Microscale Resolution Research Articles

Massively Parallel Machine Learning Driven Multiscale Simulations to Explore RAS-RAF Biology

Presented by H.I. Ingólfsson for the Joint Design of Advanced Computing Solutions for Cancer, Pilot 2 Team. Molecular dynamics simulations can provide detailed insights into biological mechanisms. However, even on the largest supercomputers available today micro-scale simulations cannot reach experimentally relevant time- and length-scales which significantly lessens their potential impact. Yet, macro-scale simulations that can cover experimental conditions normally do not provide the necessary detail to directly observe phenomena such as protein bonding or signaling. The multi-resolution framework uses machine learning, to couple a macro-scale model at experimentally relevant time- and length-scales with a vast ensemble of successively higher resolution micro-scale simulations. Given sufficient resources, the system will converge to macro-scale results at micro-scale resolution and provide fundamentally new insights. Here we present an extension to the massively parallel Multiscale Machine-Learned Modeling Infrastructure (MuMMI) incorporating the third (all-atom) resolution scale, multilevel feedback, higher fidelity macro model and support for different protein types. We are currently running a large-scale simulation campaign of RAS and RAF proteins on a complex plasma membrane using Summit, the second fastest supercomputer in the world, and will present results on the performance of the infrastructure and interactions of RAS, RAF and the lipid bilayer. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Release number: LLNL-ABS-815242.

Pore‐scale modeling of microbial activity: What we have and what we need

AbstractUnder water‐unsaturated conditions, environmental factors (e.g., substrate, nutrients, O2, water activity, and porous structure) controlling microbial activity are highly variable in space and time. The physical structure of porous media (pore size distribution and pore arrangements) has been considered to have a significant role in nutrient (either substrate and/or its reaction partners like O2) fluxes. Understanding what eventually controls microbial activity thus requires a sound description of the physical and chemical conditions in the pore space down to the microscale, as well as knowledge on how microorganisms respond to the energy and matter fluxes in their microscale environment. Microscale modeling has the advantage to resolve the processes down to their fundamental level. In recent years, microscale models describing the physical setting in unsaturated porous media such as soils, as well as growth and functional performance of microorganisms, have become increasingly established and are supported by new experimental techniques resolving the distribution of solid, liquid, and gaseous phases at microscale resolution. Still, integration of these components for simulation of microbial activities in a soil system at the microscale is scarce. This mini‐review highlights the available approaches for pore‐scale modeling of flow and transport processes in both saturated and unsaturated porous media, and for modeling the dynamics of microbial activity constrained by different soil boundary conditions. Further, it is discussed what is required in terms of model development and improved modeling concepts to achieve a holistic reactive transport modeling approach describing microbial activity at the pore scale.

Geochemical investigations of noble metal-bearing ores: Synchrotron-based micro-analyses and microcosm bioleaching studies

Auriferous sulphide ores often incorporate micro-fine (or invisible) gold and silver particles in a manner making their extraction difficult. Nobel metals are lost in the tailings due to the refractory nature of these ores. Bioleaching is an environment-friendly alternative to the commonly used and toxic cyanidation protocols for gold extraction from refractory ores. In this paper, we investigate gold and silver bioleaching from porphyry and epithermal mineralisation systems, using iron-oxidizing bacteria Acidithiobacillus ferrooxidans. The invisible Au, sequestered in refractory ores, was characterised in situ by synchrotron micro X-Ray Fluorescence (SR-μ-XRF) and X-ray Absorption Spectroscopy (XAS), offering information on Au unaltered speciation at the atomistic level within the ore matrices and at a micro-scale spatial resolution. The SR-μ-XRF and XAS results showed that 10–20 μm sized elemental Au(0) nuggets are sequestered in pyrite, chalcopyrite, arsenopyrite matrices and at the interface of a mixture of pyrite and chalcopyrite. Moreover, the preliminary bioleaching experiments of the two types of ores, showed that Acidithiobacillus ferrooxidans can catalyse the dissolution of natural heterogeneous Fe-rich geo-matrices, sequestering Au and Ag and releasing particulate phases or partially solubilising them within 60 days. These results provide an understanding of noble metal sequestration and speciation within natural ores and a demonstration of the application of synchrotron-based micro-analysis in characterizing economic trace metals in major mineral structures. This work is a contribution to the ongoing efforts towards finding feasible and greener solutions of noble metal extraction protocols.

