Microscale Precision Research Articles

Engineering programmable material-to-cell pathways via synthetic notch receptors to spatially control differentiation in multicellular constructs

Synthetic Notch (synNotch) receptors are genetically encoded, modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control, applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus, we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally, we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues.

Additive manufacturing multiscale ultrahigh strength and high accuracy nanocrystalline nickel structures and components using ultrafine anode scanning through-mask electrodeposition

Through-mask electrodeposition (TM-ECD) is a widely-used technique for manufacturing mesoscale and microscale precision metal features and components in a mass-production manner. However, in most cases, the traditional TM-ECD processes are very hard to fabricate high-accuracy multiscale (macroscale, mesoscale, microscale) features simultaneously due to the inherent nonuniform distribution of the applied energy fields including electric field, mass transportation field, and temperature field during electrodeposition. Furthermore, nanocrystalline microstructures have not been realized by TM-ECD without additional nonmetallic additives or mechanical stirring. Here, a ultrafine anode scanning through-mask electrodeposition (UASTM-ECD) process operated at DC mode is developed to further trigger the formation of nanocrystalline without additives, in which a ultrafine wire-shaped inert anode embedded in a stirring paddle reciprocates over the top surfaces of the through-masks with a spacing of less than 100 μm. Experimental investigations into this unique TM-ECD were systemically carried out by examining the geometrical profile, surface morphologies, forming accuracy, grain size, and mechanical properties of the fabricated nanocrystalline structures and components. Our findings showed that the UASTM-ECD can facilitate to achieve a significantly improved thickness distribution uniformity and considerably fine nanocrystalline ultrapure microscale and mesoscale complex cross-sectional arrays components over the traditional TM-ECD processes even though the multiscale features and components are simultaneously deposited on the same substrate. These result from the unique deposition manner of the UASTM-ECD in which the metal materials are intermittently filled into the patterned photoresist cavity molds very locally and at the extremely high current densities. This versatile approach eliminates the possible contamination by additives and integration difficulty of mechanical stirring, which can be used to fabricate the multiscale ultrahigh strength and high accuracy ultrapure nanostructured-material features.

Independent microscale sensing of phase interface and surface temperature during droplet evaporation

Despite the important and pervasive nature of evaporative heat transfer phenomena, fundamental questions still remain about the microscopic processes that occur in and around individual droplets on a surface. In order to understand the underlying physics behind evaporative heat transfer, it is critical to have information at the individual droplet level regarding the surface temperature distribution with time as well as the location and speed of the moving multiphase contact line (MCL). In this work, a multifunctional microscale device comprised of a resistance heater, an array of spatially distributed thin-film resistance temperature detectors, and a phase interface sensing capacitance micro-sensor array has been utilized to measure the local heat transfer characteristics and MCL behavior simultaneously for the evaporation of individual sessile water droplets on a horizontal heated surface. The resistance- and capacitance-based operating principles of the micro-device means that it is capable of detecting temperature changes and tracking MCL at the microscale in real time even for applications with limited or no visibility such as within thermal management hardware or processing equipment. Importantly, having knowledge of the MCL’s location and speed with microscale precision allows for its influence on surface temperature and heat transfer to be directly studied rather than inferred. Results show that the MCL passage precedes the change in local surface temperature and the duration of the time difference between these events depends on the MCL’s speed. In addition, the passage of the MCL accounts for more than 70% of the overall temperature change during the evaporation process.

Nanozyme-Based Robotics Approach for Targeting Fungal Infection.

Fungal pathogens have been designated by the World Health Organization as microbial threats of the highest priority for global health. It remains a major challenge to improve antifungal efficacy at the site of infection while avoiding off-target effects, fungal spreading, and drug tolerance. Here, a nanozyme-based microrobotic platform is developed that directs localized catalysis to the infection site with microscale precision to achieve targeted and rapid fungal killing. Using electromagnetic field frequency modulation and fine-scale spatiotemporal control, structured iron oxide nanozyme assemblies are formed that display tunable dynamic shape transformation and catalysis activation. The catalytic activity varies depending on the motion, velocity, and shape providing controllable reactive oxygen species (ROS) generation. Unexpectedly, nanozyme assemblies bind avidly to fungal (Candida albicans) surfaces to enable concentrated accumulation and targeted ROS-mediated killing in situ. By exploiting these tunable properties and selective binding to fungi, localized antifungal activity is achieved using in vivo-like cell spheroid and animal tissue infection models. Structured nanozyme assemblies are directed to Candida-infected sites using programmable algorithms to perform precisely guided spatial targeting and on-site catalysis resulting in fungal eradication within 10 min. This nanozyme-based microrobotics approach provides a uniquely effective and targeted therapeutic modality for pathogen elimination at the infection site.

