 2008, Ultramicroscopy, 108, 1586. 6. The authors would like to thank Dr. Peter Ellis for a standard sample of cobalt blue. 17:37 - 17:38

136 Probing electric polarization at solid-liquid and solid-solid interfaces in van der Waals heterostructures

Ms Harriet Nevison-Andrews, Dr Pablo Ares, Mr James Dougherty, Dr Rui Wang, Dr Rene Fabregas, Dr Thomas Dufils, Dr Laura Fumagalli University of Manchester, Manchester, United Kingdom

Abstract Text

Electric polarization is a fundamental physical property, represented by the dielectric constant, or permittivity, which quantifies how materials respond to an electric field. It is of great significance when seeking to understand the properties of materials, and has major technological applications in a variety of fields, including energy storage, electronics, electrochemistry, biochemistry, micro/nanofluidics, and molecular sensing. However, our understanding of polarization has traditionally been limited to bulk systems, with sizes down to the microscale, as such systems can be probed by standard dielectric spectroscopy equipment. Probing electric polarization at the nano- and atomic scale, particularly at the interface between materials and liquids, has remained a technical challenge due to the lack of sufficiently sensitive tools.

In this work, we used advanced scanning probe methods to probe electric polarization at the atomic scale, focusing on solid/liquid and solid/solid interfaces in van der Waals (vdW) heterostructures. First, we followed up our recent study [1] in which we applied scanning dielectric microscopy [2] to nanoslits constructed from vdW crystals and filled with water [3]. We succeeded in probing the dielectric polarization properties of water confined inside slits made of various vdW crystals. Our experiments revealed an anomalously low out-of-plane polarization of water at the solid-liquid interface with vdW crystals, irrespective of the composition of the slits. Secondly, by using electrostatic force microscopy and Kelvin probe force microscopy, we probed the electric polarization properties of solid-solid interfaces in vdW heterostructures. We found that heterostructures made of twisted hBN crystals - that is, two crystals stacked at a small twist angle - show ferroelectric-like domains arranged in triangular superlattices, which we attributed to out-of-plane dipoles forming at the interface of the two crystals [4]. These findings open up interesting new avenues for the design of heterostructures with ferroelectric properties, based on vdW crystals and confined water.

Keywords

Scanning probe microscopy, scanning dielectric microscopy, electrostatic force microscopy, Kelvin probe force microscopy, 2D materials, permittivity, interfacial water, hexagonal boron nitride

References

[1] L. Fumagalli, A. Esfandiar, R. Fabregas, S. Hu, P. Ares, A. Janardanan, Q.Yang, B. Radha, T. Taniguchi, K. Watanabe, G. Gomila, K. S. Novoselov, A. K. Geim Anomalously low dielectric constant of confined water. Science 360, 1339-1342 (2018). [2] L. Fumagalli, D. Esteban-Ferrer, A. Cuervo, J.L. Carrascosa, G. Gomila Label-free identification of single dielectric nanoparticles and viruses with ultraweak polarization forces. Nature Mater. 11, 808-816 (2012). [3] B. Radha, A. Esfandiar, F. C. Wang, A. P. Rooney, K. Gopinadhan, A. Mishchenko, A. Janardanan, P. Blake, L. Fumagalli, M. Lozada-Hidalgo, S. J. Haigh, I. V. Grigorieva, H. A. Wu, A. K. Geim Designer capillaries made with atomic scale precision. Nature 538, 222 (2016). [4] C. R. Woods, P. Ares, H. Nevison-Andrews, M. J. Holwill, R. Fabregas, F. Guinea, A. K. Geim, K. S. Novoselov, N. R. Walet, L. Fumagalli Charge-polarized interfacial superlattices in marginally twisted hexagonal boron nitride Nat. Commun. 12, 347 (2021). 17:38 - 17:39

156 High-Speed AFM as a Quality Control Tool: Measuring the Roughness Variability of SiC Monofilaments for Metal Matrix Composites

Mr Dhilan Devadasan1, Mr Nathan Sutemire1,2, Dr Mark Baker1, Professor John Watts1 1University of Surrey, Guildford, United Kingdom. 2TISICS, Farnborough, United Kingdom

Abstract Text

The coating system on silicon carbide (SiC) monofilaments, manufactured using a chemical vapour deposition (CVD) process, plays an important role in the reinforcement of metal matrix composites (MMCs). The interface between the matrix and the ceramic fibre is critical for the toughening mechanisms in MMCs. However, currently there are very few methods of testing how the coating will perform in the composite prior to MMC fabrication. One hypothesis is that the coating roughness may influence the fracture mechanics at this interface and therefore, the performance of the composite. Hence, the nanoscale roughness of these monofilaments and its variability is of interest. There has been significant research comparing the roughness of fibres produced by different manufacturers or processes using AFM, however, few have investigated the variation from a single manufacturing process, particularly for quality control applications. The significant advances in image acquisition speeds over the years, in the evolution of HS-AFM from AFM, has enabled many measurements to be collected for the necessary statistical power to differentiate very similar samples. In this paper, a batch of SiC monofilaments fabricated using identical CVD processes (for use as the reinforcement in MMCs) were characterised using HS-AFM to help identify whether a correlation could be seen between the roughness and the bend strain test results. The goal of the work is to demonstrate HS-AFM as a possible quality control tool, and additionally to identify whether the roughness was related to bend strain.

A Bristol Nano Dynamics HS-AFM was used to obtain high resolution 1000 x 1000 pixel images taken at 2 frames per second using a semi-automated, unbiased method that recorded measurements every 25 μm over lengths of several millimetres, resulting in hundreds of images for each fibre. Standard third-party software (Pygwy within Gwyddion) was used to post-process and extract roughness data. The limited 5 μm frame size, relative to the large 140 μm diameter monofilaments, in combination with the relatively large features, meant that it was impossible to detect the form of the fibre. Therefore, the paper analyses and compares two methods:

1. Measuring area roughness parameters by first flattening the image to remove the background curvature (Figure 1c), retaining the 2D information of the surface; 2. Measuring line roughness from horizontal line profiles obtained from each image to remain independent of the curvature (Figure 1d), at the cost of losing a large portion of the surface information.

HS-AFM roughness data was tested to see how accurate and reliable it was (Figure 2) before being compared with the mechanical bend strain results using correlation tests. A robust method of fibre characterisation has been demonstrated through the significant increase in the confidence of roughness mean values, as a result of hundreds of measurements. This increased accuracy and repeatability signifies that HS-AFM has the potential to be a useful quality control tool for statistical measurements of fibre roughness. Bend strain results for each monofilament were obtained from the manufacturer’s in-house testing. These bend strain results had large uncertainties, and when plotted against HS-AFM roughness results, the correlation results were not totally conclusive, and further investigations are required. On the basis of the statistical treatment of large datasets from this paper, and the superior image acquisition speeds, HS-AFM has a promising future in the field of quality control in material science.

Keywords

High-Speed AFM; SiC Monofilaments; Metal Matrix Composites; Quality Control; Large Data; Statistical Measurements; Roughness

References

Vanswijgenhoven, E., Lambrinou, K., Wevers, M. & Van Der Biest, O. Comparative study of the surface roughness of Nicalon and Tyranno silicon carbide fibres. Compos. Part A Appl. Sci. Manuf. 29A, 1417–1423 (1998).

Chawla, N., Holmes, J. W. & Mansfield, J. F. Surface roughness characterization of NicalonTM and HI-NicalonTM ceramic fibers by atomic force microscopy. Mater. Charact. 35, 199–206 (1995). Wastl, D. S., Weymouth, A. J. & Giessibl, F. J. Atomically resolved graphitic surfaces in air by atomic force microscopy. ACS Nano 8, 5233–5239 (2014). Ražić, S. E., Čunko, R., Svetličić, V. & Šegota, S. Application of AFM for identification of fibre surface changes after plasma treatments. Mater. Technol.26, 146–152 (2011). Clyne, T., & Withers, P. (1993). The interfacial region. In An Introduction to Metal Matrix Composites (Cambridge Solid State Science Series, pp. 166-217). Cambridge: Cambridge University Press. doi:10.1017/CBO9780511623080.007 Rix, M. Development of Silicon Carbide Monofilaments for the Reinforcement of Metal Matrix Composites. (University of Surrey, 2018).

17:39 - 17:40

158 Using the Mesolens to observe structural changes in E. coli mature colony biofilms under different nutrient availability

Ms Beatrice Bottura, Prof. Paul Hoskisson, Prof. Gail McConnell University of Strathclyde, Glasgow, United Kingdom

Abstract Text

Escherichia coli exists in planktonic form as rod-shaped cells measuring 1 μm x 2 μm, which can autonomously aggregate in a compact and dynamic structure called a biofilm, held together by a self-secreted extracellular matrix. Biofilms grant protection from external stresses and can lead to antibiotic resistance, and, as such, are a major health concern. A deeper understanding of biofilm structure and growth is needed to tackle these issues. Bacterial growth is exponential and occurs by binary splitting, but the growth rate and final bacterial biomass depend on growth medium composition - in particular, the type and amount of nutrients available to the cells during growth (Paliy and Gunasekera 2007; Shehata and Marr 1971). Carbon (C) and nitrogen (N) are both essential nutrients for E. coli, and are best provided by glucose and ammonia respectively (Bren et al. 2016). Microbial growth requires an appropriate C/N ratio, and a deviation from this ratio can lead to dramatic changes in the chemical composition of the bacterial surface, in turn affecting bacteria’s ability to adhere to other surfaces (McEldowney & Fletcher, 1985). While biofilm morphology for various levels of nutrients has been investigated by simulations and experiments (Mimura et al. 2000, Matsushita et al. 1990), these studies only investigated the overall 2D topology of the colony, and did not include changes in internal structure at depth. We have used the Mesolens, a custom-built optical microscope with unusual combination of numerical aperture (0.47) and magnification (4x), to image the structure of whole mature colony biofilms under different nutrient conditions. The Mesolens, which can image a volume of 100 mm3 with subcellular resolution throughout, has previously been used to investigate the internal structure of E. coli biofilms (Rooney et al. 2020), revealing a network of intra-colony channels involved in nutrient uptake. Here we have extended this initial observation to understand the role of carbon and nitrogen nutrient availability in the structure and distribution of intra-colony channels. We used a non-pathogenic E. coli strain that expressed fluorescence by green fluorescent protein DNA insertion, selected by addition of the antibiotic gentamicin. Cells in mid-exponential growth phase were resuspended in M9 minimal medium with either 10 mM or 80 mM nitrogen (and nominal carbon concentration) or 10 mM or 200 mM carbon (and nominal nitrogen concentration). Cells were inoculated onto M9 agar medium with nutrient concentration matching that of the liquid culture and incubated at 37⁰C for 2 to 3 days (until they reached maturation). Mature colony images were acquired on the Mesolens in both widefield and confocal laser scanning mode, using water immersion for optimum matching of the refractive index of the specimen. Fluorescence was excited by a 490 nm LED (widefield) or a 488 nm laser (confocal), and detected by a sensor-shifting CCD camera or a PMT respectively. FIJI (Schindelin et al. 2019) was used to calculate colony diameter of up to 20 colonies for each nutrient concentration. Intra-colony channels were observed under all nutrient conditions, and filled the colony volume entirely. In general, colonies grown on minimal medium substrates were noted to have wider channels than those grown on rich medium. We hypothesise this is because bacterial cells struggle more to extract nutrients from minimal medium than from a rich medium, and hence the channels may have adapted to facilitate nutrient uptake. We found that colonies grown on nutrient-rich substrates were almost double the size than colonies grown on nutrient-deprived conditions. We measured a colony diameter of 2.07 ± 0.15 mm for 200 mM carbon (n=7 colonies) and 1.73 ± 0.17 mm for 80 mM nitrogen (n=7 colonies), while the colonies grown on low-nutrient substrates measured 1.19 ± 0.28 mm for 10 mM nitrogen (n=10 colonies) and 0.69 ± 0.28 mm for 10 mM carbon (n=20 colonies). On low carbon substrates, the colony structure differs greatly, with small, irregularly- shaped aggregates (Figure 1), or into colonies with a linear structure as long as 3 mm (Figure 2). On the other hand, substrates with nitrogen excess led to sectoring (large non-fluorescent portions) (Figure 3). Further work will confirm whether these non-fluorescent regions comprise non-viable cells or are filled with medium. Figure 1: Colony grown on carbon-deprived substrate displaying irregular shape.

