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Instruments / Technical Area	Publication Details	Published
E01-JEM ARM 200CF	Trainable segmentation for transmission electron microscope images of inorganic nanoparticles Cameron G. Bell , Kevin P. Treder , Judy S. Kim , Manfred E. Schuster , Angus I. Kirkland , Thomas J. A. Slater Journal Of Microscopy DOI: 10.1111/jmi.13110 Diamond Proposal Number(s): [26559] Show Abstract ▼ Abstract: We present a trainable segmentation method implemented within the python package ParticleSpy. The method takes user labelled pixels, which are used to train a classifier and segment images of inorganic nanoparticles from transmission electron microscope images. This implementation is based on the trainable Waikato Environment for Knowledge Analysis (WEKA) segmentation, but is written in python, allowing a large degree of flexibility and meaning it can be easily expanded using other python packages. We find that trainable segmentation offers better accuracy than global or local thresholding methods and requires as few as 100 user-labelled pixels to produce an accurate segmentation. Trainable segmentation presents a balance of accuracy and training time between global/local thresholding and neural networks, when used on transmission electron microscope images of nanoparticles. We also quantitatively investigate the effectiveness of the components of trainable segmentation, its filter kernels and classifiers, in order to demonstrate the use cases for the different filter kernels in ParticleSpy and the most accurate classifiers for different data types. A set of filter kernels is identified that are effective in distinguishing particles from background but that retain dissimilar features. In terms of classifiers, we find that different classifiers perform optimally for different image contrast; specifically, a Random Forest classifier performs best for high-contrast ADF images, but that QDA and Gaussian Naïve Bayes classifiers perform better for low-contrast TEM images.	May 2022
	Analysing neutron radiation damage in YBa2Cu3O7-x high temperature superconductor tapes Y. Linden , W. R. Iliffe , G. He , M. Danaie , D. X. Fischer , M. Eisterer , S. C. Speller , C. R. M. Grovenor Journal Of Microscopy DOI: 10.1111/jmi.13078 Show Abstract ▼ Abstract: Superconducting windings will be necessary in future fusion reactors to generate the strong magnetic fields needed to confine the plasma, and these superconducting materials will inevitably be exposed to neutron damage. It is known that this exposure results in the creation of isolated damage cascades, but the presence of these defects alone is not sufficient to explain the degradation of macroscopic superconducting properties and a quantitative method is needed to assess the subtle lattice damage in between the clusters. We have studied REBCO coated conductors irradiated with neutrons to a cumulative dose of 3.3×1022 n*m−2 that show a degradation of both Tc and Jc values, and use HRTEM analysis to show that this irradiation introduces ∼10 nm amorphous collision cascades. In addition we introduce a new method for the analysis of these images to quantify the degree of lattice disorder in the apparently perfect matrix between these cascades. This method utilises Fast Fourier and Discrete Cosine Transformations of a statistically-relevant number of HRTEM images of pristine, neutron-irradiated, and amorphous samples, and extracts the degree of randomness in terms of entropy values. Our results show that these entropy values in both mid-frequency band FFT and DCT domains correlate with the expected level of lattice damage, with the pristine samples having the lowest and the fully amorphous regions the highest entropy values. Our methodology allows us to quantify ‘invisible’ lattice damage to and correlate these values to the degradation of superconducting properties, and also has relevance for a wider range of applications in the field of electron microscopy where small changes in lattice perfection need to be measured.	Dec 2021
B24-Cryo Soft X-ray Tomography	Correlative imaging using super‐resolution fluorescence microscopy and soft X‐ray tomography at cryogenic temperatures provides a new way to assess virosome solutions for vaccine development Chidinma A. Okolo , Archana Jadhav , Patrick Phillips , Maud Dumoux , Amanda A. Mcmurray , Vishwas D. Joshi , Claire Pizzey , Maria Harkiolaki Journal Of Microscopy DOI: 10.1111/jmi.13054 Diamond Proposal Number(s): [24622] Open Access Show Abstract ▼ Abstract: Active Virosomes (AVs) are derivatives of viruses, broadly similar to ‘parent’ pathogens, with an outer envelope that contains a bespoke genome coding for 4–5 viral proteins capable of eliciting an antigenic response. AVs are essentially novel vaccine formulations that present on their surface selected viral proteins as antigens. Once administered, they elicit an initial “anti-viral” immune response. AVs are also internalised by host cells where their cargo viral genes are used to express viral antigen(s) intracellularly. These can then be transported to the host cell surface resulting in a second wave of antigen exposure and a more potent immuno-stimulation. A new 3D correlative microscopy approach is used here to provide a robust analytical method for characterisation of Zika and Chikungunya-derivatised AV populations including vesicle size distribution and variations in antigen loading. Manufactured batches were compared to assess the extent and nature of batch-to-batch variations. We also show preliminary results that verify antigen expression on the surface of host cells. We present here a reliable and efficient high-resolution 3D imaging regime that allows the evaluation of the microstructure and biochemistry of novel vaccine formulations such as AVs.	Aug 2021
