Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.
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Jul 2022
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E01-JEM ARM 200CF
I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[29256, 30666]
Open Access
Abstract: Dental caries is a widespread disease that damages teeth by heterogeneous dissolution. Conventional histology identifies different zones within carious lesions by their optical appearance, but fails to quantify the underlying nanoscale structural changes as a function of specific location, impeding better understanding of the demineralisation process. We employ detailed collocative analysis using different imaging modalities, resolutions and fields of view. Focused ion beam-scanning electron microscopy (FIB-SEM) reveals subsurface 3D nanostructure within milled micro-sized volumes, whilst X-ray tomography allows less destructive 3D imaging over large volumes. Correlative combination of these techniques reveals fine detail of enamel rods, inter-rod substance, sheaths, crystallites and voids as a function of location. The degree of enamel demineralisation within the body of the lesion, near its front, and at the surface is visualized in 3D. We thus establish the paradigm of dental 3D nano-histology as an advanced platform for quantitative evaluation of caries-induced structural modification.
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Jun 2022
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[20274]
Open Access
Abstract: Integrating physiology with structural insights of the same neuronal circuit provides a unique approach to understanding how the mammalian brain computes information. However, combining the techniques that provide both streams of data represents an experimental challenge. When studying glomerular column circuits in the mouse olfactory bulb, this approach involves e.g., recording the neuronal activity with in vivo 2-photon (2P) calcium imaging, retrieving the circuit structure with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) and/or serial block-face scanning electron microscopy (SBEM) and correlating these datasets. Sample preparation and dataset correlation are two key bottlenecks in this correlative workflow. Here, we first quantify the occurrence of different artefacts when staining tissue slices with heavy metals to generate X-ray or electron contrast. We report improvements in the staining procedure, ultimately achieving perfect staining in ∼67% of the 0.6 mm thick olfactory bulb slices that were previously imaged in vivo with 2P. Secondly, we characterise the accuracy of the spatial correlation between functional and structural datasets. We demonstrate that direct, single-cell precise correlation between in vivo 2P and SXRT tissue volumes is possible and as reliable as correlating between 2P and SBEM. Altogether, these results pave the way for experiments that require retrieving physiology, circuit structure and synaptic signatures in targeted regions. These correlative function-structure studies will bring a more complete understanding of mammalian olfactory processing across spatial scales and time.
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Jun 2022
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I12-JEEP: Joint Engineering, Environmental and Processing
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Fabio
Arzilli
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Margherita
Polacci
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Giuseppe
La Spina
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Nolwenn
Le Gall
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Edward W.
Llewellin
,
Richard A.
Brooker
,
Rafael
Torres-Orozco
,
Danilo
Di Genova
,
David A.
Neave
,
Margaret E.
Hartley
,
Heidy M.
Mader
,
Daniele
Giordano
,
Robert
Atwood
,
Peter D.
Lee
,
Florian
Heidelbach
,
Mike R.
Burton
Diamond Proposal Number(s):
[16188]
Open Access
Abstract: The majority of basaltic magmas stall in the Earth’s crust as a result of the rheological evolution caused by crystallization during transport. However, the relationships between crystallinity, rheology and eruptibility remain uncertain because it is difficult to observe dynamic magma crystallization in real time. Here, we present in-situ 4D data for crystal growth kinetics and the textural evolution of pyroxene during crystallization of trachybasaltic magmas in high-temperature experiments under water-saturated conditions at crustal pressures. We observe dendritic growth of pyroxene on initially euhedral cores, and a surprisingly rapid increase in crystal fraction and aspect ratio at undercooling ≥30 °C. Rapid dendritic crystallization favours a rheological transition from Newtonian to non-Newtonian behaviour within minutes. We use a numerical model to quantify the impact of rapid dendritic crystallization on basaltic dike propagation, and demonstrate its dramatic effect on magma mobility and eruptibility. Our results provide insights into the processes that control whether intrusions lead to eruption or not.