Figure 2: Colony grown on carbon-deprived substrate showing linear colony morphology.

Figure 3: Colony grown on nitrogen excess substrate showing sectoring. In conclusion, mesoscale imaging allowed us to observe the phenotypical differences brought by varying nutrient concentrations, all while preserving intra-colony channel detail. The high resolution of the acquired images, together with the large volume of capture, enabled the simultaneous study of microscopic and mesoscopic features of bacterial colonies. Measuring the size of whole bacterial colonies revealed a strong dependence of colony size on nutrient availability, and at the same time we confirmed the presence of intra- colony channels at these nutrient concentrations.

Keywords

Biofilms, light microscopy, E. coli

References

1. Paliy O, Gunasekera TS. Growth of E. coli BL21 in minimal media with different gluconeogenic carbon sources and salt contents. Appl Microbiol Biotechnol. 2007 Jan;73(5):1169-72 2. Shehata TE, Marr AG. Effect of nutrient concentration on the growth of Escherichia coli. J Bacteriol. 1971 Jul;107(1):210-6. 3. Bren, A., Park, J., Towbin, B. et al. Glucose becomes one of the worst carbon sources for E.coli on poor nitrogen sources due to suboptimal levels of cAMP. Sci Rep 6, 24834 (2016) 4. S. McEldowney, M. Fletcher, Effect of growth conditions and surface characteristics of aquatic bacteria on their attachment to solid surfaces, J. Gen. Microbiol. 132 (1986) 513/523 5. Mimura, M. et al. Reaction-diffusion model of bacterial colony patterns, Phys. A. Stat. Mech. Appl. 2000, 282(1-2):283-3033 6. Matsushita, M., Fujikawa, H. Diffusion-limited growth in bacterial colony formation, Phys. A. Stat. Mech. Appl. 1990, 168(1) 7. Rooney, L.M., Amos, W.B., Hoskisson, P.A. et al. Intra-colony channels in E. coli function as a nutrient uptake system, ISME J 14, 2461–2473 (2020) 8. Schindelin, J., Arganda-Carreras, I., Frise, E. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012) 17:40 - 17:41

164 Using XANES and EELS to analyse space weathered Apollo lunar samples: Preparation for Hayabusa2 Samples

Dr Leon Hicks1, Prof. John Bridges1, Prof. Takaaki Noguchi2, Prof. Hiroshi Hidaka3, Mr Jack Piercy1 1University of Leicester, Leicester, United Kingdom. 2Kyushu University, Kyushu, Japan. 3Nagoya University, Nagoya, Japan

Abstract Text

Introduction: Airless planetary bodies with surfaces exposed to the space environment are bombarded by electrons and protons from the solar wind and cosmic rays, as well as micrometeorites, resulting in space weathering [1]. Features of space weathering include partially amorphised grain surface rims, measuring up to ~100 nm thick, containing nanophase Fe metal (npFe0) particles, vesicular blistering, and solar flare tracks [1,2]. Space weathered samples collected by the JAXA Hayabusa spacecraft from asteroid Itokawa have previously been analysed using the I14 X-ray nanoprobe beamline at Diamond Light Source synchrotron, measuring Fe-K X-ray absorption near-edge spectroscopy (XANES), and revealing an increased ferric-ferrous ratio (Fe3+/ΣFe) relative to their respective host grain mineralogy [3]. In this study, we seek to better understand the formation of space weathered lunar surface soil samples collected during the Apollo 17 mission, investigating the Fe-redox variations observed in the dominant silicate phase and the nano-grains of the space weathered rims using Fe-K XANES and EELS, with high-resolution STEM imaging. Methods/Materials: The lunar sample number is 78481,29 - a surface sample collected from the top 1 cm of trench soils at Station 8 of Apollo 17 [4]. Three FIB lift-out sections have been extracted successfully from lunar grains identified to have space weathered surfaces. Two of the lunar grains were augite pyroxene, En81Fs16 and En85Fs12, and one olivine, Fa39. Using the I14 X-ray Nanoprobe Beamline at Diamond, Fe-Kα XAS spectra are obtained from a series of XRF maps over the samples, with energies typically in the range 7000-7300 eV, with a higher energy resolution range over the XANES features (~7100-7150 eV). The XANES maps are processed using Mantis 2.3.02 [5], and isolated spectra normalized in Athena 0.8.056 [6]. By observing increasing shifts in the 1s→3d transition pre- absorption-edge peak centroid energy positions, the Fe-redox variations can be estimated between the sample host mineralogy and the space weathered zone, when compared to reference minerals of known ferric-ferrous ratio (Fe3+/ΣFe). A JEOL ARM200CF and JEOL JEM-ARM300CF instrument was used for EELS analyses and high-resolution STEM imaging respectively, at ePSIC in Diamond. EELS are performed using an accelerating voltage of 200 keV, current 15 µA, and 0.25eV/ch with a 5 mm EELS aperture, measuring linescans from the host to the space weathered zones to provide verification of the Fe-redox variation by observing the shifts in the Fe-Lα peaks.

Results: HR-STEM imaging confirmed the expected partial amorphisation and npFe0 particles (Fe0 metal confirmed observing lattice fringe spacings of ~2.06 Å) in the space weathered zones of all three lunar samples. XANES mapping was able to identify the space weathered zone separate from the host grain mineralogy, and analyses of the XANES spectra from each revealed a consistent positive shift in the 1s→3d pre-edge centroid energy positions for the space weathered zone when compared to the host. Increases of up to ~0.23 eV in the space weathered (SW) zone, compared to the substrate host mineralogy of augite or olivine, suggests an increase in ferric content in the space weathered zones up to ΔFe3+/ΣFe ~0.14 ±0.03. Positive shifts in the absorption edge positions for SW zones also support these results. A total of 18 EELS linescans measured, at various locations along the space weathered rims in all three lunar samples, also shows a consistent increased shift in the Fe-L peak energy position. A positive shift in the EELS Fe-L peak position is indicative of increased ferric content, as shown by reference minerals measured including Fe-rich olivine (Fe3+/ΣFe = 0.00) and magnetite (Fe3+/ΣFe = 0.67) with average EELS Fe-L peak positions of ~712.1 eV and ~713.6 eV respectively. Conclusion and Discussion: Fe-redox variations have been analysed using Fe-K XANES and EELS measurements with consistent results that suggest increased oxidation in the space weathered surfaces. Fe-K XANES analyses suggest oxidation increases of up to ΔFe3+/ΣFe ~0.14 ±0.03 occurring in the dominant silicate phase of the space weathered rims. These results are consistent with previously analysed space weathered asteroid Itokawa samples, which had shown increased ferric contents ranging ΔFe3+/ΣFe ~0.02-0.14 ±0.03. This is likely the result of the implanted solar wind H+ ions reacting with the segregated ferrous Fe in the surface material, where the iron disproportionation reaction (3Fe2+ → Fe0 + 2Fe3+), via the formation of water vapour, results in npFe0 condensates and oxidised Fe in the remaining partially amorphised silicate phase, as well as H2 gas released [3]. Isolating an Fe metal spectrum in the lunar sample Fe-K XANES mapping measurements, in particular analyses of the EXAFS regions of the spectra measured, and further investigation of oxidation within individual npFe particles, are future aims of this work. However, current results suggests that oxidation of silicate material exposed on the surfaces of airless bodies in the space environment is a key part of space weathering effects [1]. This work is providing useful insights for future sample return analyses, notably asteroid Ryugu and Bennu samples returned by the Hayabusa2 and OSIRIS-REx spacecrafts respectively.

Keywords

Apollo lunar; Space weathering; XANES; EELS; HR-TEM

References

[1] Pieters C. A. and Noble S. K. (2016) JGR: Planets, 121, 1865-1884. [2] Hapke B. (2001) JGR, 106, 10039- 10073. [3] Hicks L. J. et al. (2020) Meteorit. Planet., Sci. 55, 2599–2618. [4] Butler P. (1973) MSC 03211 Curator’s Catalog. pp 447. [5] Lerotic M. et al. (2014) J. Synchrotron Radiat., 21, 1206–1212. [6] Ravel B. and Newville M. (2005) J. Synchrotron Radiat., 12, 537–541. 17:41 - 17:42

171 Nanoscale chemical imaging and spectro-microscopy of engineered nanomaterials after interaction with aquatic environmental media and microorganisms

Dr. Miguel Gomez-Gonzalez1, Mr. Yunyang Wang2, Dr. Fang Xie2, Dr. Mohamed Koronfel1, Prof. Mary Ryan2, Prof. Marian Yallop3, Prof. Alexandra Porter2, Dr. Julia Parker1, Dr. Paul Quinn1 1Diamond Light Source Ltd., Didcot, United Kingdom. 2Imperial College London, London, United Kingdom. 3Bristol University, Bristol, United Kingdom

Abstract Text

X-ray-based methodologies at Synchrotron facilities are versatile characterisation techniques for a wide range of scientific fields, offering a high penetration capability with relatively low cross sections when interacting with the analysed sample, making them suitable as non-destructive imaging and characterization methods at the (sub)micrometre scale. Scanning X-ray microscopy has become a widely applicable technique for evaluating the morphology of unknown samples (e.g. phase contrast, ptychography) as well as their chemical composition (e.g. fluorescence microscopy) and speciation (e.g. X-ray absorption spectroscopy, diffraction).

The incubation of engineered nanomaterials (ENMs) within aquatic environmental media and their potential internalisation in microorganisms (e.g. algae) are research areas which have benefited from the recent advances in nano spectro-microscopy. Spatially resolved transformations and speciation changes of nanomaterials can be studied by multi-energy X-ray absorption near-edge spectroscopy (XANES) analysis. In nano-XANES, the region of interest is repeatedly imaged while the energy in the incident beam is sequentially increased over the maximum of the absorption edge of the target element. This methodology provides the ability to generate image-stacks on the fluorescence detector, which can be translated into X-ray absorption spectra at each pixel.