Shining new light into soil systems: Spectroscopy in microfluidic soil chips reveals microbial biogeochemistry

Microfluidic soil chips render optical access to the naturally opaque soil systems and enable direct investigation of microbial growth and interactions in micro-structurally and chemically controlled environments. However, chemical analyses of these interactions at high spatial and temporal resolution are still lacking. Here we propose that the use of advanced microspectroscopy techniques, namely infrared absorption, Raman scattering and synchrotron radiation based X-ray microspectroscopy, in microfluidic soil chips would make it possible to approach these phenomena. They allow monitoring biogeochemical processes in and around soil microbial cells growing in the reproducibly designed microenvironments within the chips at (sub)micrometer scale. Complementary use of several of the microspectroscopy techniques is beneficial for obtaining information about both molecular and elemental composition, oxidation states and local structure of the elements in the sample. Ultimately, we argue that microspectroscopy in microfluidic chips can lead to relevant breakthroughs in frontier research areas in soil science, such as (1) analysis of chemical responses of microbes to environmental triggers at micro-scale spatial resolution, (2) phenotypical identification and phylogenetic classification of single cells of soil microbes in situ, (3) determining spatially and time resolved effects of heavy metals and organic pollutants, including microplastics, on soils and (4) spatially resolved analysis of soil organic matter dynamics for better understanding of soil carbon storage. Tailoring the chip design to achieve optical transparency to the radiation type used by the different microspectroscopy methods is crucial to achieve this; therefore, we expect that this perspective will inspire the scientific community to use the proposed approaches and thus push both the technical development of the microspectroscopy suitable soil chips and the research frontier in soil science.

Unravelling the deposition mechanism of brittle particles in metal matrix composites fabricated via cold spray additive manufacturing

Cold spray deposition (CSD) emerged as an additive manufacturing process in recent years. It features the highest efficiency among all metal additive manufacturing processes. In this work, the deposition mechanism of brittle reinforcing particles during the CSD of metal matrix composites (MMCs) was investigated. The rebound and fragmentation of brittle microparticles during deposition were investigated at nanosecond level and with microscale resolution through in-situ observation. For the first time, the velocity that determines the transition from particle rebound to particle fragmentation was identified. Herein, this velocity is defined as “fragmentation velocity”. The X-ray computed tomography on CSD deposits reveal that most of the diamonds in the deposits were fragments of the original particles and their volume fraction significantly depended on their impact velocities relative to the fragmentation velocity. Based on these findings along with microstructure characterizations, the deposition mechanism of brittle reinforcing particles during the CSD of MMCs is proposed.

Subtractive patterning: High-resolution electrohydrodynamic jet printing with solvents

Advancements in 3D printing have initiated a paradigm in device fabrication. Electrohydrodynamic jet (e-jet) printing is a high-resolution 3D printing method that enables customizable patterning of thin-film structures, while reducing fabrication complexity and achieving high-resolution patterns with a wide variety of materials. However, to date, e-jet printing has focused on additive material deposition, rather than patterning through material subtraction. This work proposes displacement-based e-jet printing using solvent inks for subtractive patterning of polymer thin films, with microscale resolution in the x–y plane and nanoscale control in the z (dissolving) direction. The behavior of displacement-based e-jet printing is characterized using atomic force microscopy, and two methodologies are developed for controlling the linewidth and displaced depth. An example of area-selective thin film deposition on displacement-based e-jet patterns is provided to demonstrate the applicability of this patterning technique for printable microscale devices.