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Jun 2022
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[16205]
Open Access
Abstract: Gas bubble in aquatic sediments has a significant effect on its geophysical and geomechanical properties. Recent studies have shown that methane gas and hydrate can coexist in gas hydrate–bearing sediments. Accurate calibration and understanding of the fundamental processes regarding such coexisting gas bubble dynamics is essential for geophysical characterization and hazard mitigation. We conducted high-resolution synchrotron imaging of methane hydrate formation from methane gas in water-saturated sand. While previous hydrate synchrotron imaging has focused on hydrate evolution, here we focus on the gas bubble dynamics. We used a novel semantic segmentation technique based on convolutional neural networks to observe bubble dynamics before and during hydrate formation. Our results show that bubbles change shape and size even before hydrate formation. Hydrate forms on the outer surface of the bubbles, leading to reduction in bubble size, connectivity of bubbles, and the development of nano-to micro-sized bubbles. Interestingly, methane gas bubble size does not monotonously decrease with hydrate formation; rather, we observe some bubbles being completely used up during hydrate formation, while bubbles originate from hydrates in other parts. This indicates the dynamic nature of gas and hydrate formation. We also used an effective medium model including gas bubble resonance effects to study how these bubble sizes affect the geophysical properties. Gas bubble resonance modeling for field or experimental data generally considers an average or equivalent bubble size. We use synchrotron imaging data to extract individual gas bubble volumes and equivalent spherical radii from the segmented images and implement this into the rock physics model. Our modeling results show that using actual bubble size distribution has a different effect on the geophysical properties compared to the using mean and median bubble size distributions. Our imaging and modeling studies show that the existence of these small gas bubbles of a specific size range, compared to a bigger bubble of equivalent volume, may give rise to significant uncertainties in the geophysical inversion of gas quantification.
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Jun 2022
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[26307]
Open Access
Abstract: X-ray computed tomography (XCT) is regularly employed in geomechanics to non-destructively measure the solid and pore fractions of soil and rock from reconstructed 3D images. With the increasing availability of high-resolution XCT imaging systems, researchers now seek to measure microfabric parameters such as the number and area of interparticle contacts, which can then be used to inform soil behaviour modelling techniques. However, recent research has evidenced that conventional image processing methods consistently overestimate the number and area of interparticle contacts, mainly due to acquisition-driven image artefacts. The present study seeks to address this issue by systematically assessing the role of XCT acquisition parameters in the accurate detection of interparticle contacts. To this end, synchrotron XCT has been applied to a hexagonal close-packed arrangement of glass pellets with and without a prescribed separation between lattice layers. Different values for the number of projections, exposure time, and rotation range have been evaluated. Conventional global grey value thresholding and novel U-Net segmentation methods have been assessed, followed by local refinements at the presumptive contacts, as per recently proposed contact detection routines. The effect of the different acquisition set-ups and segmentation techniques on contact detection performance is presented and discussed, and optimised workflows are proposed.
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May 2022
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[22346]
Open Access
Abstract: Friction surfacing is an emerging solid-state coating technology based on frictional heat induced plastic deformation at the tip of a consumable metallic stud that allows to deposit layers with a fine-grained recrystallized microstructure at temperatures below the melting point. The generation of sound, defect-free metallurgical joints between multiple adjacent overlapping friction surfacing deposits, also referred to as multi-track friction surfacing, from dissimilar aluminum alloys is the focus of this experimental work. An extensive volumetric defect analysis is carried out for various overlap configurations, including post-processing strategies in order to assess the inter-track bonding integrity using microscopic characterization techniques and micro-computed tomography. The effect of layer arrangement and overlap distance on the volumetric defect formation in both inter-track and layer-to-substrate interface is quantified and discussed. Post-processing via hybrid friction diffusion bonding process demonstrates a significant reduction in defect volume ratio, proving higher material efficiency. The gained knowledge was used to successfully build a multi-track multi-layer friction surfacing stack, demonstrating the suitability of this process for large-scale additive manufacturing components. The subsequent mechanical analysis reveals excellent homogeneous isotropic tensile properties of the additive structure in the range of the base material tensile strength.