Direct Photo-Patterning of Efficient and Stable Quantum Dot Light-Emitting Diodes via Light-Triggered, Carbocation-Enabled Ligand Stripping.

Next generation displays based on quantum dot light-emitting diodes (QLEDs) require robust patterning methods for quantum dot layers. However, existing patterning methods mostly yield QLEDs with performance far inferior to the state-of-the-art individual devices. Here, we report a light-triggered, carbocation-enabled ligand stripping (CELS) approach to pattern QLEDs with high efficiency and stability. During CELS, photogenerated carbocations from triphenylmethyl chlorides remove native ligands of quantum dots, thereby producing patterns at microscale precision. Chloride anions passivate surface defects and endow patterned quantum dots with preserved photoluminescent quantum yields. It works for both cadmium-based and heavy-metal-free quantum dots. CELS-patterned QLEDs show remarkable external quantum efficiencies (19.1%, 17.5%, 12.0% for red, green, blue, respectively) and a long operation lifetime (T95 at 1000 nits up to 8700 h). Both are among the highest for patterned QLEDs and approach the records for nonpatterned devices, which makes CELS promising for building high-performance QLED displays and related integrated devices.

Abstract 15845: Microfabricated Anisotropic Tissue Bundles for the Production of 3D Cardiac Tissue at Scale

Rationale: Following myocardial infarct, tissue ischemia and cardiomyocyte (CM) death trigger inflammation followed by extracellular matrix (ECM) remodeling mediated by cardiac fibroblasts (CF). The cardiac tissue engineering field aims to replace damaged myocardium and restore normal cardiac function. The ECM’s high degree of structural anisotropy and the organization of resident cell populations are critical features necessary to recapitulate healthy myocardial function. Thus, there is a critical need for biomanufacturing technologies that can organize ECM and multiple, distinct cell types with microscale precision in tissue grafts of translationally relevant scale. Here, we propose a scalable material strategy for fabricating individual myofiber-like tissues with anisotropic organization and relevant cell populations comparable to native myocardium. Methods and Results: Microchannels of varying widths were perfused with fibrin (20mg/mL fibrinogen with 1U/mL thrombin supplemented with 0.05mg/mL aprotinin for the first 3 days of culture) or a mixture of fibrin and collagen (10mg/ml fibrinogen with 1U/mL thrombin and 2mg/ml collagen). Hydrogels contained up to 40 million/mL cells (25% CFs and 75% CMs) and 8 vol% RGD-functionalized dextran-vinyl sulfone (DVS) electrospun fibers. Microchannel perfusion of composite hydrogel solutions resulted in flow-induced alignment of DVS fibers across all channel sizes (200, 300, 400, and 500 μm), although fiber alignment decreased with increasing channel size. Tissue size, cell spreading, CM contraction, and myofibril development and alignment varied with channel size, cell density, and hydrogel formulation over two weeks inculture. Overall, hydrogel composites with aligned fibers and a combination of CMs and CFs resulted in anisotropic, myofiber-like tissue structures. Conclusion: In this study, we developed a microfabrication approach to produce anisotropic myofiber-like bundles using composites of naturally derived ECM and synthetic polymeric fibers that can be flow-aligned within microfabricated microchannels. This approach allows us to fabricate unit cell tissues of desired size and alignment that can be combined into a larger tissue construct for therapeutic applications.

Surface Topography-Adaptive Robotic Superstructures for Biofilm Removal and Pathogen Detection on Human Teeth.

The eradication of biofilms remains an unresolved challenge across disciplines. Furthermore, in biomedicine, the sampling of spatially heterogeneous biofilms is crucial for accurate pathogen detection and precise treatment of infection. However, current approaches are incapable of removing highly adhesive biostructures from topographically complex surfaces. To meet these needs, we demonstrate magnetic field-directed assembly of nanoparticles into surface topography-adaptive robotic superstructures (STARS) for precision-guided biofilm removal and diagnostic sampling. These structures extend or retract at multilength scales (micro-to-centimeter) to operate on opposing surfaces and rapidly adjust their shape, length, and stiffness to adapt and apply high-shear stress. STARS conform to complex surface topographies by entering angled grooves or extending into narrow crevices and “scrub” adherent biofilm with multiaxis motion while producing antibacterial reagents on-site. Furthermore, as the superstructure disrupts the biofilm, it captures bacterial, fungal, viral, and matrix components, allowing sample retrieval for multiplexed diagnostic analysis. We apply STARS using automated motion patterns to target complex three-dimensional geometries of ex vivo human teeth to retrieve biofilm samples with microscale precision, while providing “toothbrushing-like” and “flossing-like” action with antibacterial activity in real-time to achieve mechanochemical removal and multikingdom pathogen detection. This approach could lead to autonomous, multifunctional antibiofilm platforms to advance current oral care modalities and other fields contending with harmful biofilms on hard-to-reach surfaces.

