I13-2-Diamond Manchester Imaging
|
Abstract: Cell behaviour and tissue development are inherently sensitive to morphological features of tissue-engineered scaffolds. Traditionally, imaging techniques such as SEM, TEM, AFM, and CLSM provide high-resolution 2D images to characterise scaffold morphology. However, these techniques have poor penetration and low resolution transversely to the sliced planes. In contrast, synchrotron radiation X-ray micro-computed tomography (SR-µCT) enables 3-D imaging of large volumes with submicron isotropic resolution.
We used SR-µCT at beamline I13-2 (Diamond Light Source) to image jet-sprayed nonwoven fibrous scaffolds used in the Harefield Valve, both with and without human adipose-derived stem cells preserved in ethanol to maintain native wet conditions. Large-volume imaging was achieved by stitching 2x2 tiled datasets and reconstructing them into 1 mm³ volumes at 0.325 µm voxel size, enabling clear scaffold.
The scaffold exhibited a layered, transversely isotropic structure, with additional in-plane anisotropy observed when using high-speed drum fabrication. SR-µCT revealed significantly higher scaffold porosity compared to SEM analysis, which consistently underestimates porosity due to limited depth and connectivity information. Cell distribution and morphology showed that cells preferentially adhered and proliferated along in-plane structures at full scaffold colonisation. We hypothesise that the cells minimise energy expenditure by expanding in directions of least resistance.
|
Sep 2025
|
|
B16-Test Beamline
|
Matthew
Donoghue
,
Hongchang
Wang
,
Daniel
O'Toole
,
Charles E.
Connelly
,
Shahd
Horie
,
Peter
Woulfe
,
Cornelio
Salinas
,
Brid
King
,
Brendan
Tuohy
,
Evan
Kiely
,
Kazimir
Wanelik
,
Kawal
Sawhney
,
Christoph
Kleefeld
Diamond Proposal Number(s):
[24649]
Open Access
Abstract: The study of biological soft tissue structures at the micron scale details the function of healthy and pathological tissues, which is vital in the diagnosis and treatment of diseases. Speckle based X-ray phase contrast tomographic scans at a nanometer scale have the potential to thoroughly analyse such tissues in a quantitative and qualitative manner. Diamond light source, the UKs national synchrotron facility developed and refined a 1-D X-ray speckle-based imaging technique, referred to as Fly scan mode. This novel image acquisition technique was used to perform a rapid structural composition scan of rodent lung histology samples. The rodent samples were taken from healthy and Staphylococcus aureus induced acute respiratory distress syndrome models. The analysis and cross comparison of the fly scan method, absorption-based tomography and conventional histopathology H&E staining microscopy are discussed in this paper. This analysis and cross comparison outline the ways the speckle-based technique can be of benefit. These advantages include improved soft tissue contrast, 3-D volumetric rendering, segmentation of specific gross tissue structures, quantitative analysis of gross tissue volume. A further advantage is the analysis of cellular distribution throughout the volumetric rendering of the tissue sample. The study also details the current limitations of this technique and points to ways in which future work on this imaging modality may progress.
|
Oct 2024
|
|
I13-2-Diamond Manchester Imaging
|
Carlos
Navarrete-Leon
,
P. Stephen
Patrick
,
Adam
Doherty
,
Harry
Allan
,
Silvia
Cipiccia
,
Shashidhara
Marathe
,
Kaz
Wanelik
,
Michela
Esposito
,
Charlotte K.
Hagen
,
Alessandro
Olivo
,
Marco
Endrizzi
Diamond Proposal Number(s):
[30748]
Open Access
Abstract: Two-directional beam-tracking (2DBT) is a method for phase-contrast imaging and tomography that uses an intensity modulator to structure the X-ray beam into an array of independent circular beamlets that are resolved by a high-resolution detector. It features isotropic spatial resolution, provides two-dimensional phase sensitivity, and enables the three-dimensional reconstructions of the refractive index decrement, δ, and the attenuation coefficient, μ. In this work, the angular sensitivity and the spatial resolution of 2DBT images in a synchrotron-based implementation is reported. In its best configuration, angular sensitivities of ∼20 nrad and spatial resolution of at least 6.25 µm in phase-contrast images were obtained. Exemplar application to the three-dimensional imaging of soft tissue samples, including a mouse liver and a decellularized porcine dermis, is also demonstrated.