In this presentation, the transformation of ENMs after incubation within aquatic environmental media is described: 1) Synthetic zinc oxide (ZnO) template-growth nanorods (∼725 nm length, ∼140 nm diameter) were incubated in a range of real-world wastewater solutions. By applying in situ nano-XANES, real-time dissolution, morphological and chemical evolution of ZnO nanorods within short incubation times (1-3 hours) were observed [1] with a spatial resolution of ~100x100 nm per pixel.

2) Synthetic ceria (CeO2) nanoparticles (∼9 nm) were aged and subsequently added to the green model algal species Raphidocelis subcapitata following the OECD recommendations, at CeO2 concentrations that may be representative of hot-spots of diesel-pollution (~5 mg L-1). The algae were later fixed by applying a high pressure freezing/freeze substitution protocol and resin embedded in Quetol. Ultrathin sections (100-200 nm thickness) were analysed by nano-XANES, after screening the regions with Ce-hotspots by X-ray fluorescence mapping.

Our results revealed significant Zn- and Ce-speciation changes after incubation and interaction with organics/algae, highlighting the need for time-resolved studies of nanomaterial transformations to evaluate the impact of these transformed species at the point of exposure to microorganisms. Fundamental knowledge of how the chemistry of the individual particles change, and the heterogeneity of transformations within hydrated systems, will reveal the critical physicochemical properties determining environmental damage and deactivation of engineered nanomaterials used in consumer products.

Keywords

ZnO and CeO2 engineered nanomaterials in situ nano spectro-microscopy nano-XANES, <75nm spatial resolution ultrathin Raphidocelis subcapitata sections

References

[1] M. A. Gomez-Gonzalez et al., ACS Nano, vol. 13, no. 10, pp. 11049–11061, Oct. 2019. 17:42 - 17:43

176 Investigating the biochemistry of Alzheimer’s disease using synchrotron x- ray microscopy and spectroscopy

James Everett1,2, Jake Brooks2, Frederik Lermyte2,3, Vindy Tjendana-Tjhin2, Germán Plascencia-Villa4, Ian Hands-Portman5, Jane Donnelly2, Kharmen Billimoria2,6,7, George Perry4, Xiongwei Zhu8, Peter Sadler6, Peter O'Connor6, Joanna Collingwood2, Neil Telling1 1School of Pharmacy and Bioengineering, Keele University, Stoke-on-Trent, United Kingdom. 2School of Engineering, University of Warwick, Coventry, United Kingdom. 3Department of Chemistry, Technical University of Darmstadt, Darmstadt, Germany. 4Department of Biology and Neurosciences Institute, The University of Texas at San Antonio (UTSA), San Antonio, USA. 5School of Life Sciences, University of Warwick, Coventry, United Kingdom. 6Department of Chemistry, University of Warwick, Coventry, United Kingdom. 7LGC Ltd., Teddington, United Kingdom. 8Department of Chemistry, Case Western Reserve University, Cleveland, USA

Abstract Text

Summary: For several decades, disrupted brain metal homeostasis has been linked to the development of dementia-causing neurodegenerative disorders such as Alzheimer’s disease, which afflict millions of individuals worldwide. Despite these observations, our current knowledge of metal biochemistry in neurodegenerative disease lacks the chemical and spatial sensitivity required to understand how metals contribute to disease development. To address these shortcomings, this work employed a variety of hard and soft x-ray synchrotron techniques to characterize the metal biochemistry of Alzheimer’s brain pathologies with exceptional chemical and spatial sensitivity. These experiments demonstrated Alzheimer’s brains to harbour chemically-reactive metal forms previously undocumented in human neurobiology, opening new avenues for disease diagnosis and treatment. Introduction Metals are vital for brain well-being. However, when managed inappropriately by the brain, metals can also cause brain damage [1,2]. Brain metal imbalances have been linked to the development of numerous dementia-causing neurodegenerative diseases including Alzheimer’s (AD) and Parkinson’s [3,4], which afflict millions of individuals worldwide. Disruptions to brain metal homeostasis are commonly associated with the formation of pathological protein lesions: an example being the co-localisation of chemically-reduced and potentially toxic iron to Alzheimer’s amyloid-β pathologies [5,6]. Such metal forms are not commonplace in disease-free brains, suggesting their formation to be a result of protein lesion interaction with endogenous metals.

Metal chemistry in biological systems can vary over extremely small (< 100 nm) spatial scales [6]. Thus, to understand these systems, nanoscale resolution imaging and chemical sensitivity is essential. The current knowledge of metal biochemistry in neurodegenerative disease lacks this level of detail required to understand how metals influence disease aetiology; preventing the development of viable metal-targeting technologies for diagnosis and treatment, due to a lack of specificity [7]. To address these shortcomings, this research employed state-of-the-art synchrotron x-ray techniques to establish the nanoscale chemical composition of AD brain tissues and amyloid pathologies.

Materials and Methods Synchrotron x-ray experiments combined with electron and light microscopies were performed on isolated amyloid plaques/aggregates, cortical tissue from AD subjects, and age-matched disease-free tissue controls.

To map elemental distributions within the samples, x-ray fluorescence (XRF) mapping was performed. For broad distributions across large tissue areas, XRF mapping to a ca. 2 µm spatial resolution was conducted using Diamond Light Source (DLS) microfocus beamline I18. High resolution 100 nm XRF mapping on localized regions of interest (e.g. amyloid plaques) was performed using the hard x-ray nanoprobe on DLS beamline I14. Additional phase contrast ptychography was performed on beamline I14 to visualize nanoscale sample morphology.

To establish the chemical speciation of sample materials at ≤ 50 nm spatial resolution, soft and hard x-ray spectromicroscopy were performed using scanning transmission x-ray microscopy (STXM) at DLS beamline I08 and XANES mapping at beamline I14 respectively. X-ray absorption spectra revealing metal oxidation state and the organic (e.g. C, O and P) composition of the sample material were obtained, allowing the chemical composition of highly localised regions of interest to be determined. Magnetically-sensitive x-ray magnetic circular dichroism (XMCD) STXM measurements were also performed, enabling the magnetic characterisation of both iron compounds and elemental-metallic phases.

No dyes, contrast agents or aldehyde fixatives were used during the preparation of sample materials for synchrotron x-ray analysis, offering an unprecedented insight into the native chemistry of these biological specimens.

Results and Discussion XRF elemental mapping and STXM speciation mapping showed amyloid pathologies from AD tissues to sequester multiple metal elements (e.g. Ca, Fe, Cu, Zn), exceeding metal intensities in the surrounding neuropil where examined. Nanofocus x-ray absorption spectromicroscopy revealed dramatic nanoscale variations in metal oxidation state within the same amyloid plaques, including the discovery of chemically- reduced elemental (zero-oxidation-state) ferromagnetic iron (Fe0) (Figure 1). Metal loading within amyloid plaques corresponded with changes in amyloid organic chemistry, allowing for label-free imaging of amyloid structures against the background tissue. Furthermore, amyloid-β was shown to convert ferric (Fe3+) iron into more reactive chemically-reduced states, including Fe0, directly implicating amyloid-β in the disrupted iron homeostasis observed in AD [8].

The identification of Fe0 within AD amyloid plaques indicates that biogenic metallic elements, previously observed only in microorganisms, viruses and plants [9], can also occur in humans. The reactivity of these metallic phases differs from their metal oxide counterparts, and may redefine our understanding of metal neurochemistry and toxicity in neurodegenerative diseases. Transition metals associated with specific disease pathologies offer potential for disease diagnosis and staging, whilst chemically-reduced iron associated with amyloid structures may represent novel targets for AD therapies intended to lower metal toxicity in affected brain regions.

Conclusions These results demonstrate the power of combined synchrotron x-ray microscopy and spectroscopy to examine the biochemistry of neurodegenerative disease. The identification of metal types uniquely associated with Alzheimer’s pathology provides a step-change in our understanding of how metals contribute to AD, with potential to facilitate the development of viable therapies designed to restore metal balance in AD brains. The methodology used here can be readily applied to a myriad of biological systems, offering a new toolkit for the examination of biological materials. Figure 1. STXM examination of a human Alzheimer’s disease amyloid plaque containing metallic iron (Fe0). (a) 350 eV image showing overall plaque morphology. (b) Carbon K-edge protein map. (c) Iron L-edge map. (d) Calcium L-edge map. (e) Carbon K-edge carbonate map. (f) Oxygen K-edge carbonate map (g) Composite image showing: protein (green), calcium (blue), oxygen K-edge carbonate (sky blue), and iron (red) content of the plaque. (h) Iron L2,3-edge x-ray absorption spectra from the iron areas highlighted in (c). Data from Everett et al. Nanoscale (2018) [6].

Keywords

Synchrotron, x-rays, microscopy, spectroscopy, metals, neurobiology, neurodegenerative disease, Alzheimer's disease

References

1. A. I. Bush, Trends Neurosci. 26, 207-214 (2003); 2. J. Prousek, Pure Appl. Chem. 79, 2325-2338 (2007); 3. R. Giampietro, et al., Mol. Pharm. 15, 808-820 (2018); 4. H. Kozlowski, et al., Coord. Chem. Rev. 256, 2129-2141 (2012); 5. C. J. Maynard, et al., Int. J. Exp Pathol. 86, 147-159 (2005); 6. J. Everett, et al., Nanoscale 10, 11782- 11796 (2018); 7. M. T. Nuñez and P. Chana-Cuevas, Pharmaceuticals. 11, 109 (2018); 8. J. Everett, et al., Sci Rep. 10, 10332 (2020); 9. J. Huang, et al., Chem. Soc. Rev. 44, 6330-6374 (2015). 17:43 - 17:44

185 Single-cell live microscopy studies uncover the harmful impact of human genomic variations in a kinetochore protein, Astrin.

Dr Asifa Islam, Prof Viji Draviam Queen Mary University of London, London, United Kingdom

Abstract Text

During cell division, chromosomes are captured by microtubules via a multiprotein complex called the kinetochore. Taking advantage of single-cell live microscopy methods, we investigate the impact of human genomic variations in kinetochore proteins that can cause pregnancy loss and developmental defects such as primary microcephaly (MCPH) and cancer-susceptible disorder mosaic variegated aneuploidy (MVA). The kinetochore protein Astrin is specifically recruited to kinetochores following their attachment to microtubule- ends, and its arrival stabilizes chromosome-microtubule attachments. Human genomic variations in Astrin are known, but their impact on chromosome segregation has not been studied. We have used a combination of cell biology techniques to study the subcellular localization and cell cycle impact of Astrin variants- p.Q1012* and p.L7Qfs*21 - identified in a screen of healthy individuals and Astrin p.E755K found in cancer cells. We will present our quantitative cell biology findings that explain the occurrence of Astrin p.L7Qfs*21, but not p.Q1012, homozygotes within a healthy general population.