Detrimental effects of sand ingression in jet engine ceramic coatings captured with Raman-based 3D rendering

The infiltration of molten calcium, magnesium, and alumino-silicates (CMAS) into jet engine thermal barrier coatings (TBCs) causes an increased stiffness and a destructive phase destabilization eventually leading to thermomechanical failure. Using 3D confocal Raman spectroscopy, the extent of thermochemical degradation was identified by mapping the monoclinic phase volume fraction (mPVF) throughout a standard EB-PVD 7YSZ TBC coating. The mPVF is characterized as a function of depth and infiltration time with microscale resolution. Results show that phase destabilization was most intense along the column's perimeter whereas the core of the column retained most of its original phase. The greatest amount of phase destabilization occurs within the first hour of the interaction after which the average minimum mPVF is 32 %. This value increases to 42 % after a ten-fold increase in interaction time. This ability to quantitatively and non-destructively characterize degradation of CMAS infiltrated TBCs will accelerate development of degradation resistant coatings.

Scale-free vertical tracking microscopy.

The behavior and microscale processes associated with freely suspended organisms, along with sinking particles underlie key ecological processes in the ocean. Mechanistically studying such multiscale processes in the laboratory presents a considerable challenge for microscopy: how to measure single cells at microscale resolution, while allowing them to freely move hundreds of meters in the vertical direction? Here we present a solution in the form of a scale-free, vertical tracking microscope, based on a 'hydrodynamic treadmill' with no bounds for motion along the axis of gravity. Using this method to bridge spatial scales, we assembled a multiscale behavioral dataset of nonadherent planktonic cells and organisms. Furthermore, we demonstrate a 'virtual-reality system for single cells', wherein cell behavior directly controls its ambient environmental parameters, enabling quantitative behavioral assays. Our method and results exemplify a new paradigm of multiscale measurement, wherein one can observe and probe macroscale and ecologically relevant phenomena at microscale resolution. Beyond the marine context, we foresee that our method will allow biological measurements of cells and organisms in a suspended state by freeing them from the confines of the coverslip.

Micro-rocket robot with all-optic actuating and tracking in blood

Micro/nanorobots have long been expected to reach all parts of the human body through blood vessels for medical treatment or surgery. However, in the current stage, it is still challenging to drive a microrobot in viscous media at high speed and difficult to observe the shape and position of a single microrobot once it enters the bloodstream. Here, we propose a new micro-rocket robot and an all-optic driving and imaging system that can actuate and track it in blood with microscale resolution. To achieve a high driving force, we engineer the microrobot to have a rocket-like triple-tube structure. Owing to the interface design, the 3D-printed micro-rocket can reach a moving speed of 2.8 mm/s (62 body lengths per second) under near-infrared light actuation in a blood-mimicking viscous glycerol solution. We also show that the micro-rocket robot is successfully tracked at a 3.2-µm resolution with an optical-resolution photoacoustic microscope in blood. This work paves the way for microrobot design, actuation, and tracking in the blood environment, which may broaden the scope of microrobotic applications in the biomedical field.

Multimodal quantitative optical elastography of the crystalline lens with optical coherence elastography and Brillouin microscopy.

Assessing the biomechanical properties of the crystalline lens can provide crucial information for diagnosing disease and guiding precision therapeutic interventions. Existing noninvasive methods have been limited to global measurements. Here, we demonstrate the quantitative assessment of the elasticity of crystalline lens with a multimodal optical elastography technique, which combines dynamic wave-based optical coherence elastography (OCE) and Brillouin microscopy to overcome the drawbacks of individual modalities. OCE can provide direct measurements of tissue elasticity rapidly and quantitatively, but it is a challenge to image transparent samples such as the lens because this technique relies on backscattered light. On the other hand, Brillouin microscopy can map the longitudinal modulus with micro-scale resolution in transparent samples. However, the relationship between Brillouin-deduced modulus and Young's modulus is not straightforward and sample dependent. By combining these two techniques, we can calibrate Brillouin measurements with OCE, based on the same sample, allowing us to completely map the Young's modulus of the crystalline lens. The combined system was first validated with tissue-mimicking gelatin phantoms of varying elasticities (N = 9). The OCE data was used to calibrate the Brillouin shift measurements and subsequently map the Young's modulus of the phantoms. After validation, OCE and Brillouin measurements were performed on ex-vivo porcine lenses (N = 6), and the Young's modulus of the lenses was spatially mapped. The results show a strong correlation between Young's moduli measured by OCE and longitudinal moduli measured by Brillouin microscopy. The correlation coefficient R was 0.98 for the phantoms and 0.94 for the lenses, respectively. The mean Young's modulus of the anterior and posterior lens was 1.98 ± 0.74 kPa and 2.93 ± 1.13 kPa, respectively, and the Young's modulus of the lens nucleus was 11.90 ± 2.94 kPa. The results presented in this manuscript open a new way for truly quantitative biomechanical mapping of optically transparent (or low scattering) tissues in 3D.