|
Sep 2024
|
|
I13-1-Coherence
|
Diamond Proposal Number(s):
[19194, 21309]
Open Access
Abstract: We apply X-ray ptycho-tomography to perform high-resolution, non-destructive, three-dimensional (3D) imaging of Fe-rich inclusions in paleomagnetically relevant materials (zircon single crystals from the Bishop Tuff ignimbrite). Correlative imaging using quantum diamond magnetic microscopy combined with X-ray fluorescence mapping was used to locate regions containing potential ferromagnetic remanence carriers. Ptycho-tomographic reconstructions with voxel sizes 85 nm and 21 nm were achievable across a field-of-view > 80 µm; voxel sizes as small as 5 nm were achievable over a limited field-of-view using local ptycho-tomography. Fe-rich inclusions 300 nm in size were clearly resolved. We estimate that particles as small as 100 nm—approaching single-domain threshold for magnetite—could be resolvable using this “dual-mode” methodology. Fe-rich inclusions (likely magnetite) are closely associated with apatite inclusions that have no visible connection to the exterior surface of the zircon (e.g., via intersecting cracks). There is no evidence of radiation damage, alteration, recrystallisation or deformation in the host zircon or apatite that could provide alternative pathways for Fe infiltration, indicating that magnetite and apatite grew separately as primary phases in the magma, that magnetite adhered to the surfaces of the apatite, and that the magnetite-coated apatite was then encapsulated as primary inclusions within the growing zircon. Rarer examples of Fe-rich inclusions entirely encapsulated by zircon are also observed. These observations support the presence of primary inclusions in relatively young and pristine zircon crystals. Combining magnetic and tomography results we deduce the presence of magnetic carriers that are in the optimal size range for carrying strong and stable paleomagnetic signals but that remain below the detection limits of even the highest-resolution X-ray tomography reconstructions. We recommend the use of focused ion beam nanotomography and/or correlative transmission electron microscopy to directly confirm the presence of primary magnetite in the sub 300 nm range as a necessary step in targeted paleomagnetic workflows.
|
Mar 2024
|
|
I13-2-Diamond Manchester Imaging
|
Diamond Proposal Number(s):
[29256]
Open Access
Abstract: High-resolution spatial and temporal analysis and 3D visualization of time-dependent processes, such as human dental enamel acid demineralization, often present a challenging task. Overcoming this challenge often requires the development of special methods. Dental caries remains one of the most important oral diseases that involves the demineralization of hard dental tissues as a consequence of acid production by oral bacteria. Enamel has a hierarchically organized architecture that extends down to the nanostructural level and requires high resolution to study its evolution in detail. Enamel demineralization is a dynamic process that is best investigated with the help of in situ experiments. In previous studies, synchrotron tomography was applied to study the 3D enamel structure at certain time points (time-lapse tomography). Here, another distinct approach to time-evolving tomography studies is presented, whereby the sample image is reconstructed as it undergoes continuous rotation over a virtually unlimited angular range. The resulting (single) data set contains the data for multiple (potentially overlapping) intermediate tomograms that can be extracted and analyzed as desired using time-stepping selection of data subsets from the continuous fly-scan recording. One of the advantages of this approach is that it reduces the amount of time required to collect an equivalent number of single tomograms. Another advantage is that the nominal time step between successive reconstructions can be significantly reduced. We applied this approach to the study of acidic enamel demineralization and observed the progression of demineralization over time steps significantly smaller than the total acquisition time of a single tomogram, with a voxel size smaller than 0.5 μm. It is expected that the approach presented in this paper can be useful for high-resolution studies of other dynamic processes and for assessing small structural modifications in evolving hierarchical materials.
|
Feb 2024
|
|
E01-JEM ARM 200CF
I08-1-Soft X-ray Ptychography
I13-2-Diamond Manchester Imaging
I14-Hard X-ray Nanoprobe
I18-Microfocus Spectroscopy
|
Cyril
Besnard
,
Ali
Marie
,
Sisini
Sasidharan
,
Petr
Buček
,
Jessica M.
Walker
,
Julia E.
Parker
,
Matthew C.
Spink
,
Robert A.
Harper
,
Shashidhara
Marathe
,
Kaz
Wanelik
,
Thomas E. J.
Moxham
,
Enrico
Salvati
,
Konstantin
Ignatyev
,
Michal M.
Klosowski
,
Richard M.
Shelton
,
Gabriel
Landini
,
Alexander M.