Keywords

Live-cell microscopy Mitosis Kinetochore Genomic variation 17:44 - 17:45

188 Cryo Soft X-ray Microscopy for Whole Cell Imaging: Progress in the Development of a Commercial Laboratory Scale Device

Kenneth Fahy1, Tony McEnroe1, Dunja Skoko1, William Fyans1, Fergal O'Reilly2,1, Paul Sheridan1 1SiriusXT, Dublin, Ireland. 2University College Dublin, Dublin, Ireland

Abstract Text

SiriusXT is developing a commercial bench-top cryo soft X-ray microscope for 3D cryo-soft X-ray tomography (cryo-SXT). Cryo-SXT uses X-rays in the ‘water window’ that extends from the K-absorption edge of carbon to the K-edge of oxygen, that is from about 282 eV (λ = 4.4 nm) to 533 eV (λ = 2.3 nm). Water is transparent to these X-rays, but organic molecules are absorbing. Therefore, these X-rays can be used as the basis for microscopy of whole cells in their near-native (frozen) state, without need for any contrast enhancing agents. A 3D tomogram with resolution between 25 nm to 60 nm (full pitch) is produced by rotating the cell over a range of angles, with an image acquired at each tilt angle [1, 2, 3]. The concept is equivalent to a medical CT scan applied at the nanoscale. Similar to Hounsfield units in medical CT, cellular organelles within the cell can be discernible from each other by their respective x-ray linear absorption coefficient values. While great progress has been made over the last two decades in developing cryo-SXT as an imaging technique on synchrotron hosted microscopes [4-7], only two laboratory soft x-ray microscopy systems have been reported so far [8-10]. The SiriusXT approach is novel insofar as no commercial lab-scale soft X-ray microscope has been available up to now, which is capable of delivering the necessary image quality and throughput required by the biomedical community. Bringing cryo-SXT capabilities to the laboratories as a table-top solution will promote development of unique flexible sample handling systems for imaging of adherent and in suspension cells, thus accelerating the establishment of integrated multiscale hybrid microscopy methods that could benefit by combining cryo- SXT with light and electron microscopy techniques [11, 12]. For example, cryo-SXT could be used as a fast pre-screening tool for EM imaging, where whole single cell SXT data is used to select the region of interest to be further imaged with electron microscopy or correlated light and electron microscopy. We will present our cryo correlative workflow in detail, including results of cryo-SXT as applied to a variety of biological specimens.

Keywords structural biology, cell imaging, microscopy, nano tomography, soft x-ray, water window, 3D cell imaging, whole cell imaging

References

[1] Schneider G, Guttmann P, Heim S et al 2010 Nature Methods 7 985-987 [2] Müller WG, Heymann JB, Nagashima K et al 2012 J Struct Biol 177 179 [3] McDermott G, Fox DM, Epperly L et al 2012 BioEssays 34 320 [4] Carrascosa JL, Chichon FJ, Pereiro E et al 2009 Journal of Structural Biology 168 234 [5] Larabell CA & Nugent KA 2010 Current Opinion in Structural Biology 20 623 [6] Schneider G, Guttman P, Heim S et al 2010 Nature Methods 7 985 [7] Carzaniga R, Domart MC, Collinson LM et al 2014 Protoplasma 251 449 [8] Kördel M, Dehlinger A, Seim C et al 2020 Optica 7 (6) 658-674 [9] Fogelqvist E, Kördel M, Carannante V et al 2017 Scientific Reports 7 [10] Legall H, Blobel G, Stiel H et al 2012 Optics Express 20 (16) 18362-18369 [11] Zeev-Ben-Mordehai T, Hagen C, Grunewald K 2014 Current Opinion in Virology 5 42-49 [12] Dent K, Hagen C, Grunewald K 2014 Critical Step-by-Step Approaches Toward Correlative Fluorescence/ Soft X-Ray Cryo-Microscopy of Adherent Mammalian Cells Methods in Cell Biology 124, 179-216 17:45 - 17:46

191 XRnanotech: Nanostructured Diffractive X-Ray Optics

Dr. Florian Döring1,2, Dr. Gergely Huszka1,2, Dr. Adam Kubec1,2 1XRnanotech, Untersiggenthal, Switzerland. 2Paul Scherrer Institut, Villigen-PSI, Switzerland

Abstract Text

X-rays offer elemental and chemical sensitivity along with high penetration depth. They represent excellent probes for research and investigation of matter. The ongoing development of accelerator-based photon sources like synchrotrons or X-ray free-electron lasers (XFELs) led to a strong increase in X-ray brilliance over the last decades and enabled ever-new experimental techniques yielding unprecedented spatial, temporal and spectral resolution. Alongside the development of X-ray sources, detector technology improved continuously and, together with the increase in computational power, now enables new scientific applications. At the same time, it was important that progress in the field of optics kept pace with this development. At XRnanotech, which is a spin-off company from the Paul Scherrer Institut in Switzerland, we develop nanostructured diffractive optical elements to enable experiments at many large-scale research facilities. Our goal is to push the limits of diffractive X-ray optics by continuously, improving the resolution and efficiency for applications in microscopy, scattering and spectroscopy. Moreover, we expand the range of applications of diffractive optics from vacuum ultraviolet to hard X-rays by exploring new fabrication materials, processes, and designs. We exploit the fact that diffractive optics have a fundamental advantage over other kinds of X-ray optics like mirrors and refractive lenses, which is the possibility to precisely control and manipulate the optical wave front. This allows realizing complex optical functionalities like efficient beam-shaping optics for transmission X-ray microscopes, spiral zone plates for generating beams with an orbital angular momentum, off axis zone plates in order to combine microscopy and spectroscopy or beam-splitting zone plates that combine focusing and beam-splitting for shot-to-shot normalization schemes at XFELs [1-5]. In this contribution, we will highlight the latest developments of diffractive optical elements at XRnanotech in terms of resolution, efficiency and optical functionality.

Fig. 1. Examples of nanostructured diffractive optical elements and components available from XRnanotech.

Keywords

X-ray optics Fresnel zone plates Diffraction gratings X-ray microscopy

References

[1] P R Ribič et al. Phys. Rev. X 24 p. 296 (2018). [2] F. Döring et al. Optica 7 (8), 1007-1014 (2020). [3] B Rösner, et al., Optics Express 25 p. 30686 (2017). [4] I Vartiainen, et al., Optics Express 23 p. 13278 (2015). [5] B.Rösner et al. Optica 7 (11), 1602-1608 (2020). 17:46 - 17:47

201 Investigation of life science samples using an annular silicon drift detector at low beam currents

Max Patzschke1, Dr Nils Schlüter2, Dr Andrew Menzies1 1Bruker Nano, Berlin, Germany. 2Museum für Naturkunde, Berlin, Germany

Abstract Text

Silicon drift detectors (SDD) have become state of the art technology in the field of EDS microanalysis. The accuracy and precision which SDDs offer is comparable or better than WDS (Ritchie et al., 2012; Cubukcu et al., 2008). Special detector designs and concepts have immensely improved the performance of SDDs and their applications to complex samples. The XFlash® FlatQUAD (Fig. 1) has been developed for applications that are limited by normal EDS, for example, shadow effects and low count rates when applying low beam currents. The XFlash® FlatQUAD is inserted between the pole piece and the sample, and ideally suited for the analysis of topographically complex samples. Shadowing effects are minimized by collecting X-rays using four separate detector segments. This detector covers a very large solid angle (1.1 sr) and allows sufficient data collection at low beam currents on beam sensitive samples. Even at beam currents in the pA range, a sample can be investigated without carbon coating under high vacuum. Compared to low vacuum analysis, this results in higher spatial resolution of the SEM images and X-ray element mappings.

In this study a sea urchin (Fig. 2) and a microbial mat of diatoms were investigated without the requirement of any sample preparation. The EDS map shows a sea urchin (Paracentrotus lividus) collected in the Aegean sea near Athens, Greece. The bumps on the right are the sockets where the urchins long spines are attached. They are being held in place by muscles and collagen so that they can be moved if the urchin wants to hide in a crevasse and they can be held rigid in defense mode, e.g. when we step on it. In the center of the micrograph individual sand grains are discernable in red. The skeleton (Figure 3) of the sea urchin is produced by biomineralization, consisting of high-magnesium calcite. The architecture of the skeleton reveals a sponge-like lightweight structure (Figure 3), which can bare extremely high weights. Such information is only possible with the annular design, avoiding shadowing and collecting highest count rates at low beam currents. For element distribution mappings of complex structures like these and other beam-sensitive life science samples, the XFlash® FlatQUAD, is unmatched.

Figure 1: The XFlash® FlatQUAD

Figure 2: Sea urchin Figure 3: Skeleton of the sea urchin

Keywords

Sea urchin, low kV, analysis, diatome, microbial mat, FlatQUAD, life science, annular silicon drift detector, shadow effects, Bruker, SDD, XFlash® FlatQUAD, ultra low beam current, SEM, EDS,

References

Ritchie et al., 2012; Cubukcu et al., 2008 17:47 - 17:48

208 Capturing the intracellular universe at near-native states and in 4D: the many uses of cryo-soft X-ray tomography for in-depth investigations of biological systems

Dr Ilias Kounatidis1, Dr Mohamed Koronfel1, Dr Archana Jadhav1, Dr Chidinma Okolo1, Mr Jeffrey Irwin2, Dr Maria Harkiolaki1 1Diamond Light Source, Didcot, United Kingdom. 2Zeiss, California, USA

Abstract Text

Summary

Cryo-Soft X-ray Tomography (cryo-SXT) is a 3D imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for sectioning, staining, or chemical modification. The power of the technique is demonstrated here through a series of current biomedical projects with an emphasis on cellular interactions and their structural responses to environmental challenges within a near-physiological context. The potential of cryo-SXT as a part of emerging advanced multimodal imaging techniques is also presented.

Introduction

Soft X-ray imaging has earned its place in the 3D high-resolution imaging hall of fame in recent years through the accumulated body of work that has given us a greater insight into the structures and processes that individual cells and cell populations employ in their maintenance and response mechanisms. Cryo-SXT allows imaging of whole cryo-preserved cells in the water-window X-ray energy range (284-543 eV) in which carbon- rich biological structures absorb X-rays more than the oxygen-rich media that surrounds them resulting in natural absorption contrast imaging through direct detection. At the UK synchrotron correlative cryo-imaging beamline B24 cryoSXT has matured into a well-documented, semi-automated, and amenable method that can generate 4D imaging data that faithfully captures spatial and temporal information within in vitro biological systems. The main technological challenges that present themselves within a facility once the microscopy physical infrastructure is optimised invariably involve applicability and in particular ease-of-access and ease-of- use. These parameters have been satisfied at B24 through the production of robust sample preparation protocols, intelligent data collection strategies and the development of user-friendly interfaces and in silico processes 1. These advances have been exploited in a number of projects of biomedical relevance. In this work, we will present, as a case study for the benefits affronted by the B24 developments in SXT, advances in our understanding of the immune response with special emphasis. The ability of cryoSXT to interface with other imaging methods and provide correlated high-content composites that cannot be attained otherwise will also be presented.