Microrobotics for Precision Biofilm Diagnostics and Treatment

Advances in small-scale robotics and nanotechnology are providing previously unimagined opportunities for new diagnostic and therapeutic approaches with high precision, control, and efficiency. We designed microrobots for tetherless biofilm treatment and retrieval using iron oxide nanoparticles (NPs) with dual catalytic-magnetic functionality as building blocks. We show 2 distinct microrobotic platforms. The first system is formed from NPs that assemble into aggregated microswarms under magnetic fields that can be controlled to disrupt and retrieve biofilm samples for microbial analysis. The second platform is composed of 3-dimensional (3D) micromolded opacifier-infused soft helicoids with embedded catalytic-magnetic NPs that can be visualized via existing radiographic imaging techniques and controlled magnetically inside the root canal, uninterrupted by the soft and hard tissues surrounding the teeth in an ex vivo model. These microrobots placed inside the root canal can remove biofilms and be efficiently guided with microscale precision. The proof-of-concept paradigm described here can be adapted to target difficult-to-reach anatomical spaces in other natural and implanted surfaces in an automated and tether-free manner.

Multiscale, multi-perspective imaging assisted robotic microinjection of 3D biological structures.

Microinjection is a widely used technique employed by biologists with applications in transgenesis, cryopreservation, mutagenesis, labeling/dye injection and in-vitro fertilization. However, microinjection is an extremely laborious manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. Here, we present a computer-guided robot that automates the targeted microinjection of Drosophila melanogaster and zebrafish (Danio rerio) embryos, two important model organisms in biological research. The robot uses a series of cameras to image an agar plate containing embryos at multiple magnifications and perspectives. This imaging is combined with machine learning and computer vision algorithms to pinpoint a location on the embryo for targeted microinjection with microscale precision. We demonstrate the utility of this microinjection robot to successfully microinject Drosophila melanogaster and zebrafish embryos. Results obtained indicate that the robotic microinjection approach can significantly increase the throughput of microinjection as compared to manual microinjection while maintaining survival rates comparable to human operators. In the future, this robotic platform can be used to perform high throughput microinjection experiments and can be extended to automatically microinject a host of organisms such as roundworms (Caenorhabditis elegans), mosquito (Culicidae) embryos, sea urchins (Echinoidea) and frog (Xenopus) oocytes.

CNN-based CP-OCT sensor integrated with a subretinal injector for retinal boundary tracking and injection guidance.

.Significance: Subretinal injection is an effective way of delivering transplant genes and cells to treat many degenerative retinal diseases. However, the technique requires high-dexterity and microscale precision of experienced surgeons, who have to overcome the physiological hand tremor and limited visualization of the subretinal space.Aim: To automatically guide the axial motion of microsurgical tools (i.e., a subretinal injector) with microscale precision in real time using a fiber-optic common-path swept-source optical coherence tomography distal sensor.Approach: We propose, implement, and study real-time retinal boundary tracking of A-scan optical coherence tomography (OCT) images using a convolutional neural network (CNN) for automatic depth targeting of a selected retinal boundary for accurate subretinal injection guidance. A simplified 1D U-net is used for the retinal layer segmentation on A-scan OCT images. A Kalman filter, combining retinal boundary position measurement by CNN and velocity measurement by cross correlation between consecutive A-scan images, is applied to optimally estimate the retinal boundary position. Unwanted axial motions of the surgical tools are compensated by a piezoelectric linear motor based on the retinal boundary tracking.Results: CNN-based segmentation on A-scan OCT images achieves the mean unsigned error (MUE) of () using an ex vivo bovine retina model. GPU parallel computing allows real-time inference () and thus real-time retinal boundary tracking. Involuntary tremors, which include low-frequency draft in hundreds of micrometers and physiological tremors in tens of micrometers, are compensated effectively. The standard deviations of photoreceptor (PR) and choroid (CH) boundary positions get as low as when the depth targeting is activated.Conclusions: A CNN-based common-path OCT distal sensor successfully tracks retinal boundaries, especially the PR/CH boundary for subretinal injection, and automatically guides the tooltip’s axial position in real time. The microscale depth targeting accuracy of our system shows its promising possibility for clinical application.