Korsunsky
Diamond Proposal Number(s):
[27749, 30684, 30691, 31005, 29256, 23873]
Open Access
Abstract: Caries, a major global disease associated with dental enamel demineralization, remains insufficiently understood to devise effective prevention or minimally invasive treatment. Understanding the ultrastructural changes in enamel is hampered by a lack of nanoscale characterization of the chemical spatial distributions within the dental tissue. This leads to the requirement to develop techniques based on various characterization methods. The purpose of the present study is to demonstrate the strength of analytic methods using a correlative technique on a single sample of human dental enamel as a specific case study to test the accuracy of techniques to compare regions in enamel. The science of the different techniques is integrated to genuinely study the enamel. The hierarchical structures within carious tissue were mapped using the combination of focused ion beam scanning electron microscopy with synchrotron X-ray tomography. The chemical changes were studied using scanning X-ray fluorescence (XRF) and X-ray wide-angle and small-angle scattering using a beam size below 80 nm for ångström and nanometer length scales. The analysis of XRF intensity gradients revealed subtle variations of Ca intensity in carious samples in comparison with those of normal mature enamel. In addition, the pathways for enamel rod demineralization were studied using X-ray ptychography. The results show the chemical and structural modification in carious enamel with differing locations. These results reinforce the need for multi-modal approaches to nanoscale analysis in complex hierarchically structured materials to interpret the changes of materials. The approach establishes a meticulous correlative characterization platform for the analysis of biomineralized tissues at the nanoscale, which adds confidence in the interpretation of the results and time-saving imaging techniques. The protocol demonstrated here using the dental tissue sample can be applied to other samples for statistical study and the investigation of nanoscale structural changes. The information gathered from the combination of methods could not be obtained with traditional individual techniques.
|
Jul 2023
|
|
I13-2-Diamond Manchester Imaging
|
Diamond Proposal Number(s):
[28574]
Open Access
Abstract: We present a flyscan compatible acquisition scheme for three-modal X-Ray Computed Tomography (CT) with two-dimensional phase sensitivity. Our approach is demonstrated using a “beam tracking” setup, through which a sample’s attenuation, phase (refraction) and scattering properties can be measured from a single frame, providing three complementary contrast channels. Up to now, such setups required the sample to be stepped at each rotation angle to sample signals at an adequate rate, to prevent resolution losses, anisotropic resolution, and under-sampling artefacts. However, the need for stepping necessitated a step-and-shoot implementation, which is affected by motors’ overheads and increases the total scan time. By contrast, our proposed scheme, by which continuous horizontal and vertical translations of the sample are integrated with its rotation (leading to a “cycloidal-spiral” trajectory), is fully compatible with continuous scanning (flyscans). This leads to greatly reduced scan times while largely preserving image quality and isotropic resolution.
|
Dec 2022
|
|
E01-JEM ARM 200CF
I13-2-Diamond Manchester Imaging
|
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.
|
Jun 2022
|
|
I13-2-Diamond Manchester Imaging
|
Diamond Proposal Number(s):
[21081, 24233]
Abstract: Soft porous matter is commonly encountered in artificial tissue applications, pharmaceuticals delivery systems and in cosmetic and food products. These materials are typically opaque and tend to deform under very small levels of shear; this makes the characterization of their microstructure very challenging, particularly in the native state. Air-in-oil systems (oleofoams) are an emerging type of soft material with promising applications in cosmetics and foods, which contain air bubbles stabilized by Pickering fat crystals dispersed in a liquid oil phase. Synchrotron radiation X-ray computed tomography (SR - XCT) is a non-invasive, non-destructive technique increasingly used to investigate multiphasic, porous materials, owing to its high flux which enables sub-micron resolution and significant statistics at rapid acquisition speed. While the penetration of high energy X-rays can provide high resolution images and allows the reconstruction of the 3D structure of samples, the experimental setup and measuring parameters need to be carefully designed to avoid sample deformation or beam damage.
In this work, a robust methodology for investigating the 3D microstructure of soft, porous matter was developed. Sample preparation and experimental setup were chosen to allow synchrotron tomographic analysis of soft oleofoams with a low melting point (<30 °C). In particular, the use of cryogenic conditions (plunge-freeze in liquid nitrogen) provided stability against melting during the acquisition. Additionally, an image processing workflow was designed for analysing the 3D microstructure of the samples using ImageJ. Hence, the size and shape distribution of the air phase, as well as the thickness of the continuous gel phase could be determined for samples with significantly different microstructures (fresh vs. heated). Furthermore, the use of time-resolved X-ray radiography (XRR) allowed to study dynamic changes in the microstructure of the samples during thermal destabilization, visualizing bubble coalescence and growth in optically opaque foam samples with a sub-second timescale.
|
Aug 2021
|
|
I13-2-Diamond Manchester Imaging
|
Diamond Proposal Number(s):
[12538, 14907, 23866]
Open Access
Abstract: Thousands of soft tissue microtomography experiments are conducted at synchrotrons around the world each year, and the quality of results varies widely. Soft biological tissues pose a particular challenge for synchrotron tomography, owing to poor contrast and susceptibility to deformation and beam damage artefacts. The rationale behind the choice of sample preparation methods, imaging parameters and reconstruction strategy is not always reported in articles, and so we conducted a systematic investigation of these aspects of experimental design for central nervous system samples. Computational segmentation can be particularly challenging for soft-tissue tomograms, and so we demonstrate the use of supervoxel-based machine-learning segmentation of our data.
|
Jul 2021
|
|