Methods/Materials

Cryo-SXT imaging at beamline B24 is performed with an UltraXRM-S220C microscope (Carl Zeiss X-ray Microscopy) following an experimental workflow based on the following sequential steps: i) culture or deposition of cells on perforated carbon film-coated EM Grids, ii) exposure of cells to biological or/and chemical triggers, iii) addition of gold nanoparticles fiducials necessary for projection alignment, v) plunge freezing with the use of a Leica EM GP model. The sample preparation is followed by: vi) mapping and samples quality evaluation with a conventional microscope (Axioimager M2, Zeiss, coupled to a Linkam cryostage), vii) loading of samples into the X-ray microscope, viii) acquisition of 2D X-ray mosaics, ix) acquisition of tilt series projections and ix) X-ray data reconstruction into 3D tomographic volumes using the IMOD software. Visualisation, analyses and correlation is achieved through a number of freely available software packages such as Fiji, ICY and Chimera. Results Understanding the structural manifestations of cellular responses and how these propagate within a population of cells is an absolute requirement when trying to understand aspects of the immune response. Here we present a select panel of projects that take us through (a) a pathogenic challenge in a cell population and two of the ways professional killer immune cells can react to clear perceived threats, (b) the production of cytotoxic substances and (c) the engulfment of pathogens. The infection process we present here involves a member of the Herpesviridae family which induces Marek’s disease in avian species. The virus propagates through cell contacts and is highly tumourogenic. We have used cryoSXT to document massive virus-induced membrane remodeling in host cells and track the progress of the virus from the nucleus of infected cells through a number of envelopment stages in the cytoplasm and finally its cross-over to healthy cells where a new infection is established. We will present new structural data that links this virus with clear cytopathic events in the host cell population. As an exemplar of the use of cryoSXT in the understanding of cytotoxic substance generation, we will present work that resulted in the discovery of a new membrane-less organelle that functions as a molecular bomb on the surface of infected cells 2. And finally, the act of close-contact clearance through phagocytosis will be documented in high-resolution 3D snapshots of primary professional macrophages responding to pathogens and exhausting themselves while ingesting perceived assailants for the benefit of their community. The correlative potential of the method will be discussed and explore through its use in existing projects that harness the power of more than one high-resolution multidimensional imaging methods such as electron tomography and hard X-ray elemental analysis. In specific a recent SARS-Cov2 project that brought together cryoSXT, serial cryoFIB/SEM volume imaging and cell lamellae-based cryo-electron tomography (cryoET) will be highlighted 3. Conclusion Here, we present a wide range of biomedical projects that have benefited from the use of cryo-SXT at the correlative cryo-imaging beamline B24. This technology is now fully commissioned, easily accessible and serving the wider biomedical community.

Keywords

Cryo-soft X-ray tomography, host-pathogen interaction, cytotoxicity, biomedical imaging, multimodal imaging

References

1. Kounatidis I, Stanifer ML, Phillips MA, Paul-Gilloteaux P, Heiligenstein X, Wang H, Okolo CA, Fish TM, Spink MC, Stuart DI, Davis I, Boulant S, Grimes JM, Dobbie IM, Harkiolaki M. 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway. Cell. 2020 Jul 23;182(2):515-530.e17. doi: 10.1016/j.cell.2020.05.051. 2. Bálint Š, Müller S, Fischer R, Kessler BM, Harkiolaki M, Valitutti S, Dustin ML. Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells. Science. 2020 May 22;368(6493):897-901. doi: 10.1126/science.aay9207. 3. Mendonca L, Howe A, Gilchrist J, Sun D, Knight M, Zanetti-Domingues L, Bateman B, Krebs AS, Chen L, Radecke J, Sheng Y, Li V, Ni T, Kounatidis I, Koronfel M, Szynkiewicz M, Harkiolaki M, Martin-Fernandez M, James W. Zhang P. Correlative Multi-scale Cryo-imaging Unveils SARS-CoV-2 Assembly and Egress. Res Sq [Preprint]. 2021 Jan 19:rs.3.rs-134794. doi: 10.21203/rs.3.rs-134794/v1. 17:48 - 17:49

229 The use of through the length scale chemical analysis in steels used in hostile environments

Mr Sharhid Jabar1, Dr Sam Marks2, Dr Geoff West1 1WMG, University of Warwick, Coventry, United Kingdom. 2Oxford Instruments Nanoanalysis, High Wycombe, United Kingdom

Abstract Text

In this work the need for a through-the-length-scale analytical approach to adequately describe the microstructure in creep strength enhanced ferritic (CSEF) steels will be shown. At the millimetre scale, micro-X- ray Fluorescence (µ-XRF) is used to quantify chemical variations due to micro-segregation effects. This enables the identification of locations with extreme composition within the steel. This is important as the material is more likely to fail in a compositionally extreme region rather than in ‘random’ or areas with an ‘average’ composition. In these targeted locations, the microscale analysis of precipitated features and inclusions was undertaken, highlighting significant differences in the area fraction and number per unit area of these features between negatively and positively segregated areas. To analyse the nanoscale, precipitation-strengthening phases, it is normal to use Transmission Electron Microscopy (TEM), however the standard ways of doing this does have significant limitations. Carbon- extraction replicas allow large areas of the sample to be analysed but are not site specific and cannot easily be referenced back to the segregation behaviour of the bulk material. Meanwhile FIB-prepared TEM lamellas are site specific but it is hard to analyse sufficiently large areas within a reasonable timeframe. To speed up sample investigations, it is highly desirable to perform as much of the analysis as possible within the SEM/FIB-SEM platform. This is achieved by utilising different available SEM modes to optimise spatial resolution, resulting in accurate high-resolution nanoscale characterisation. With respect to chemical characterisation the spatial resolution is governed by the interaction volume, a measure of electron penetration and X-ray generation. The interaction volume can be reduced to improve spatial resolution in 2 ways. The first is by utilising low accelerating voltages to perform surface sensitive Energy Dispersive Spectroscopy (EDS) with X-rays generated from a sub-surface volume < 100 nm deep. The second approach is to utilise STEM-SEM analysing an electron transparent sample that is < 100 nm thick. This greatly improves spatial resolution whilst allowing users to operate at higher accelerating voltages. Data was acquired using a windowless Oxford Instruments Extreme detector on a Zeiss Merlin SEM and a large area Oxford Instruments XmaxN 150 on a Thermo Fisher Scios. In this paper we will highlight the need for through-the-length-scale analysis to gain a full understanding of a material. The advantages and disadvantages of both high spatial resolution low kV EDS and STEM-SEM EDS are compared, Figure 1, and discussed in detail. We will also compare theoretical X-ray yields at a range of accelerating voltages, to highlight the advantages of STEM-SEM over conventional TEM EDS. Keywords

Steel, segregation, high spatial resolution EDS in SEM 17:49 - 17:50

241 The combined application of optical tweezers and advanced microscopy for examination of mutual interaction of red blood cells influenced by nano- materials

Prof. Igor Meglinski1, Miss Tatiana Avsievich2, Ms Ruixue Zhu2, Prof. Alexander Yatskovskiy3, Dr Alexander Bykov2 1Aston University, Birmingham, United Kingdom. 2University of Oulu, Oulu, Finland. 3I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation

Abstract Text

Application of red blood cells (RBC) as natural transport courier for systemic drug delivery is considered a new paradigm in modern medicine and possesses a great potential. n the frame of this paradigm, the interaction of nano- or sub-micro-particles with the RBC as a drug carrier has recently been studIn frame of this paradigm drugs, re-presented as nano- or sub-micro-particles of various volume fractions, are considered to be placed on the surface of RBC for further transportation. In this point of view ultimate understanding of mutual interaction of RBC influenced by nano-materials composed the drugs is required. Optical tweezers (OT) is widely used to explore mechanisms of cells’ interaction with the ability to trap non-invasively, manipulate and displace living cells with a notably high accuracy. In the current report, we present the results of the study of mutual interaction of RBC with various nano-particles and polymeric nano-capsules by using utilizing a two-channel OT system. Scanning electron microscopy is utilized for direct observation of nanoparticles localization on the RBC membranes. The obtained results suggest that, in the presence of nano-capsules, the RBC aggregation in plasma satisfies the ‘cross-bridges’ model. We also examined the influence of pulsed He-Ne laser radiation on the mutual RBC interaction. The combined application of OT and advanced microscopy approaches brings new insights into the conception of direct observation of cells interaction influenced by nano-materials for the estimation of possible cytotoxic effects. The experiments are performed in a platelet-free blood plasma mimicking the RBC natural environment. We show that nano-diamonds influence mutual RBC interactions more antagonistically than other nanoparticles, resulting in higher aggregation forces and the formation of larger cell aggregates. In contrast, polymeric particles do not cause anomalous RBC aggregation. The results emphasize the application of optical tweezers for the direct quantitative assessment of the mutual interaction of RBC influenced by nanomaterials.

Keywords optical tweezers, advanced microscopy, red blood cells (RBC), nano-particles, nano-capsules, scanning electron microscopy, blood aggregation

References

R. Zhu, T. Avsievich, A. Popov, A. Bykov, and I. Meglinski, “In vivo nano-biosensors of red blood cell-mediated delivery”, Biosensors & Bioelectronics, Vol.175, 112845 (2021) T. Avsievich, R. Zhu, A. Popov, A. Bykov, and I. Meglinski, “The advancement of blood cell research by optical tweezers”, Reviews in Physics, Vol.5, 100043 (2020) R. Zhu, T. Avsievich, A. Popov, and I. Meglinski, “Optical Tweezers in the Studies of Red Blood Cells”, Cells, Vol.9, No.3, 545 (2020) R.Zhu, T. Avsievich, A. Popov, A. Bykov, and I. Meglinski, “Influence of Pulsed He-Ne Laser Irradiation on the Red Blood Cell Interaction Studied by Optical Tweezers”, Micromachines, Vol.10, No.12, 853 (2019) T. Avsievich, A. Popov, A. Bykov, and I. Meglinski, “Mutual interaction of red blood cells influenced by nanoparticles”, Scientific Reports, Vol.9, 5147 (2019) T. Avsievich, A. Popov, A. Bykov, I. Meglinski, “Mutual interaction of red blood cells assessed by optical tweezers and scanning electron microscopy imaging”, Optics Letters, Vol.43, No.16, 3921 – 3924 (2018) 17:50 - 17:51

246 Experimental Solid Angle of the X-ray Perimeter Array Detector (XPAD) on the Argonne PicoProbe Analytical Electron Microscope

Nestor J. Zaluzec Photon Science Directorate, Argonne National Laboratory, Argonne, Illinois 60439, USA

Abstract Text

For the last few decades research efforts at Argonne have been focused upon the optimization of hardware and software for x-ray energy dispersive spectroscopy (XEDS) to improve the performance of microanalytical sensitivity in the Analytical Electron Microscope [1-3]. The Argonne X-ray Perimeter Array Detector (XPAD) is our latest innovation and is interfaced to the prototype ANL 30-300 kV PicoProbe Analytical Electron Microscope engineered by ThermoFisher Scientific Instruments [4]. This unique XEDS configuration consists of a linear array of detectors which judiciously circumscribes the specimen. It’s implementation is designed to maximize the collection solid angle and minimizes the penumbra and system peak effects of the instrument [5].

To measure and qualify the performance of this system we use a commercially available test specimen of nanocrystalline Ge on microporous SiNx [6]. This uniformly thick 20 nm Ge film on top of 20 nm of SiNx has an regular array of two micron holes which also facilitates accurate measurement of the relevant parameters needed to assess the functionality of the microanalytical system. In figure 1 is plotted the penumbra optimized performance of this detector measured using the Ge/SiNx test specimen at 200 kV for a holder tilt range of + 45 degrees. The experimental variation in the angle corrected intensity is ~ 10%. Non-optimized commercial holders will nearly always have greater variation.

Figure 1. ) Experimental Penumbra Profile illustrating 10% variation due to geometrical shadowing of the detector as a function of primary holder tilt. We note that the individual detectors of the array will show greater variation, however, the symmetry of the configuration mitigates this variation when all detectors are summed.