Dynamic Photo-cross-linking of Native Silk Enables Macroscale Patterning at a Microscale Resolution.

Light-based structuring methods have shown reconstituted silk to be a versatile and appropriate material for a range of optical and biomaterial-based applications. However, without an understanding of how an unmodified, native, silk responds to photoprocessing, the full potential of this material cannot be realized. Here, we show that the use of native silk enables the production of compound patterns with improved resolution and image quality when quantitatively compared to standard reconstituted silk, which we link directly to the influence of molecular weight. Further insights into the mechanism behind silk structure development are provided through mechanical (rheological) and structural (FTIR) measurements and results show that processing can tune properties over several orders of magnitude, enabling potential replication of several soft tissue types. Finally, broadening our application perspective, this combination of mask-less lithography and native silk resulted in the fabrication of transparent optical elements for data storage and labeling.

Hybrid Modeling of Electrohydrodynamic Jet Printing

Additive manufacturing (AM) has grown quickly in popularity as a fast, flexible fabrication technique. However, the physical complexity of most AM processes makes it difficult to produce dynamical models that capture the system evolution from the beginning to the end of the process. This difficulty in model creation inhibits the application of many sophisticated control techniques that may improve process performance and reliability. This work uses a hybrid dynamical system structure and the combination of physics- and data-driven partial process models to address the modeling challenge for a specific AM technology—a microscale resolution technique called electrohydrodynamic jet printing—in which electric field is used to eject droplets from the meniscus at the end of a fluid-filled nozzle. Specifically, modeling of the fluid meniscus as a paraboloidal cap instead of a spherical cap extends the range of meniscus deformation that can be captured by the physics-driven model. In addition, the physics-driven model fidelity is improved by using equilibrium information and two-parameter correction coefficients identified from data to relax assumptions on the electric field and nozzle geometry models. Then, the maximum stable meniscus deformation is leveraged to define the limit of the physics-driven model’s applicability, and for the remaining segment of the process, data-driven time-delayed linear time invariant models are identified. Finally, the complete hybrid model is validated against empirical time series data.

Modeling Microgels with a Controlled Structure across the Volume Phase Transition.

Thermoresponsive microgels are soft colloids that find widespread use as model systems for soft matter physics. Their complex internal architecture, made of a disordered and heterogeneous polymer network, has been so far a major challenge for computer simulations. In this work, we put forward a coarse-grained model of microgels whose structural properties are in quantitative agreement with results obtained with small-angle X-ray scattering experiments across a wide range of temperatures, encompassing the volume phase transition. These results bridge the gap between experiments and simulations of individual microgel particles, paving the way to theoretically address open questions about their bulk properties with unprecedented nano- and microscale resolution.

Three-Dimensional Laser Printing of Macro-Scale Glass Objects at a Micro-Scale Resolution.

Three-dimensional (3D) printing has allowed for the production of geometrically complex 3D objects with extreme flexibility, which is currently undergoing rapid expansion in terms of materials, functionalities, as well as areas of application. When attempting to print 3D microstructures in glass, femtosecond laser-induced chemical etching (FLICE)—which is a subtractive 3D printing technique—has proved itself a powerful approach. Here, we demonstrate the fabrication of macro-scale 3D glass objects of large heights up to ~3.8 cm with an identical lateral and longitudinal feature size of ~20 μm. The remarkable accomplishment is achieved by revealing an unexplored regime in the interaction of ultrafast laser pulses with fused silica, which results in depth-insensitive focusing of the laser pulses inside fused silica.

Light-Responsive, Shape-Switchable Block Copolymer Particles.

