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High-speed liquid micro-jets are used to rapidly and repeatedly deliver protein microcrystals to focused and pulsed X-ray beams in the method of serial femtosecond crystallography. However, the current continuous flow of crystals is mismatched to the arrival of X-ray pulses, wasting vast amounts of an often rare and precious sample. Here, we introduce a method to address this problem by periodically trapping and releasing crystals in the liquid flow, creating locally concentrated crystal bunches, using an optical trap integrated in the microfluidic supply line. We experimentally demonstrate a 30-fold increase of particle concentration into 10 Hz bunches of 6.4 µm diameter polystyrene particles. Furthermore, using particle trajectory simulations, a comprehensive description of the optical bunching process and parameter space is presented. Adding this compact optofluidics device to existing injection systems would thereby dramatically reduce sample consumption and extend the application of serial crystallography to a greater range of protein crystal systems that cannot be produced in high abundance. Our approach is suitable for other microfluidic systems that require synchronous measurements of flowing objects.
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In order to enable advanced technological applications of nanocrystal composites, e.g., as functional coatings and layers in flexible optics and electronics, it is necessary to understand and control their mechanical properties. The objective of this study was to show how the elasticity of such composites depends on the nanocrystals' dimensionality. To this end, thin films of titania nanodots (TNDs; diameter: ~3-7 nm), nanorods (TNRs; diameter: ~3.4 nm; length: ~29 nm), and nanoplates (TNPs; thickness: ~6 nm; edge length: ~34 nm) were assembled via layer-by-layer spin-coating. 1,12-dodecanedioic acid (12DAC) was added to cross-link the nanocrystals and to enable regular film deposition. The optical attenuation coefficients of the films were determined by ultraviolet/visible (UV/vis) absorbance measurements, revealing much lower values than those known for titania films prepared via chemical vapor deposition (CVD). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed a homogeneous coverage of the substrates on the µm-scale but a highly disordered arrangement of nanocrystals on the nm-scale. X-ray photoelectron spectroscopy (XPS) analyses confirmed the presence of the 12DAC cross-linker after film fabrication. After transferring the films onto silicon substrates featuring circular apertures (diameter: 32-111 µm), freestanding membranes (thickness: 20-42 nm) were obtained and subjected to atomic force microscopy bulge tests (AFM-bulge tests). These measurements revealed increasing elastic moduli with increasing dimensionality of the nanocrystals, i.e., 2.57 ± 0.18 GPa for the TND films, 5.22 ± 0.39 GPa for the TNR films, and 7.21 ± 1.04 GPa for the TNP films.
RESUMO
Serial femtosecond crystallography is a new method for protein structure determination utilizing intense and destructive X-ray pulses generated by free-electron lasers. The approach requires the means to deliver hydrated protein crystals to a focused X-ray beam and replenish them at the repetition rate of the pulses. A liquid-jet sample delivery system where a gas dynamic virtual nozzle is printed directly on a silicon-glass microfluidic chip using a 2-photon-polymerization 3D printing process is implemented. This allows for rapid prototyping and high-precision production of nozzles to suit the characteristics of a particular sample and opens up the possibility for high-throughput and versatile sample delivery systems that can integrate microfluidic components for sample detection, characterisation, or control. With the hybrid system described here, stable liquid jets with diameters between 1.5 µm at liquid flow rate of 1.5 µl/min and more than 20 µm at liquid flow rate of 100 µl/min under atmospheric and vacuum conditions are generated. The combination of 2D lithography with direct 3D printing may streamline the integration of free-form-features and also facilitate scale-up production of such integrated microfluidic devices that may be useful in many other applications such as flow cytometry and optofluidics.
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Engineered heart tissue (EHT) has proven as valuable tool for disease modelling, drug safety screening, and cardiac repair. Especially in combination with the stem cell technology, these in vitro models of the human heart have generated interest not only of basic cardiovascular researchers but also of regulatory authorities responsible for drug safety. A main limitation of 3D-based assays for evaluating cardiotoxicity is their limited throughput. We integrated piezo-bending actuators in a 24-well system for the generation of strip-like rat and human EHT attached to hollow, elastic silicone posts. Muscle contractions of EHTs induced a measurable electrical current in the piezo-bending actuators that could be analysed for contraction amplitude, frequency, and contraction and relaxation kinetics. Compared with the standard video-optical analysis of contractile activity, the new system allows for (a) the analysis of several tissues in parallel, (b) switching between auxotonic and isometric contractions by inserting a stiff metal post in the silicone post opposing the piezo actuator, (c) continuous measurement over days with low data volume (megabyte), (d) automated measurement without the necessity of adjustment of tissue position for video-optical analysis, (e) reduced complexity and costs, (f) high sensitivity of contraction detection, (g) calculation of absolute contraction force, and (h) suitability for variable tissue geometries. The new set-up for contraction analysis based on piezo-bending actuators is a promising new method for the parallel screening of EHT for pharmacological drug effects and other applications of muscle tissue engineering (e.g., skeletal muscle engineering or cardiac repair).
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Engenharia Tecidual , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , RatosRESUMO
Traumatic spinal cord injuries result in impairment or even complete loss of motor, sensory and autonomic functions. Recovery after complete spinal cord injury is very limited even in animal models receiving elaborate combinatorial treatments. Recently, we described an implantable microsystem (microconnector) for low-pressure re-adaption of severed spinal stumps in rat. Here we investigate the long-term structural and functional outcome following microconnector implantation after complete spinal cord transection. Re-adaptation of spinal stumps supports formation of a tissue bridge, glial and vascular cell invasion, motor axon regeneration and myelination, resulting in partial recovery of motor-evoked potentials and a thus far unmet improvement of locomotor behaviour. The recovery lasts for at least 5 months. Despite a late partial decline, motor recovery remains significantly superior to controls. Our findings demonstrate that microsystem technology can foster long-lasting functional improvement after complete spinal injury, providing a new and effective tool for combinatorial therapies.