The modeled geometrical solid angle of the XPAD was calculated to be 4.47 sR. In order to confirm this we have conducted experimental measurements of the solid angle by quantitatively measuring the x-ray emission from the Ge/SiNx test specimen. Using the microporous holes in the test specimen we are able to measure the incident beam current using an in-situ Faraday cup without removing the specimen and only translating the specimen by ~ 1 micron. Figure 2 compares the normalized intensity variation for the Ge Ka emission with theoretical ionization cross-section. As expected the beam normalized intensity increases with decreasing accelerating voltage and follows the expected dependence of the relativistically corrected cross- section. A nominal 40% increase from 300 to 60 kV has been confirmed. [7-8]

Figure 2, Comparison of variation in cross-section with x-ray emission as a function of accelerating voltage (values normalized at 200 kV) . Solid line is the model, squares are experimental values.

Using our custom/optimized specimen holder an experimental measurement of the absolute intensity as a function of beam current was performed to measure the solid angle to compare with the design. To do such we utilize the relationship shown in following equation.

th here Iα = measured x-ray intensity (Integral counts) per unit area, σα(Eo, Z) = α -shell ionization cross th th section, Eo = incident electron energy, ωa = a -shell fluorescence yield, Γα = α -shell radiative partition function, Wz and CZ the atomic weight and composition, Z = atomic number, No = Avogadro’s number, ρ = local specimen density, ξo = the incident electron beam current, t = acquisition (live) time, εα = the detector energy efficiency and Ωα = the detector collection solid angle. Of all these parameters, the accuracy in our measurement of Ω is most affected by the value of the least known parameter, namely the K- shell ionization cross-section [7-8].

In table 1 we list the range of values of Ω based upon our experimental measurements conducted at 60, 200 and 300 kV. Ideally all the solid angle measurements in this table would be identical, however, owing to the uncertainity in the values of σα (Eo, Z) this is not achieved. Ω is thus evaluated for two different K-shell cross- section models to give a realistic estimate of the uncertainties. In table 1, it is clear that the experimental values for Ω are well within the range expected by the design parameters for XPAD of 4.47 sR.

Average Ge Ka Intensity Theoretical Accelerating Voltage Experimental (Integrated counts/nA- Cross-section (kV) Ω (sR) sec) (Barns) 60 21370 232.2-261.6 4.28-3.80 200 15570 157.8-182.7 4.59-3.97 300 15160 144.0-167.5 4.90-4.21

Table 1: Experimentally determined solid angle for XPAD on the ANL PicoProbe. The modeled solid angle was 4.47 sR and is within the errors of the cross-section models used herein. Further work is in progress to assess the functionality of XPAD. This work was supported by the Photon Science Directorate and Laboratory Directed Research and Development (LDRD) funding from Argonne National Laboratory, provided by the Director, as well as the Office of Science, of the U.S. Department of Energy under Contract No. DE-AC02-06CH11357. It was also supported in part by CRADA #01300701 between Argonne National Laboratory and ThermoFisher Scientific Instruments

Keywords

AEM, TEM, STEM, Microanalysis, XEDS, EDS, Spectroscopy, SDD, SiLi, X-ray, Quantitation,

References

[1] Zaluzec N.J.; (2004), “XEDS Systems for the Next Generation Analytical Electron Microscope”,Micro. Microanal., 10, S2, 122 [2] Zaluzec, N.J. (2009). “Innovative instrumentation for analysis of nanoparticles: The π steradian detector”. MicroscopyToday 17(4), 56. [3] Argonne National Laboratory. (2010). High collection efficiency X-ray spectrometer system with integrated electron beam stop, electron detector and X-ray detector for use on electron-optical beam lines and microscopes, US Patent 8,314,386, [4] Zaluzec, N.J. Microscopy & Microanalysis (2021), Pittsburg Pa, August 2021 [5] Zaluzec N.J., Wen J., Wang J. , Miller D.J.; (2016) “ Quantitative Measurements of the Penumbra of XEDS Systems in an AEM “ Microsc. Microanal. 22 (Suppl 3), 278- [6] http://www.temwindows.com/category_s/55.htm [7] Zaluzec N.J. , (1984) “K and L Shell Cross Sections for X-ray Microanalysis in an AEM”, Analytical Electron Microscopy, ed. D. Williams, San Francisco Press, 279-284, [8] Llovet, X, Powell, C.J., Salvat, F. and Joblonski A, (2014) “Cross Sections for Inner-Shell Ionization by Electron Impact, Journal of Physical and Chemical Reference Data 43, 13102 17:51 - 17:52

250 CHEMICAL DECORATION OF GRAPHENE AND 2D-MATERIALS: AN AFM OUTLOOK

Dr Vladimir Korolkov Park Systems UK Ltd, Nottingham, United Kingdom

Abstract Text

The formation of two-dimensional (2D) supramolecular arrays has been proven as a highly versatile route to the control of the spatial organization, down to the molecular scale, of the chemical functionality of a surface. These molecular networks, which can be formed through self-assembly processes on a variety of different substrates, including semiconductors, metals, insulators and layered materials, are, in almost all cases, limited to surfaces of bulk crystals. Progress towards chemical decoration of graphene and other 2D-materials, that have a thickness of an atom, has been quite limited so far. Likewise, the growth of higher layers on such materials has not even been reported. Although, similar inorganic structures – Van-der-Waals heterostructures – are a very popular research subject. Specifically, the additional functional control, which may be achieved through the formation of heterostructures realized by placing one supramolecular layer on another to result in growth into the third dimension perpendicular to the substrate, has not been widely explored for these materials. Here we describe the successful formation of heterostructures formed by the sequential growth of distinct 1D and 2D arrays on graphene. It is possible, using high-resolution atomic force microscopy (AFM), to determine an epitaxial alignment between successive layers. We chose to investigate a combination of a bicomponent hexagonal network (CAM) formed by cyanuric acid (CA) and melamine (M), and monocomponent honeycomb and linear arrays formed by, respectively, trimesic acid (TMA) and 2,6-Naphthalenedicarboxylic acid (NDA). The heterostructures are formed by first depositing a CAM monolayer that is then used as the substrate for a further deposition cycle whereby monolayers of TMA or NDA are adsorbed to form a heterostructure. The layers are deposited via sequential immersion in solutions, and we have investigated heterostructure formation on the surface of graphene. We use ambient AFM to acquire images of the molecular arrangements in adsorbed networks. This work represents a significant advance in chemical decoration of graphene and the application of AFM to imaging such networks through the acquisition of images, under ambient conditions, with sufficiently high resolution to identify the relative placement of molecules in different layers of the resulting heterostructures. In the future, we envisage that this approach to chemical decoration of graphene and building supramolecular heterostructures will provide the foundation for the growth of much more complex materials in which multiple layers can be deposited sequentially with the possibilities to tune the chemical, optical and electronic properties of the resulting organic/inorganic heterostructure. The exploitation of hydrogen bonding stabilizes the growth of 2D sheets that have highly parallel interfaces. The use of graphene as a substrate suggests that this approach can be combined with 2D materials to introduce molecular functionality into stacked device architectures, and may also provide a complete molecular analogue approach to the stacking of layered materials.

Keywords graphene, self-assembly, molecules, high resolution, supramolecular, surface, 2D-materials 17:52 - 17:53

251 X-ray Ptychography Imaging of Human Chromosomes After Low-dose Irradiation

Ms Archana Bhartiya1,2,3, Dr Darren Batey4, Dr silvia cipiccia4, Dr Xiaowen Shi5, Dr Christoph Rau4, Prof Stanley Botchway3, Dr Mohammed Yusuf1,3,6, Prof Ian Robinson1,3,7 1London Centre for Nanotechnology,University College London, London, United Kingdom. 2Department of Chemistry, University College London, London, United Kingdom. 3Research Complex at Harwell, Didcot, United Kingdom. 4Diamond Light Source, Didcot, United Kingdom. 5Department of Physics,New Mexico State University, Las Cruces, USA. 6Centre for Regenerative Medicine and Stem Cell Research,Aga Khan University, Karachi, Pakistan. 7Condensed Matter Physics and Materials Science Division, Brookhaven National Lab, New York, USA

Abstract Text

Studies into investigating the structure and composition of human chromosomes in the field of cytogenetics have spanned over several decades. In this work, we take advantage of the coherent X-rays available at the synchrotron facility at Diamond Light Source, Didcot, UK to extract individual masses (including DNA content and associated chromosomal proteins) of all 46 chromosomes. The experiments were performed, using the phase-sensitive X-ray ptychography method at the I-13 beamline. We have produced ‘X-ray karyotypes’ of unstained chromosome spreads (Fig.1) to determine the gain or loss of genetic material upon low-level X-ray irradiation doses due to radiation damage [1]. Two observations were made: first, the mass of each chromosome is 6-times higher than its theoretical DNA content. First, this suggests the presence of possible unknown nucleoproteins contributing to the excess mass. Secondly, the mass of the chromosomes is seen to increase upon low-level irradiation, suggesting the onset of the cell cycle repair mechanism. We show that the X-ray ptychography technique is useful for determining the total genome mass of species at the different stages of the cell cycle without invasive staining as well as the effects of ionizing radiation on the genome. a) b)

Figure 1: Ptychographic reconstructed unstained chromosome spread obtained from non-irradiated T cells, a) phase-retrieval image, FoV: 32 µm × 32 µm, scale bar = 10 µm, b) X-ray karyotype of the same spread, with a best-linear-fit.

[1] A. Bhartiya, D. Batey, S. Cipiccia, X. Shi, C. Rau, S.W. Botchway, M. Yusuf, and I.K. Robinson. “X-ray Ptychography Imaging of Human Chromosomes after Low-dose Irradiation”. Chromosome Research, (2021). DOI : 10.1007/s10577-021-09660-7. Keywords

X-ray microscopy, Karyotype, Irradiation, Chromosome structure, Mass determination

References

A. Bhartiya, D. Batey, S. Cipiccia, X. Shi, C. Rau, S.W. Botchway, M. Yusuf, and I.K. Robinson. “X-ray Ptychography Imaging of Human Chromosomes after Low-dose Irradiation”. Chromosome Research, (2021). DOI : 10.1007/s10577-021-09660-7. 17:53 - 17:54

268 3D multi-modal imaging of demineralised dentine using combinedscanning transmission X-ray microscopy (STXM-CT) and micro-X-ray diffraction (µ-XRD-CT) tomography techniques

Mr Nathanael Leung1, Dr Jingyi Mo1, Dr Robert Harper2, Dr Stuart Bartlett3, Dr Konstantin Ignatyev3, Dr Richard Shelton2, Prof Gabriel Landini2, Dr Tan Sui1 1University of Surrey, Guildford, United Kingdom. 2University of Birmingham, Birmingham, United Kingdom. 3Diamond Light Source, Didcot, United Kingdom

Abstract Text

Objectives Dental caries and dental erosion are common oral conditions caused by chronic exposure to acids that are bacterial or non-bacterial in origin, respectively. Continuous exposure of teeth to acid causes structural and compositional changes that can lead to the irreversible loss of dental hard tissues with potential pulpal involvement, altered vertical occlusion and poor aesthetics. Considerable efforts have been aimed at studying natural and artificial carious lesions in dentine. However, most studies have been limited to 2D analyses or were unable to achieve fine resolutions in 3D, therefore, lacking critical insights into the microstructural changes that occur during demineralisation. This investigation aimed to study the 3D changes that occur in dentine, at the lattice- and microscale, due to acid demineralisation.