	Correlative X‐ray and neutron tomography of root systems using cadmium fiducial markers T. Clark , G. Burca , R. Boardman , T. Blumensath Journal Of Microscopy 55 DOI: 10.1111/jmi.12831 Show Abstract ▼ Abstract: The interactions between plant roots and soil are an area of active research, particularly in terms of water and nutrient uptake. Because noninvasive, in vivo studies are required, tomographic imaging appears an obvious method to use, but no one imaging modality is well suited to capture the complete system. X‐ray imaging gives clear insight to soil structure and composition; however, water is comparatively transparent to X‐rays and biological matter also displays poor contrast with respect to the pores between soil particles. Neutron imaging presents a complementary view where water and biological matter are better distinguished but the soil minerals are not imaged as clearly as they would be with X‐rays. This work aims to develop robust methods for complementary X‐ray/neutron tomographic imaging of plant root samples which should lead to new insight into water and nutrient transport in soil. The key challenges of this project are to develop experiments that will meet the requirements of both imaging modalities as well as the biological requirements of the plant samples and to develop ways to register a pair of reconstructed volume images of a sample that will typically have been produced with entirely separate facilities. The use of cadmium fiducial markers for registration has been investigated. Simulations were conducted to investigate the expected registration accuracy as the quantity and distribution of the markers varied. The findings of these simulations were then tested experimentally as plant samples were grown and imaged using neutrons with the IMAT instrument at ISIS Neutron and Muon Source at the STFC Rutherford Appleton Laboratory in Harwell, and with X‐rays at µ‐VIS X‐ray Imaging Centre at the University of Southampton.	Sep 2019
I13-2-Diamond Manchester Imaging	Imaging techniques for observing laminar geometry in the feather shaft cortex Christian Laurent , Sharif Ahmed , Richard Boardman , Richard Cook , Gareth Dyke , Colin Palmer , Philipp Schneider , Roland De Kat Journal Of Microscopy DOI: 10.1111/jmi.12820 Show Abstract ▼ Abstract: Bird feather shafts are light, stiff, and strong, but the fine details of how their structure, mechanics and function relate to one another remains poorly understood. The missing piece in our understanding may be the various fibrous layers that make up the shaft's cortex. Detailed imaging techniques are needed to enable us to capture, analyse and quantify these layers before we can begin to unravel the relationship between their structure, mechanics and function. We show that Serial‐Block‐Face scanning electron microscopy, scanning confocal polarised microscopy and synchrotron‐based computed tomography are three suitable techniques to investigate layer thickness and fibre orientation in the feather cortex. These techniques and other are discussed in terms of their ability to resolve the fibrous laminar structure of the feather cortex, on sample preparation, and on throughput. Annotated images are presented for each and less suitable techniques are presented in the supplement.	Jun 2019
I13-2-Diamond Manchester Imaging	Optimization of digital volume correlation computation in SR-microCT images of trabecular bone and bone-biomaterial systems M. Peña Fernández , A. H. Barber , G. W. Blunn , G. Tozzi Journal Of Microscopy 373 DOI: 10.1111/jmi.12745 Diamond Proposal Number(s): [14080] Show Abstract ▼ Abstract: A micromechanical characterization of biomaterials for bone tissue engineering is essential to understand the quality of the newly regenerated bone, enabling the improvement of tissue regeneration strategies. A combination of microcomputed tomography in conjunction with in situ mechanical testing and digital volume correlation (DVC) has become a powerful technique to investigate the internal deformation of bone structure at a range of dimensional scales. However, in order to obtain accurate three‐dimensional strain measurement at tissue level, high‐resolution images must be acquired, and displacement/strain measurement uncertainties evaluated. The aim of this study was to optimize imaging parameters, image postprocessing and DVC settings to enhance computation based on ‘zero‐strain’ repeated high‐resolution synchrotron microCT scans of trabecular bone and bone‐biomaterial systems. Low exposures to SR X‐ray radiation were required to minimize irradiation‐induced tissue damage, resulting in the need of advanced three‐dimensional filters on the reconstructed images to reduce DVC‐measured strain errors. Furthermore, the computation of strain values only in the hard phase (i.e. bone, biomaterial) allowed the exclusion of large artefacts localized in the bone marrow. This study demonstrated the suitability of a local DVC approach based on synchrotron microCT images to investigate the micromechanics of trabecular bone and bone‐biomaterial composites at tissue level with a standard deviation of the errors in the region of 100 microstrain after a thorough optimization of DVC computation.	Jul 2018