Microfluidic droplets as structural templates for Matrigel to enable 1-week large organoid modeling

Organoid technology has proved promising for personalized cancer therapy and assessing drug efficacy and toxicity. For many applications, the organoids are required to possess enough volume to recapitulate mass transport efficiency, be reproducible and rapid to model. However, these features cannot be satisfied by using the current batch culture protocols. Herein, we present a microfluidic droplet templating method that fulfills these requirements. The organoids derived from cell-laden Matrigel droplets are uniform, and reach the target sizes immediately after Matrigel solidification. Large organoids (diameter ~ 500 μm or above) are obtained within 1-week of in vitro culture development. This approach also enables rapid and easy access to asymmetric tissue assemblies with microscale precision, which can be used to study cancer cell metastasis.

A dual nozzle 3D printing system for super soft composite hydrogels

Due to their inability to sustain their own weight, 3D printing materials as soft as human tissues is challenging. Hereby we describe the development of an extrusion additive manufacturing (AM) machine able to 3D print super soft hydrogels with micro-scale precision. By designing and integrating new subsystems into a conventional extrusion-based 3D printer, we obtained hardware that encompasses a range of new capabilities. In particular, we integrated a heated dual nozzle extrusion system and a cooling platform in the new system. In addition, we altered the electronics and software of the 3D printer to ensure fully automatized procedures are delivered by the 3D printing device, and super-soft tissue mimicking parts are produced. With regards to the electronics, we added new devices to control the temperature of the extrusion system. As for the software, the firmware of the conventional 3D printer was changed and modified to allow for the flow rate control of the ink, thus eliminating overflows in sections of the printing path where the direction/speed changes sharply.

Extracellular scaffold design for ultra-soft microtissue engineering

Soft and ultra-soft extracellular scaffolds constitute a major fraction of most human internal organs, except for the skeletal system. Modelling these organs in vitro requires a comprehensive understanding of their native scaffolding materials and proper engineering approaches to manufacture tissue architectures with microscale precision. This review focuses on the properties of soft and ultra-soft scaffolds, including their interactions with cells, mechanical properties (e.g. viscoelasticity), and existing microtissue engineering techniques. It also summarises challenges presented by the conflict between the properties of the materials demanded by cell behaviours and the capacities of engineering techniques. It proposes that leveraging the engineering ability of soft and ultra-soft scaffolds will promote therapeutic advances and regenerative medicine.

Influence of dielectric thickness and electrode structure on the ion wind generation by micro fabricated plasma actuators

Surface dielectric barrier discharges are investigated in order to explore the combined effects of barrier thickness and microstructure of the exposed electrode on the ion wind generation. Actuators with straight and structured high voltage electrodes with characteristic sizes of 200 and 250 µm and dielectric thicknesses of 0.5, 1 and 2 mm are compared. It is observed that: i) actuator efficiency of ion wind generation strongly depends on the applied voltage amplitude; ii) operation voltage depends on the dielectric thickness logarithmically; iii) electrode microstructure slightly increases the dynamic pressure (few percent in maximum), however the effect decreases with thicker dielectrics and smaller electrode structures; iv) the pattern of the most intensive discharge parts as well as the dielectric erosion repeats the regular structure of the electrodes down to 200 µm. Several identical samples are tested during different days to estimate the impact of the air humidity and the degradation of the dielectric. The microscale precision of the sample manufacture was accomplished by a commercial facility for printed circuit boards.

Graphene/high-oriented polypyrrole foam enables new-type ultrasensitive micro-distance detection

Micro-resolution range finder (MRRF) is an essential tool in various cutting-edge fields such as shooting macro, precision instrument, vibration monitoring and micro-collision-avoidance systems. Desirable MRRF requires micro-scale precision, high accuracy, rapid response, and long-term cycling stability. Yet conventional MRRFs based on optical feedback, precise machining, piezoelectric effects and magnetostriction are still limited by complicated designs, precise sequence of tasks, and cumbersome device volume. As such, for the first time, we present a new-type of micro-distance sensor consisting of reduced graphene oxide/high-oriented polypyrrole foam (rGO/HOPPyF) for MRRF. Based on the distance-resistance property of the as-prepared foam, the micro-distance sensor has shown advantages of easy operation, low cost, miniaturized device. Here, large-rate resistance change and great mechanical stability are realized in rGO/HOPPyF, thus allowing precise and accurate micro-distance detection. The micro-distance sensor demonstrates ultrahigh sensitivities of 386.7, 1260 and 546.7 m−1 within the step-length regions of 10 ~ 80, 80 ~ 290, 290 ~ 570 µm, respectively. Remarkably, both the precision and accuracy of our sensor are superior over those of the commercial micrometer and laser rangefinders, providing a new technological advance towards practical MRRF.