Methods Combined µ-XRD-CT and STXM-CT with 2 µm resolution was used to study the crystal structure and microstructure of dentine samples with different degrees of artificial demineralisation (0 vol.%, 30 vol.%, and 50 vol.%), in comparison with dentine samples containing natural caries. XRD analysis was focussed on the (002) lattice plane, looking at the d-spacing, peak intensity and peak width to assess the changes in crystallite quality with demineralisation.

Results Reconstructions of the sample cross-sections showed distributions of crystallite size and peak intensity. The observed decrease in crystallite size and diminishing peak intensity correlated with diminishing crystallite quality as a result of demineralisation.

Conclusion Insights obtained from the morphological characterisation of the dentine demineralisation process would help understand the process and potentially aid the development of restorative dentine treatments.

Acknowledgements Diamond Light Source provided access to the I18 facilities under allocation SP23724-1. Project funded by the EPSRC project (EP/S022813/1) “Understanding and enhancing the mechanical performance of bioinspired zirconia-based dental materials” and the EPSRC PhD studentship (2119900).

Keywords

Acid demineralisation; Caries; Synchrotron X-ray diffraction tomography; Synchrotron scanning transmission tomography 17:54 - 17:55

310 Use of FIB-DIC to measure the residual stress of a SnO2:F based coating on glass

Jauffrey Lescoffit1,2, Dr Liam Dwyer2, Dr Irina Gordovskaya2, Dr Tan Sui1, Dr Mark Baker1 1University of Surrey, Guildford, United Kingdom. 2NSG Pilkington, Lathom, United Kingdom

Abstract Text

Focused ion beam – digital image correlation (FIB-DIC) is a recently introduced residual stress measurement technique which could be relevant for many industries. This work presents promising results regarding its applicability to thin-films deposited on glass substrates. Thin-films are applied on glass to improve the optical, electrical and mechanical properties of innovative products. With the evolution of coating technologies, these films now consist of stacks of layers in which residual stresses systematically arise. Assessing these stresses is challenging as they can only be measured indirectly or using diffraction techniques in crystalline materials. FIB-DIC is a residual stress measurement technique relying on focused ion beam (FIB) milling and digital image correlation (DIC). This technique could be well-suited for coatings on glass as it can be applied to measuring the residual stress of non-crystalline materials. The strain data were calculated by applying DIC analysis to a set of SEM micrographs acquired at regular intervals during the milling procedure. Relaxation curves can then be obtained by plotting the relaxation strain ε as a function of the relative milling depth h/D. A generalised form of the curves typically obtained by FIB-DIC has been determined [1] and can be fitted to the experimental data (Fig. 1). The stresses can then be calculated using the elastic constant of the coating material and Hooke’s law.

Fig. 1: Principles of the FIB-DIC residual stress measurement technique: sets of SEM images acquired during strain relaxation induced by double-slot FIB milling are analysed by DIC and the stress is calculated via Hooke’s law.

In this work, FIB-DIC was applied to a 0.4 µm-thick SnO2:F based coating on soda lime glass supplied by NSG. Double-slots were milled at the micro-scale (two 1 µm-wide slots spaced by 3 to 10 µm) using a Nova NanoLab 600 DualBeam FIB-SEM (FEI, US) with a Ga+ source operating at 30 kV and 30 pA to locally induce the stress relaxation of the as-deposited coating (Fig. 2). The film was sufficiently conductive for FIB-SEM characterisation without requiring an additional conductive coating which may have affected the stress profile. Two different DIC analysis programs were used and the results compared: an open-source MATLAB code called Digital Image Correlation and Tracking [2] and the commercially available Vic-2D (Correlated Solutions Inc., US). Fig. 2: SEM micrograph of a double-slot after complete milling through the coating. In this example, the slots consisted of two 12 µm-long and 1 µm-wide trenches spaced by 3 µm. The central rectangular area was imaged by SEM and used for DIC strain analysis. For double slots of different dimensions, DIC windows corresponding to 75% of the slot spacing D were used. In this work, each milling step corresponded to a depth of around 0.1 µm and 5 consecutive SEM images were recorded. Both DIC analysis programs gave comparable and consistent strain results for the individual milling steps (Fig. 3). As expected, the relaxation strains were found to be higher for shorter double-slot spacings. Considering the thickness of the thin film, the shortest spacing used in this work (3 µm) would allow a good approximation of the residual stress at full relaxation using FIB-DIC. Three double-slots were milled using this spacing and analysed by the two DIC programs. The recorded data were averaged for each milling step and plotted as a function of the relative milling depth h/D (Fig. 4). The relaxation strain determined from curve --3 fitting was around 4 × 10 . Assuming elastic constants of E = 230 GPa and = 0.25 for SnO 2, this corresponds to a compressive residual stress of -1.1 ± 0.1 GPa. This value is in good agreement with the range of -1.1 to -1.3 GPa typically measured for such coatings using diffraction techniques such as XRD. Fig. 3: Horizontal relaxation plotted for each SEM micrograph acquired during the milling procedure. The strains measured by DIC are very consistent for each milling step and increase for shorter spacings D between the slots.

Fig. 4: Horizontal strain plotted as a function of the milling depth and the corresponding curves fitted by least squares regression. The data shown corresponds to results from FIB-DIC applied to three separate 3 µm-spaced double-slots on the 0.4 µm-thick SnO2:F based coating on glass. FIB-DIC has been successfully applied to measure the residual stresses of a coating deposited on glass. Shorter spacings could be used during the double-slot milling to obtain a more accurate estimation of the strain at full relaxation. Other coatings should now be used to further validate the technique. More complex stacks of different layers would be particularly interesting as FIB-DIC also enables stress depth profiling. This versatile technique is very promising and will offer a better understanding of residual stresses. If a reliable protocol is tailored for non-conductive samples, it could considerably speed up the development of cutting-edge glass products.

Keywords

Residual stress, strain relaxation, focused ion beam milling, FIB, scanning electron microscopy, SEM, digital image correlation, DIC, FIB-DIC, thin film, coating, glass, FTO.

References

[1] Lord et al. (2018). A Good Practice Guide for Measuring Residual Stresses using FIB-DIC, NPL good practice guide N° 148. [2] Senn (2015). Digital Image Correlation and Tracking. https://uk.mathworks.com/matlabcentral/fileexchange/50994-digital-image-correlation-and-tracking 17:55 - 17:56

318 Noble Gas Bubbles in Thin Films

Rebecca B. Cummings1, Riccardo Bassiri2, Iain W. Martin1, Ian MacLaren1 1University of Glasgow, Glasgow, United Kingdom. 2Stanford University, Stanford, USA

Abstract Text

The noble gas content of two different thin films was quantified in a spatially resolved way using DualEELs and high angle annular dark field (HAADF) images. DualEELS is a form of electron energy loss spectroscopy (EELS) that takes two spectra, one from the low-loss region and one from the high-loss region, in quick succession. Absolute quantification of the noble gas areas is possible using the process proposed by Craven in 2016 [1].

This contribution will apply a modified version of this technique to two noble gases, argon and xenon. These two cases have applications in increasing the sensitivity of gravitational wave detectors and better understanding the behaviour of nuclear reactor cladding, respectively. The argon gas is in titanium dioxide doped tantalum pentoxide and the xenon in Zircaloy-4. The argon content in the Ta2O5 films comes from the deposition process, which is argon ion-beam deposition. For the Zircaloy-4 films the xenon was deliberately implanted using Xe-ion irradiation.

Figure 1: A map of the xenon content in one of the datasets of the Zircaloy-4 sample. The xenon is visible in the lighter regions and the intensity shows the number of xenon atoms in the columns represented by the pixels. The small scale demonstrates the sensitivitity of the technique. The colourmap is inferno from matplotlib [2].

Tantalum pentoxide is used as an optical coating for mirrors in gravitational wave detectors. Fully understanding the structure of the coatings is the first step towards reducing their noise contributions, thus improving the sensitivity of the detector. Lower noise will increase the sensitivity of the detector. Xenon bubbles in the cladding of nuclear reactors are well documented; xenon is a fission product and the bubbles are formed after it implants into the cladding as ions and then captures electrons. The sample investigated was made by a process that directly mimics the that which occurs within reactors. On a small scale, such as in these samples, the bubbles can affect the optical and physical properties of the films; on a large scale, such as in a reactor, they may weaken the reactor cladding.

Three Ta2O5 films were investigated to see if there was a relationship between annealing temperature and bubble formation. It was shown using HAADF imaging that after annealing at 400 ºC the argon in the film started to coalesce into bubbles and after annealing at 600°C these bubbles grew larger and more defined. A semi-empirical standard was created for the quantification using argon data from the EELS Atlas [3] and experimental data scaled using a Hartree Slater cross-section. The density and pressure of argon within the bubbles was calculated for 35 bubbles in the 600 °C sample. The pressures were calculated using the Van der Waals equation. The bubbles had a mean diameter, density and pressure of 22 Å, 870 kg/m3 and 400 MPa, respectively. The bubbles are thus a high-density liquid [4].

The same spatially resolved quantification technique was then applied to the Zircaloy-4 film. It was once again shown that xenon was present in the film using HAADF images, and that it had grouped into bubbles. A semi- empirical standard was made using the EELS Atlas [3] and experimental data that was scaled using a Hartree Slater cross-section. The densities and pressures of 244 xenon bubbles were calculated, the pressures using the appropriate form of Jelea's equation of state for xenon/krypton mixtures confined in nuclear fuels [5]. On average, the xenon bubbles had a diameter of 21.4 Å, a density of 2333 kg/m3 and a pressure of 113 GPa.

This spatially resolved quantification technique is capable of isolating a very small fraction of the total EELS signal and using it to calculate the densities and pressures of nanoscale bubbles. It can be readily applied to various materials that contain small gas bubbles, so long as an EELS edge in the right energy range is present for the gas of interest.

