The Dual Imaging and Diffraction(DIAD)beamline at Diamond Light Source(Didcot,U.K.)implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using c...The Dual Imaging and Diffraction(DIAD)beamline at Diamond Light Source(Didcot,U.K.)implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using complementary X-ray modalities to reveal structural detail at various length scales.Namely,the underlying beamline principle and its practical implementation allow the collocation of chosen regions within the sample and their interrogation using real-space imaging(radiography and tomography)and reciprocal space scattering(diffraction).The switching between the two principal modes is made smooth and rapid by design,so that the data collected is interlaced to obtain near-simultaneous multimodal characterization.Different specific photon energies are used for each mode,and the interlacing of acquisition steps allows conducting static and dynamic experiments.Building on the demonstrated realization of this state-of-the-art approach requires further refining of the experimental practice,namely,the methods for gauge volume collocation under different modes of beam−sample interaction.To address this challenge,experiments were conducted at DIAD devoted to the study of human dental enamel,a hierarchical structure composed of hydroxyapatite mineral nanocrystals,as a static sample previously affected by dental caries(tooth decay)as well as under dynamic conditions simulating the process of acid demineralization.Collocation and correlation were achieved between WAXS(wide-angle X-ray scattering),2D(radiographic),and 3D(tomographic)imaging.While X-ray imaging in 2D or 3D modes reveals real-space details of the sample microstructure,X-ray scattering data for each gauge volume provided statistical nanoscale and ultrastructural polycrystal reciprocal-space information such as phase and preferred orientation(texture).Careful registration of the gauge volume positions recorded during the scans allowed direct covisualization of the data from two modalities.Diffraction gauge volumes were identified and visualized within the tomographic data sets,revealing the underlying local information to support the interpretation of the diffraction patterns.The present implementation of the 4D microscopy paradigm allowed following the progression of demineralization and its correlation with time-dependent WAXS pattern evolution in an approach that is transferable to other material systems.展开更多
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 require...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 timestepping 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 0.5μm.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.展开更多
To reflect human development,it is critical to create a substrate that can support long-term cell survival,differentiation,and maturation.Hydrogels are promising materials for 3D cultures.However,a bulk structure cons...To reflect human development,it is critical to create a substrate that can support long-term cell survival,differentiation,and maturation.Hydrogels are promising materials for 3D cultures.However,a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction.Herein,granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell(hiPSC)-derived neural networks.A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules.Cells and hydrogel granules were combined using a weaker secondary gelation step,forming self-supporting cell laden scaffolds.At three and seven days,granular scaffolds supported higher cell viability compared to bulk hydrogels,whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions(65.52±11.59μm)after seven days compared to bulk hydrogels(22.90±4.70μm).Long-term(three-month)cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold.This approach is significant as it provides a simple,rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.展开更多
基金funded by The Engineering and Physical Sciences Research Council(EPSRC)entitled“Tackling human dental caries by multi-modal correlative microscopy and multiphysics modelling”(EP/P005381/1)“Rich Nonlinear Tomography for advanced materials”(EP/V007785/1).
文摘The Dual Imaging and Diffraction(DIAD)beamline at Diamond Light Source(Didcot,U.K.)implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using complementary X-ray modalities to reveal structural detail at various length scales.Namely,the underlying beamline principle and its practical implementation allow the collocation of chosen regions within the sample and their interrogation using real-space imaging(radiography and tomography)and reciprocal space scattering(diffraction).The switching between the two principal modes is made smooth and rapid by design,so that the data collected is interlaced to obtain near-simultaneous multimodal characterization.Different specific photon energies are used for each mode,and the interlacing of acquisition steps allows conducting static and dynamic experiments.Building on the demonstrated realization of this state-of-the-art approach requires further refining of the experimental practice,namely,the methods for gauge volume collocation under different modes of beam−sample interaction.To address this challenge,experiments were conducted at DIAD devoted to the study of human dental enamel,a hierarchical structure composed of hydroxyapatite mineral nanocrystals,as a static sample previously affected by dental caries(tooth decay)as well as under dynamic conditions simulating the process of acid demineralization.Collocation and correlation were achieved between WAXS(wide-angle X-ray scattering),2D(radiographic),and 3D(tomographic)imaging.While X-ray imaging in 2D or 3D modes reveals real-space details of the sample microstructure,X-ray scattering data for each gauge volume provided statistical nanoscale and ultrastructural polycrystal reciprocal-space information such as phase and preferred orientation(texture).Careful registration of the gauge volume positions recorded during the scans allowed direct covisualization of the data from two modalities.Diffraction gauge volumes were identified and visualized within the tomographic data sets,revealing the underlying local information to support the interpretation of the diffraction patterns.The present implementation of the 4D microscopy paradigm allowed following the progression of demineralization and its correlation with time-dependent WAXS pattern evolution in an approach that is transferable to other material systems.
基金done as part of“Tackling human dental caries by multi-modal correlative microscopy and multi-physics modelling”(EP/P005381/1)and“Rich Nonlinear Tomography for advanced materials”(EP/V007785/1)funded by The Engineering and Physical Sciences Research Council(EPSRC)+2 种基金the support of the Health Research Bridging Salary Scheme(BRR00060-DF02 and BRR00060-DF03,respectively)the Medical Science Divisions,University of Oxford.Synchrotron tomography data were collected on I13-2 beamline in Diamond Light Source(Diamond Light Source Ltd.,Didcot,Oxfordshire,OX110DE,U.K.)under the proposal mg29256-1additional data beamtime facilitated by Dr.Andrew Bodey of Diamond Light Source.The authors wish to thank Dr.Jonathan D.James(School of Dentistry,University of Birmingham)for the support in preparing the dental sample.Prof.Jin-Chong Tan(University of Oxford,U.K.)is thanked for the additional supervision of the study.
文摘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 timestepping 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 0.5μm.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.
基金This study was supported by funding from the Biotechnology and Biological Sciences Research Council(BB/H008527/1)(www.bbsrc.ac.uk)China Regenerative Medicine International(CRMI),Jiangsu Industrial Technology Research Institute(JITRI),and Engineering and Physical Sciences Research Council(EPSRC EP/P005381/1 and EP/V007785/1).
文摘To reflect human development,it is critical to create a substrate that can support long-term cell survival,differentiation,and maturation.Hydrogels are promising materials for 3D cultures.However,a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction.Herein,granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell(hiPSC)-derived neural networks.A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules.Cells and hydrogel granules were combined using a weaker secondary gelation step,forming self-supporting cell laden scaffolds.At three and seven days,granular scaffolds supported higher cell viability compared to bulk hydrogels,whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions(65.52±11.59μm)after seven days compared to bulk hydrogels(22.90±4.70μm).Long-term(three-month)cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold.This approach is significant as it provides a simple,rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.