Abstract IA10: Multiscale simulations of k-Ras on lipid bilayers

Abstract Simulating proteins on lipid membranes could provide unprecedented insights into cancer biology and a host of other phenomena. However, such simulations face conflicting computational constraints: reaching biologically relevant time and length scales (milliseconds and microns) requires continuum-level models but understanding the processes of interest requires molecular level detail. I will present a new type of massively parallel, multiscale simulation framework that brings together these two modeling paradigms. Using a machine learning algorithm, we couple a novel continuum model to an ensemble of molecular dynamics simulations. The result is a simulation at macro length- and time-scales that incorporates microscale precision. We demonstrate this capability with a large-scale simulation of several hundred k-Ras proteins on a realistic lipid bilayer spanning 1 μm2. Citation Format: Frederick H. Streitz. Multiscale simulations of k-Ras on lipid bilayers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA10.

High-compact picosecond laser functionalizes surfaces with microscale precision

Diffractive optics allows 500-watt pulses to rapidly functionalize surfaces in up to 16 parallel beamlets, each controlled by an acousto-optical modulator.

Catalytic antimicrobial robots for biofilm eradication.

Magnetically driven robots can perform complex functions in biological settings with minimal destruction. However, robots designed to damage deleterious biostructures could also have important impact. In particular, there is an urgent need for new strategies to eradicate bacterial biofilms as we approach a post-antibiotic era. Biofilms are intractable and firmly attached structures ubiquitously associated with drug-resistant infections and destruction of surfaces. Existing treatments are inadequate to both kill and remove bacteria leading to reinfection. Here we design catalytic antimicrobial robots (CARs) that precisely and controllably kill, degrade and remove biofilms with remarkable efficiency. CARs exploit iron oxide nanoparticles (NPs) with dual catalytic-magnetic functionality that (i) generate bactericidal free radicals, (ii) breakdown the biofilm exopolysaccharide (EPS) matrix, and (iii) remove the fragmented biofilm debris via magnetic field driven robotic assemblies. We develop two distinct CAR platforms. The first platform, the biohybrid CAR, is formed from NPs and biofilm degradation products. After catalytic bacterial killing and EPS disruption, magnetic field gradients assemble NPs and the biodegraded products into a plow-like superstructure. When driven with an external magnetic field, the biohybrid CAR completely removes biomass in a controlled manner, preventing biofilm regrowth. Biohybrid CARs can be swept over broad swathes of surface or can be moved over well-defined paths for localized removal with microscale precision. The second platform, the 3D molded CAR, is a polymeric soft robot with embedded catalytic-magnetic NPs, formed in a customized 3D printed mold to perform specific tasks in enclosed domains. Vane-shaped CARs remove biofilms from curved walls of cylindrical tubes, and helicoid-shaped CARs drill through biofilm clogs, while simultaneously killing bacteria. In addition, we demonstrate applications of CARs to target highly confined anatomical surfaces in the interior of human teeth. These 'kill-degrade-and-remove' CARs systems could have significant impact in fighting persistent biofilm-infections and in mitigating biofouling of medical devices and diverse surfaces.

Nanoliter Fluid Handling for Microbiology Via Levitated Magnetic Microrobots

Microbiological environments are a clear application area for microscale robotics, but targeted delivery of biochemical cues to particular cells or tissues remains a significant challenge. This challenge is compounded by the sheer number of cells that compose tissues. Multirobot system development has been limited due to challenges in discrete operation of several robots in a confined space. Here, we demonstrate the use of multiple diamagnetically levitated robots for delivering biochemicals dissolved in liquid in amanner compatible with light microscopy. Specifically, we show loading, transport, and diffusive release of fluorescent molecules in hydrogels with microscale precision, as well as induction of synthetically engineered bacterial cells with signaling molecules. This platform holds potential for impact and utility in fields, such as developmental biology, tissue engineering, and synthetic biology.

Laser printed flat lenses for optofluidics

Conventional three-dimensional optics requires curvature to control the wave front of light thus making it difficult to reduce the size of the optical systems. Furthermore, for correction of optical aberrations, complex optical systems comprising more than one lens are used. This adds additional bulk, mass and complexity to the optical systems. Recent development in diffractive optics has enabled new thin lightweight optical elements such as metalenses. We introduce resonant laser printing technique as a flexible photo-thermal technology for metalens fabrication with the ability to control the light with microscale precision. Our laser printed metalenses can be integrated in bio-sensors, bio-imaging systems, and optofluidical devices.