A robust strategy is developed for preparing light-responsive block copolymer (BCP) particles in which shape and color can be actively controlled with high spatial and temporal resolution. The key to achieving light-responsive shape transitions of BCP particles is the design and synthesis of surfactants containing light-active groups (i.e., nitrobenzyl esters and coumarin esters) that modulate the amphiphilicity and interfacial activity of the surfactants in response to light of a specific wavelength. These light-induced changes in surfactant structure modify the surface and wetting properties of BCP particles, affording both shape and morphological transitions of the particles, for example from spheres with an onion-like inner morphology to prolate or oblate ellipsoids with axially stacked nanostructures. In particular, wavelength-selective shape transformation of the BCP particles can be achieved with a mixture of two light-active surfactants that respond to different wavelengths of light (i.e., 254 and 420 nm). Through the use of light-emitting, photoresponsive surfactants, light-induced changes in both color and shape are further demonstrated. Finally, to demonstrate the potential of the light-triggered shape control of BCP particles in patterning features with microscale resolution, the shape-switchable BCP particles are successfully integrated into a patterned, free-standing hydrogel film, which can be used as a portable, high-resolution display.

Hybrid Laser Printing of 3D, Multiscale, Multimaterial Hydrogel Structures.

Fabrication of multiscale, multi-material three-dimensional (3D) structures at high resolution is difficult using current technologies. This is especially significant when working with hydrated and mechanically weak hydrogel materials. In this work, a new hybrid laser printing (HLP) technology is reported to print complex, multiscale, multimaterial, 3D hydrogel structures with microscale resolution. This technique of fabrication utilizes sequential additive and subtractive modes of material fabrication, that are typically considered as mutually exclusive due to differences in their material processing conditions. Further, compared to current laser writing systems that enforce stringent processing depth limits, HLP is shown to fabricate structures at any depth inside the material. As a proof-of-principle, a Mayan Pyramid with embedded cube-frame is printed using model synthetic polyethylene glycol diacrylate (PEGDA) hydrogel. Printing of ready-to-use open-well chips with embedded microchannels is also demonstrated using PEGDA and gelatin methacrylate (GelMA) hydrogels for potential applications in biomedical sciences. Next, HLP is used in additive and additive modes to print multiscale 3D structures spanning in size from centimeter to micrometers within minutes, which is followed by printing of 3D, multi-material, multiscale structures using this technology. Overall, this work demonstrates that HLP's fabrication versatility can potentially offer a unique opportunity for a range of applications in optics and photonics, biomedical sciences, microfluidics, soft robotics, etc.

Mapping the spatial variation of mitral valve elastic properties using air-pulse optical coherence elastography

The mitral valve is a highly heterogeneous tissue composed of two leaflets, anterior and posterior, whose unique composition and regional differences in material properties are essential to overall valve function. While mitral valve mechanics have been studied for many decades, traditional testing methods limit the spatial resolution of measurements and can be destructive. Optical coherence elastography (OCE) is an emerging method for measuring viscoelastic properties of tissues in a noninvasive, nondestructive manner. In this study, we employed air-pulse OCE to measure the spatial variation in mitral valve elastic properties with micro-scale resolution at 1 mm increments along the radial length of the leaflets. We analyzed differences between the leaflets, as well as between regions of the valve. We found that the anterior leaflet has a higher elastic wave velocity, which is reported as a surrogate for stiffness, than the posterior leaflet, most notably at the annular edge of the sample. In addition, we found a spatial elastic gradient in the anterior leaflet, where the annular edge was found to have a greater elastic wave velocity than the free edge. This gradient was less pronounced in the posterior leaflet. These patterns were confirmed using established uniaxial tensile testing methods. Overall, the anterior leaflet was stiffer and had greater heterogeneity in its mechanical properties than the posterior leaflet. This study measures differences between the two mitral leaflets with greater resolution than previously feasible and demonstrates a method that may be suitable for assessing valve mechanics following repair or during the engineering of synthetic valve replacements.