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May 2022
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[18925, 19958, 21485, 23508]
Open Access
Abstract: Cryo-soft-X-ray tomography is being increasingly used in biological research to study the morphology of cellular compartments and how they change in response to different stimuli, such as viral infections. Segmentation of these compartments is limited by time-consuming manual tools or machine learning algorithms that require extensive time and effort to train. Here we describe Contour, a new, easy-to-use, highly automated segmentation tool that enables accelerated segmentation of tomograms to delineate distinct cellular compartments. Using Contour, cellular structures can be segmented based on their projection intensity and geometrical width by applying a threshold range to the image and excluding noise smaller in width than the cellular compartments of interest. This method is less laborious and less prone to errors from human judgement than current tools that require features to be manually traced, and it does not require training datasets as would machine-learning driven segmentation. We show that high-contrast compartments such as mitochondria, lipid droplets, and features at the cell surface can be easily segmented with this technique in the context of investigating herpes simplex virus 1 infection. Contour can extract geometric measurements from 3D segmented volumes, providing a new method to quantitate cryo-soft-X-ray tomography data. Contour can be freely downloaded at github.com/kamallouisnahas/Contour.
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May 2022
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[10315, 16497, 22575]
Open Access
Abstract: Musculoskeletal tissues are complex hierarchical materials where mechanical response is linked to structural and material properties at different dimensional levels. Therefore, high-resolution three-dimensional tomography is very useful for assessing tissue properties at different scales. In particular, Synchrotron Radiation micro-Computed Tomography (SR-microCT) has been used in several applications to analyze the structure of bone and biomaterials. In the past decade the development of digital volume correlation (DVC) algorithms applied to SR-microCT images and its combination with in situ mechanical testing (four-dimensional imaging) have allowed researchers to visualise, for the first time, the deformation of bone tissue and its interaction with biomaterials under different loading scenarios. However, there are several experimental challenges that make these measurements difficult and at high risk of failure. Challenges relate to sample preparation, imaging parameters, loading setup, accumulated tissue damage for multiple tomographic acquisitions, reconstruction methods and data processing. Considering that access to SR-microCT facilities is usually associated with bidding processes and long waiting times, the failure of these experiments could notably slow down the advancement of this research area and reduce its impact. Many of the experimental failures can be avoided with increased experience in performing the tests and better guidelines for preparation and execution of these complex experiments; publication of negative results could help interested researchers to avoid recurring mistakes. Therefore, the goal of this article is to highlight the potential and pitfalls in the design and execution of in situ SR-microCT experiments, involving multiple scans, of musculoskeletal tissues for the assessment of their structural and/or mechanical properties. The advice and guidelines that follow should improve the success rate of this type of experiment, allowing the community to reach higher impact more efficiently.
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May 2022
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I13-2-Diamond Manchester Imaging
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Carles
Bosch
,
Tobias
Ackels
,
Alexandra
Pacureanu
,
Yuxin
Zhang
,
Christopher J.
Peddie
,
Manuel
Berning
,
Norman
Rzepka
,
Marie-Christine
Zdora
,
Isabell
Whiteley
,
Malte
Storm
,
Anne
Bonnin
,
Christoph
Rau
,
Troy
Margrie
,
Lucy
Collinson
,
Andreas T.
Schaefer
Diamond Proposal Number(s):
[20274]
Open Access
Abstract: Understanding the function of biological tissues requires a coordinated study of physiology and structure, exploring volumes that contain complete functional units at a detail that resolves the relevant features. Here, we introduce an approach to address this challenge: Mouse brain tissue sections containing a region where function was recorded using in vivo 2-photon calcium imaging were stained, dehydrated, resin-embedded and imaged with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT). SXRT provided context at subcellular detail, and could be followed by targeted acquisition of multiple volumes using serial block-face electron microscopy (SBEM). In the olfactory bulb, combining SXRT and SBEM enabled disambiguation of in vivo-assigned regions of interest. In the hippocampus, we found that superficial pyramidal neurons in CA1a displayed a larger density of spine apparati than deeper ones. Altogether, this approach can enable a functional and structural investigation of subcellular features in the context of cells and tissues.
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May 2022
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