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The objective of this study was to develop an injectable alginate based formulation for immobilizing enzymes into microfluidic systems. The gelation was induced upon lowering the pH by addition of d-glucono-δ-lactone (GDL) and release of Ca+ ions from solid CaCO3. The effects of GDL concentration on enzymatic activity and gelation time were investigated. The results indicated that increasing the GDL concentration increased both surface area and enzymatic activity. Also, chitosan was added to the formulation at different ratios to enhance the stability of enzyme during immobilization. For microfluidic application, 100µl spiral coil single channel microchip was fabricated and alginate GDL mixture containing ß-glucosidase was injected to the microchannel prior to gelation. Enzymatic conversion was performed by pumping substrate (pNPG) through the microchannel. The results indicated that the entire substrate was converted continuously during 24h without any leakage or deactivation of immobilized enzyme.
Assuntos
Alginatos/química , Hidrogéis/química , Microfluídica/métodosRESUMO
After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury. The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion. In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.
Assuntos
Axônios/fisiologia , Polietilenoglicóis/administração & dosagem , Polimetil Metacrilato/administração & dosagem , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal/fisiologia , Animais , Feminino , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/fisiopatologia , Engenharia Tecidual , Cicatrização/fisiologiaRESUMO
Base-pairing stability in DNA-gold nanoparticle (DNA-AuNP) multimers along with their dynamics under different electron beam intensities was investigated with in-liquid transmission electron microscopy (in-liquid TEM). Multimer formation was triggered by hybridization of DNA oligonucleotides to another DNA strand (Hyb-DNA) related to the concept of DNA origami. We analyzed the degree of multimer formation for a number of samples and a series of control samples to determine the specificity of the multimerization during the TEM imaging. DNA-AuNPs with Hyb-DNA showed an interactive motion and assembly into 1D structures once the electron beam intensity exceeds a threshold value. This behavior was in contrast with control studies with noncomplementary DNA linkers where statistically significantly reduced multimerization was observed and for suspensions of citrate-stabilized AuNPs without DNA, where we did not observe any significant motion or aggregation. These findings indicate that DNA base-pairing interactions are the driving force for multimerization and suggest a high stability of the DNA base pairing even under electron exposure.
Assuntos
DNA/química , Ouro/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Citratos/química , Estabilidade de Medicamentos , Modelos Moleculares , Conformação de Ácido Nucleico , Propriedades de SuperfícieRESUMO
Large-scale integrated silicon photonic circuits suffer from two inevitable issues that boost the overall power consumption. First, fabrication imperfections even on sub-nm scale result in spectral device non-uniformity that require fine-tuning during device operation. Second, the photonic devices need to be actively corrected to compensate thermal drifts. As a result significant amount of power is wasted if no athermal and wavelength-trimmable solutions are utilized. Consequently, in order to minimize the total power requirement of photonic circuits in a passive way, trimming methods are required to correct the device inhomogeneities from manufacturing and athermal solutions are essential to oppose temperature fluctuations of the passive/active components during run-time. We present an approach to fabricate CMOS backend-compatible and athermal passive photonic filters that can be corrected for fabrication inhomogeneities by UV-trimming based on low-loss amorphous-SOI waveguides with TiO2 cladding. The trimming of highly confined 10 µm ring resonators is proven over a free spectral range retaining athermal operation. The athermal functionality of 2nd-order 5 µm add/drop microrings is demonstrated over 40°C covering a broad wavelength interval of 60 nm.
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A method to compensate for fabrication tolerances and to fine-tune individual photonic circuit components is inevitable for wafer-scale photonic systems even with most-advanced CMOS-fabrication tools. We report a cost-effective and highly accurate method for the permanent trimming of hydrogenated amorphous silicon photonic devices by UV-irradiation. Microring resonators and Mach-Zehnder-interferometers were utilized as photonic test devices. The MZIs were tuned forth and back over their complete free spectral range of 5.5 nm by locally trimming the two MZI-arms. The trimming range exceeds 8 nm for compact ring resonators with trimming accuracies of 20 pm. Trimming speeds of ≥ 10 GHz/s were achieved. The components did not show any substantial device degradation.
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PURPOSE: Visual sensations in patients with blindness and retinal degenerations may be restored by electrical stimulation of retinal neurons with implantable microelectrode arrays. A prospective trial was initiated to evaluate the safety and efficacy of a wireless intraocular retinal implant (EPIRET3) in six volunteers with blindness and RP. METHODS: The implant is a remotely controlled, fully intraocular wireless device consisting of a receiver and a stimulator module. The stimulator is placed on the retinal surface. Data and energy are transmitted via an inductive link from outside the eye to the implant. Surgery included removal of the lens, vitrectomy, and implantation of the EPIRET3 device through a corneal incision. The clinical outcome after implantation and explantation of the device was determined. The implant was removed after 4 weeks, according to the study protocol. RESULTS: Implantation was successful in all six patients. While the anterior part was fixed with transscleral sutures, the stimulating foil was placed onto the posterior pole and fixed with retinal tacks. The implant was well tolerated, causing temporary moderate postoperative inflammation, whereas the position of the implant remained stable until surgical removal. In all cases explantation of the device was performed successfully. Adverse events were a sterile hypopyon effectively treated with steroids and antibiotics in one case and a retinal break in a second case during explantation requiring silicone oil surgery. CONCLUSIONS: The EPIRET3 system can be successfully implanted and explanted in patients with blindness and RP. The surgical steps are feasible, and the postoperative follow-up disclosed an acceptable range of adverse events.