Synchrotron tomography of intervertebral disc deformation quantified by digital volume correlation reveals microstructural influence on strain patterns

The intervertebral disc (IVD) has a complex and multiscale extracellular matrix structure which provides unique mechanical properties to withstand physiological loading. Low back pain has been linked to degeneration of the disc but reparative treatments are not currently available. Characterising the disc’s 3D microstructure and its response in a physiologically relevant loading environment is required to improve understanding of degeneration and to develop new reparative treatments. In this study, techniques for imaging the native IVD, measuring internal deformation and mapping volumetric strain were applied to an in situ compressed ex vivo rat lumbar spine segment. Synchrotron X-ray micro-tomography (synchrotron CT) was used to resolve IVD structures at microscale resolution. These image data enabled 3D quantification of collagen bundle orientation and measurement of local displacement in the annulus fibrosus between sequential scans using digital volume correlation (DVC). The volumetric strain mapped from synchrotron CT provided a detailed insight into the micromechanics of native IVD tissue. The DVC findings showed that there was no slipping at lamella boundaries, and local strain patterns were of a similar distribution to the previously reported elastic network with some heterogeneous areas and maximum strain direction aligned with bundle orientation, suggesting bundle stretching and sliding. This method has the potential to bridge the gap between measures of macro-mechanical properties and the local 3D micro-mechanical environment experienced by cells. This is the first evaluation of strain at the micro scale level in the intact IVD and provides a quantitative framework for future IVD degeneration mechanics studies and testing of tissue engineered IVD replacements. Statement of SignificanceSynchrotron in-line phase contrast X-ray tomography provided the first visualisation of native intact intervertebral disc microstructural deformation in 3D. For two annulus fibrosus volumes of interest, collagen bundle orientation was quantified and local displacement mapped as strain. Direct evidence of microstructural influence on strain patterns could be seen such as no slipping at lamellae boundaries and maximum strain direction aligned with collagen bundle orientation. Although disc elastic structures were not directly observed, the strain patterns had a similar distribution to the previously reported elastic network. This study presents technical advances and is a basis for future X-ray microscopy, structural quantification and digital volume correlation strain analysis of soft tissue.

Multi-species micropatterning of organic materials by liquid droplet array transfer printing

Because techniques such as photolithography for inorganic materials are yet to have their equivalents for organic materials, multispecies patterning of functional organic materials at microscale resolution remains in development. This paper reports on a liquid droplet transfer printing method, that is, used for microscale patterning of multispecies materials. A transfer printing instrument is built with a multidimensional micropositioning stage, thereby enabling the technique to be used to pattern different materials on a single surface. The size range of the transferred liquid droplets can be tuned by manipulating the surface wettability and the gap between the surfaces of the sample and the stamp. The developed technique is used to prepare red–green–blue pixel patterns and microlenses integrated with color filters.

The Perdigão: Peering into Microscale Details of Mountain Winds

AbstractA grand challenge from the wind energy industry is to provide reliable forecasts on mountain winds several hours in advance at microscale (∼100 m) resolution. This requires better microscale wind-energy physics included in forecasting tools, for which field observations are imperative. While mesoscale (∼1 km) measurements abound, microscale processes are not monitored in practice nor do plentiful measurements exist at this scale. After a decade of preparation, a group of European and U.S. collaborators conducted a field campaign during 1 May–15 June 2017 in Vale Cobrão in central Portugal to delve into microscale processes in complex terrain. This valley is nestled within a parallel double ridge near the town of Perdigão with dominant wind climatology normal to the ridges, offering a nominally simple yet natural setting for fundamental studies. The dense instrument ensemble deployed covered a ∼4 km × 4 km swath horizontally and ∼10 km vertically, with measurement resolutions of tens of meters and seconds. Meteorological data were collected continuously, capturing multiscale flow interactions from synoptic to microscales, diurnal variability, thermal circulation, turbine wake and acoustics, waves, and turbulence. Particularly noteworthy are the extensiveness of the instrument array, space–time scales covered, use of leading-edge multiple-lidar technology alongside conventional tower and remote sensors, fruitful cross-Atlantic partnership, and adaptive management of the campaign. Preliminary data analysis uncovered interesting new phenomena. All data are being archived for public use.