I13-2-Diamond Manchester Imaging	A review of techniques for visualising soft tissue microstructure deformation and quantifying strain Ex Vivo C. M. Disney , P. D. Lee , J. A. Hoyland , M. J. Sherratt , B. K. Bay Journal Of Microscopy 35 DOI: 10.1111/jmi.12701 Diamond Proposal Number(s): [12776] Show Abstract ▼ Abstract: Many biological tissues have a complex hierarchical structure allowing them to function under demanding physiological loading conditions. Structural changes caused by ageing or disease can lead to loss of mechanical function. Therefore, it is necessary to characterise tissue structure to understand normal tissue function and the progression of disease. Ideally intact native tissues should be imaged in 3D and under physiological loading conditions. The current published in situ imaging methodologies demonstrate a compromise between imaging limitations and maintaining the samples native mechanical function. This review gives an overview of in situ imaging techniques used to visualise microstructural deformation of soft tissue, including three case studies of different tissues (tendon, intervertebral disc and artery). Some of the imaging techniques restricted analysis to observational mechanics or discrete strain measurement from invasive markers. Full‐field local surface strain measurement has been achieved using digital image correlation. Volumetric strain fields have successfully been quantified from in situ X‐ray microtomography (micro‐CT) studies of bone using digital volume correlation but not in soft tissue due to low X‐ray transmission contrast. With the latest developments in micro‐CT showing in‐line phase contrast capability to resolve native soft tissue microstructure, there is potential for future soft tissue mechanics research where 3D local strain can be quantified. These methods will provide information on the local 3D micromechanical environment experienced by cells in healthy, aged and diseased tissues. It is hoped that future applications of in situ imaging techniques will impact positively on the design and testing of potential tissue replacements or regenerative therapies.	Apr 2018
	An optical configuration for fastidious STEM detector calibration and the effect of the objective-lens pre-field L. Jones , A. Varambhia , H. Sawada , P. D. Nellist Journal Of Microscopy 180 DOI: 10.1111/jmi.12672 Show Abstract ▼ Abstract: In the scanning transmission electron microscope, an accurate knowledge of detector collection angles is paramount in order to quantify signals on an absolute scale. Here we present an optical configuration designed for the accurate measurement of collection angles for both image-detectors and energy-loss spectrometers. By deflecting a parallel electron beam, carefully calibrated using a diffraction pattern from a known material, we can directly observe the projection-distortion in the post-specimen lenses of probe-corrected instruments, the 3-fold caustic when an image-corrector is fitted, and any misalignment of imaging detectors or spectrometer apertures. We also discuss for the first time, the effect that higher-order aberrations in the objective-lens pre-field has on such an angle-based detector mapping procedure.	Jan 2018
	Biological applications of cryo-soft X-ray tomography E. Duke , K. Dent , M. Razi , L. M. Collinson Journal Of Microscopy 255 (2), 65-70 DOI: 10.1111/jmi.12139 PMID: 24942982 Show Abstract ▼ Abstract: X-rays are used for imaging many different types of biological specimen, ranging from live organisms to the individual cells and proteins from which they are made. The level of detail achieved as a result of the imaging varies depending on both the sample and the technique used. One of the most recent technical developments in X-ray imaging is that of the soft X-ray microscope, designed to allow the internal structure of individual biological cells to be explored. With a field of view of ∼10–20 × ∼10–20 μm, a penetration depth of ∼10 μm and a resolution of ∼40 nm3, the soft X-ray microscope neatly fits between the imaging capabilities of light and electron microscopes.	Jun 2014
B16-Test Beamline	Region-of-interest tomography using filtered backprojection: assessing the practical limits Albrecht Kyrieleis , Valeriy Titarenko , Thomas Connolley , Mark Ibison , Philip Withers Journal Of Microscopy DOI: 10.1111/j.1365-2818.2010.03408.x PMID: 21118206 Diamond Proposal Number(s): [486] Show Abstract ▼ Abstract: There are many cases where it is desirable to reconstruct at high resolution a small volume from a larger sample. Here we describe the outcomes of a reconstruction trial based on real samples aimed at delineating the practical limits to which a small region of interest can be viewed from a large sample. Our approach has been to artificially truncate the sinograms of whole sample scans to simulate region of interest tomography. A simple filtered back projection algorithm has been applied, with the sinograms extended laterally in a simple manner to make up for the truncated portions. The impact of the degree of truncation (from 0% down to 99%), the number of projections used, as well as the position of the region of interest, on the faithfulness of the reconstruction is evaluated for a range of sample types. We have assessed the nature of, and extent to which, artefacts are introduced and the degree to which simple strategies can minimize them to an acceptable level without the need for complex reconstruction algorithms, projection stitching strategies or very large detectors. It is found that for a wide range of objects the effect of truncation on feature detection is negligible and that excellent images can be reconstructed if the number of projections is calculated not on the basis of the number of pixels on the camera, but on the number of pixels that would be required to scan the whole sample at the chosen pixel resolution. This paper demonstrates that in many cases more sophisticated reconstruction strategies are not necessary.	Jun 2010

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