Keywords

EELS, thin films, mirror coatings, noble gas bubbles, electron microscopy

References

[1] A. J. Craven, J. Bobynko, B. Sala, and I. MacLaren, “Accurate measurement of ab- solute experimental inelastic mean free paths and EELS differential cross-sections,” Ultramicroscopy, vol. 170, pp. 113 – 127, 2016. [2] J. D. Hunter, “Matplotlib: A 2D graphics environment,” Computing in Science & Engineering, vol. 9, no. 3, pp. 90–95, 2007. [3] C. Ahn and O. Krivanek, EELS Atlas: a reference guide of electron energy loss spectra covering all stable elements. Gatan, 1983. [4] R. B. Cummings, R. Bassiri, I. W. Martin, and I. MacLaren, “Argon bubble formation in tantalum oxide-based films for gravitational wave interferometer mirrors,” Opt. Mater. Express, vol. 11, pp. 707–718, Mar 2021. [5] A. Jelea, “An equation of state for xenon/krypton mixtures confined in the nuclear fuels,” Journal of Nuclear Materials, vol. 530, p. 151952, 2020. 17:56 - 17:57

322 FLIMbow: a multicolor multi-lifetime labeling method for individual cells and cell lineages

Vasilisa Polinovskaya1, Igor Kramarev1, Dmitry Gorbachev1, Ilya Solovyev2, Alexander Savitsky2, Maria Lukina3, Lyubov Shimolina3, Vadim Elagin3, Marina Shirmanova3, Konstantin Lukyanov1 1Skolkovo Institute of Science and Technology, Moscow, Russian Federation. 2A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation. 3Privolzhsky Research Medical University, Nizhny Novgorod, Russian Federation

Abstract Text

Brainbow and the related multicolor cell-labeling methods is an elegant strategy that has found numerous applications in the following research areas: cell lineages tracking in embryology and stem cell biology, connectome mapping in neuroscience, monitoring of tumor growth in cancer biology, and so on [1]–[3]. Brainbow allows for permanent markings (labeling) of target cells in multiple distinct colors by combining and expressing 3-4 fluorescent proteins (FPs) at different ratios in each cell. In general, Brainbow with three FPs can generate a palette reaching up to 100 colors. However, it is almost impossible to distinguish between the close hues and increase their due to natural experimental especially in in vivo research. Moreover, each FP occupies the corresponding fluorescence channel, leaving no spectral space for marking any objects of interest. We developed the FLIMbow method, which expanded the palette of labels in a new dimension, using two independent parameters to distinguish objects: not only the color of the fluorescence but also the lifetime. We visualized in the Fluorescence Lifetime Imaging Microscopy (FLIM) mode cells stochastically expressing FPs of a similar spectrum, but with different lifetimes. We have revealed that these cells have distinguishable pseudo colors, similar to the effect in spectral Brainbow, but only within one fluorescent channel. We have shown that lentiviral vector-mediated multi-lifetime marking remained stable after cell division, thus allowing the analysis of clonal cell fates in vitro and in vivo (Fig.1). Expansion of the pool of fluorescent proteins into two extra fluorescent channels increases the amount of information about each labeled cell, which in turn fine-tunes the accuracy of determining the cell to a particular clone. As proof of principle, we studied tumor development in mice. FLIMbow multi-parameter marking technology has shown both quantitative and qualitative superiority over the original and related Brainbow methods.

Figure 1. Hela cells labeled with green FLIMbow lentiviruses, сell clones retain their pseudo-colored labels during division.

Keywords

FLIM, Brainbow, multiparameter imaging, cell lineage

References [1] J. Livet et al., “Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system,” Nature, vol. 450, no. 7166, pp. 56–62, 2007, doi: 10.1038/nature06293. [2] K. Weber et al., “RGB marking facilitates multicolor clonal cell tracking ” Nat. Med., vol. 17, no. 4, pp. 504–509, 2011, doi: 10.1038/nm.2338. [3] S. Lamprecht et al., “Multicolor lineage tracing reveals clonal architecture and dynamics in colon cancer,” Nat. Commun., vol. 8, no. 1, pp. 1–8, 2017, doi: 10.1038/s41467-017-00976-9. 17:57 - 17:58

324 STGM studies of charge symmetry in the geometrically modulated Seebeck coefficient in encapsulated graphene nanoconstrictions

Miss Eli Castanon1,2, Dr Matthew Hamer3, Mr Andrew Niblett2, Mr Sergio Gonzalez2, Dr Johanna Zultak4, Dr Roman Gorbachev5, Dr Olga Kazakova1, Prof. Oleg Kolosov2 1National Physical Laboratory, London, United Kingdom. 2Lancaster University, Lancaster, United Kingdom. 3National Graphene Institute, Manchester, United Kingdom. 4National graphene institute, Manchester, United Kingdom. 5National Graphene institute, Manchester, United Kingdom

Abstract Text

The thermoelectric (TE) applications of graphene have generated great interest due to its extraordinary electronic and thermal properties, gate-controlled ambipolar behaviour, and competitive Seebeck coefficient (). In recent studies, the effects of nanostructuring on the local variations of the Seebeck coefficient have been explored for bare graphene samples with patterned bow-tie constrictions[1], and mono- and bi-layer junctions[2]. These represent a new paradigm in the control of the TE properties on the nanoscale, enabling the development of single metal thermocouples[3] for temperature sensing and coolers for thermal load distribution and hot-spot removal with nanoscale dimensions. Here, we study the spatial distribution of the local Seebeck domains via the thermoelectric voltage in encapsulated graphene devices with patterned constrictions. This novel approach explores two different strategies to improve the signal and control of the S domains: (1) the enhancement of the Seebeck coefficient by encapsulation, gate carrier control, and temperature gradient control; and (2) the creation of local S domains by patterning constrictions of varying geometry and size as shown in figure 1(a). To study the response of the devices, maps of the thermovoltage with nanoscale resolution were created using scanning thermal gate microscopy (STGM), a novel SPM mode in which a hot tip is employed as the local heating source and scanned over the sample in open circuit configuration, thus creating thermovoltage signal that is proportional to the Seebeck coefficient. A schematic representation of the STGM is depicted in figure 1(b). The resulting thermovoltage maps were acquired for different gate voltages and different temperature gradients between the tip and sample. In figure 1(c) and (d), maps acquired for p- and n-doped graphene, respectively, are presented. Local TE junctions with an opposite gradient of Seebeck coefficient are formed across the device. Furthermore, there is a clear almost perfect inversion of the TE effect sign with the inversion of the charge carriers. One-dimensional profiles performed across the devices (see figure 1(e)) show that the highest Seebeck coefficient gradient occurs in the central rectangular constriction due to changes of the Seebeck coefficient in this area as well as in the half-bowtie bottom constriction. At these locations the electron mean free path (EMFP) would be reduced, leading to the observed sign change in S gradient. In this study, we have demonstrated the formation of the local Seebeck domains TE junctions in graphene devices with nanopatterned constrictions of varying geometries. Means to control the intensity and sign of the thermoelectric signal have also been shown. The combination of these solutions could lead to effective thermal management in electronic graphene devices, and the development of important applications such as single material thermocouples or coolers at the nanoscale. We also demonstrate the viability of STGM as a novel visualisation and characterisation tool able to provide much higher resolution than conventional optical methods for the characterisation of local TE properties. Figure 1. Nanoscale thermoelectric characterisation of encapsulated graphene devices. (a) Schematic of the experimental set-up for STGM. The probe acts as a local heater, while the thermovoltage generated on the graphene layer is measured through the Au contacts, highlighted in yellow in the figure. (b) Topography of the device. Scale bar is . The patterned constrictions are surrounded by yellow, red and light blue squares, and shown in more detail on the right side of the topography map, highlighting the varying geometries and sizes. Representative thermovoltage maps for (c) p-doped graphene and (d) n-doped graphene, acquired at low temperature , show the presence of local domains of opposite thermovoltage signal and thus, opposite Seebeck coefficient. Furthermore, opposite polarity of the Seebeck coefficient is achieved by changing the dominant charge carrier in graphene as highlighted when comparing the images in (c) and (d). (e) Profiles comparing the thermovoltage response for n-doped and p-doped graphene along the dotted grey line shown in (c). The authors gracefully acknowledge support of Graphene Flagship core 3 and EPSRC HiWiN grant EP/V00767X/1.

Keywords

Graphene, scanning thermal gate microscopy, nanoscale thermal properties, local Seebeck coefficient References

[1] A. Harzheim, J. Spiece, C. Evangeli, E. McCann, V. Falko, Y. Sheng, J.H. Warner, G.A.D. Briggs, J.A. Mol, P. Gehring, O. V. Kolosov, Geometrically Enhanced Thermoelectric Effects in Graphene Nanoconstrictions, Nano Lett. 18 (2018) 7719–7725. https://doi.org/10.1021/acs.nanolett.8b03406. [2] A. Harzheim, C. Evangeli, O. V. Kolosov, P. Gehring, Direct mapping of local Seebeck coefficient in 2D material nanostructures via scanning thermal gate microscopy, 2D Mater. 7 (2020) 041004. https://doi.org/10.1088/2053-1583/aba333. [3] A. Harzheim, F. Könemann, B. Gotsmann, H. van der Zant, P. Gehring, Single-Material Graphene Thermocouples, Adv. Funct. Mater. 30 (2020) 1–5. https://doi.org/10.1002/adfm.202000574. 17:58 - 17:59

334 Scanning thermal microscopy of 2D materials in various environments

Dr. Khushboo Agarwal1, Sergio Gonzalez Munoz1, Eli Castanon2, Andy Niblett1, Prof. Oleg Kolosov1 1Lancaster University, Lancaster, United Kingdom. 2National Physical Laboratory, London, United Kingdom

Abstract Text

Scanning thermal microscopy of 2D materials in various environments

K. Agarwal, S. G. Munoz, E. Castanon, A. Niblett, and O. V. Kolosov

Physics Department, Lancaster University, Lancaster, LA1 4YB, UK

Abstract: The understanding of thermal transport at nanoscale level opens up new pathways in upgrading the efficiencies of nanostructured devices and materials. In addition, thin films with highly anisotropic thermal conductivities offer high potential for thermal management of the present day electronics [1]. Apart from deploying anisotropic materials in manufacturing processes, it is extremely difficult and challenging to measure and analyze even the basic thermophysical properties of materials. Scanning Thermal Microscopy (SThM) provides versatile approach for measurements of thermal conductivity of the materials and devices for various spatial geometries [2]. Whereas most of SThM studies are performed in ambient conditions in air and at room temperature (RT), these measurements suffer from spurious effects of the through-the-air heat transport and do not allow investigating the nanoscale thermal transport in the various temperatures of the sample.

Fig 1. Inse flake on Si substrate (a) Topography, (b) SThM in air at RT, (c) SThM in HV at RT, and (d) SThM in HV at cryogenic temperature (~170K). In the present study, we have performed SThM measurements of the thermal conductance in exfoliated InSe and graphene layers of thickness from few atomic layers to quasi bulk materials, in air as well as high vacuum (HV) of 10^-7 torr. The room temperature results for in air and HV measurements were compared and the effect of heat conductance in air were analyzed in detail. The HV measurements were performed both at room temperatures as well as at cryogenic temperatures to understand the thermal transport mechanisms. InSe is one of the novel van der Waals materials that promises high performance as future thermoelectrics, used often in combination with graphene electrodes. An analytical model was used to deconvolute the values of contact resistances and thermal resistances. The temperature dependent thermal conductance values for variable thickness of InSe samples gave insight into the thermal transport mechanism and possibility of its use as an active thermoelectric material. This new approach allowed us to measure anisotropic thermal conductivity and interfacial thermal conductance of the InSe thin films exfoliated on different substrates which can further be utilized to understand the interfacial thermal conductance in nanocomposites and supperlattice structures paving new routes for enhanced thermoelectric performances.

The authors acknowledge the support of Graphene Flagship core 3 grant to Lancaster University and HiWiN EP/V00767X/1 EPSRC funding.

Keywords

Thermal Transport, 2D materials, SThM, High vacuum, Cryogenic

References

1. Shi, L., et al., Evaluating Broader Impacts of Nanoscale Thermal Transport Research. Nanoscale and Microscale Thermophysical Engineering, 2015. 19(2): p. 127-165. 2. El Sachat, A., et al., Heat Transport Control and Thermal Characterization of Low-Dimensional Materials: A Review. Nanomaterials, 2021. 11(1): p. 33.