4D Printed Soft Microactuator for Particle Manipulation via Surrounding Medium Variation.

Soft actuators have assumed vital roles in a diverse number of research and application fields, driving innovation and transformative advancements. Using 3D molding of smart materials and combining these materials through structural design strategies, a single soft actuator can achieve multiple functions. However, it is still challenging to realize soft actuators that possess high environmental adaptability while capable of different tasks. Here, the response threshold of a soft actuator is modulated by precisely tuning the ratio of stimulus-responsive groups in hydrogels. By combining a heterogeneous bilayer membrane structure and in situ multimaterial printing, the obtained soft actuator deformed in response to changes in the surrounding medium. The response medium is suitable for both biotic and abiotic environments, and the response rate is fast. By changing the surrounding medium, the precise capture, manipulation, and release of micron-sized particles of different diameters in 3D are realized. In addition, static capture of a single red blood cell is realized using biologically responsive medium changes. Finally, the experimental results are well predicted using finite element analysis. It is believed that with further optimization of the structure size and autonomous navigation platform, the proposed soft microactuator has significant potential to function as an easy-to-manipulate multifunctional robot.

Stiffness-tunable substrate fabrication by DMDbased optical projection lithography for cancer cell invasion studies.

OBJECTIVE: Cancer cell invasion is a critical cause of fatality in cancer patients. Physiologically relevant tumor models play a key role in revealing the mechanisms underlying the invasive behavior of cancer cells. However, most existing models only consider interactions between cells and extracellular matrix (ECM) components while neglecting the role of matrix stiffness in tumor invasion. Here, we propose an effective approach that can construct stiffness-tunable substrates using digital mirror device (DMD)-based optical projection lithography to explore the invasion behavior of cancer cells. The printability, mechanical properties, and cell viability of three-dimensional (3D) models can be tuned by the concentration of prepolymer and the exposure time. The invasion trajectories of gastric cancer cells in tumor models of different stiffness were automatically detected and tracked in real-time using a deep learning algorithm. The results show that tumor models of different mechanical stiffness can yield distinct regulatory effects. Moreover, owing to the biophysical characteristics of the 3D in vitro model, different cellular substructures of cancer cells were induced. The proposed tunable substrate construction method can be used to build various microstructures to achieve simulation of cancer invasion and antitumor screening, which has great potential in promoting personalized therapy.

Skin-Inspired Capacitive Flexible Tactile Sensor with an Asymmetric Structure for Detecting Directional Shear Forces.

Flexible pressure sensors based on micro-/nanostructures can be integrated into robots to achieve sensitive tactile perception. However, conventional symmetric structures, such as pyramids or hemispheres, can sense only the magnitude of a force and not its direction. In this study, a capacitive flexible tactile sensor inspired by skin structures and based on an asymmetric microhair structure array to perceive directional shear force is designed. Asymmetric microhair structures are obtained by two-photon polymerization (TPP) and replication. Owing to the features of asymmetric microhair structures, different shear force directions result in different deformations. The designed device can determine the directions of both static and dynamic shear forces. Additionally, it exhibits large response scales ranging from 30 Pa to 300 kPa and maintains high stability even after 5000 cycles; the final relative capacitive change (ΔC/C0 ) is <2.5%. This flexible tactile sensor has the potential to improve the perception and manipulation ability of dexterous hands and enhance the intelligence of robots.

Correlative super-resolution bright-field and fluorescence imaging by microsphere assisted microscopy.

The resolution of fluorescence imaging has been significantly enhanced with the development of super-resolution imaging techniques, surpassing the diffraction limit and reaching sub-diffraction scales of tens of nanometers. However, the resolution of the bright-field images of cells is restricted by the diffraction limit, leading to a significant gap between the resolutions of fluorescence and bright-field imaging, which hinders the research of the precise distribution of intracellular nanostructures. A microsphere superlens offers a promising solution by providing label-free super-resolution imaging capabilities compatible with fluorescence super-resolution imaging. In this study, we used microsphere superlenses to simultaneously enhance the resolution of bright-field and fluorescence imaging, achieving correlated super-resolution bright-field and fluorescence imaging. Compared to conventional bright-field images, we improved the imaging resolution from λ/1.3 to λ/4.2. A correlative super-resolution of mouse skeletal muscle cells was achieved, enabling the clear observation of the precise distribution of nanoparticles in mouse skeletal muscle cells. Furthermore, microsphere superlenses inherit the advantages of optical imaging, which is expected to enable the capturing of ultrafast biological activity within living cells with extremely high temporal resolutions.

Fabrication of hollow microtube arrays based on a femtosecond laser double-pulse multiphoton polymerization.

Microtubes with widely varied dimensions and materials have great prospects in functional devices applied in microoptics, microrobot, and biomedicine. However, the fabrication of vertically protruding hollow microtubes with high diameter-to-thickness ratio is challenging and few reported. Femtosecond laser two-photon polymerization can solve this problem via point-by-point scanning or SLM-based parallel processing, but the low efficiency limits its high throughput fabrication. Here, we report a novel, to the best of our knowledge, femtosecond laser double-pulse multiphoton polymerization approach for high efficiency fabrication of hollow microtube arrays. We established a two-aperture laser beam reshaping system to generate a circular beam via two rounds of Fresnel diffraction. Based on the unique laser energy distribution, hollow microtubes with high diameter-to-thickness ratio can be generated by two successively laser pulses exposure, which can improve the fabrication efficiency significantly. With the optimized parameters, we can achieve repeatable and uniform microtube array fabrication in large scale, and the yield can be 94.9%. Defocus testing showed that the proposed approach has a high range of focusing tolerance. The proposed microtube fabrication approach is meaningful in providing some enlightenment for researchers in the field of microfabrication.

A Study on the Applicability of Aromatic Parameters in the Maturity Evaluation of Lacustrine Source Rocks and Oils Based on Pyrolysis Simulation Experiments.

Aromatic maturity parameters were evaluated via closed-system pyrolysis experiments using a Mesozoic lacustrine source rock from the Yingen-Ejinaqi Basin, thereby ensuring a uniform source. Pulverized rock aliquots (200 mg) were reacted with water at temperatures ranging from 250 to 550 °C at 5 °C/min, and the aromatic fractions of expelled oil and extracts of the solid residue were analyzed by GC-MS. The experiments showed that the relative abundance of aromatic hydrocarbons in the oil and extractable organic matter (EOM) of source rock had different evolutionary characteristics. With the increase in the thermal evolution degree, the relative abundance of aromatic hydrocarbons in the EOM showed the characteristics of ″increased early (Ro < 0.80), unchanged middle (Ro = 0.80-2.00%), decreased lately (Ro > 2.00%)″. While the relative abundance of aromatic hydrocarbons in the expelled oils continuously increased, as the Ro values increased from 0.62 to 2.39%, the relative abundance of aromatic hydrocarbons gradually increased from 8 to 46%. With increased maturity, the relative abundance of 1-3-ring aromatic hydrocarbons continuously decreased, as observed in the phenanthrene homologs. Meanwhile, the relative abundance of 4+-ring aromatic hydrocarbons continuously increased, as seen in chrysene homologs. It was suggested that the effects of maturity on the composition of aromatic hydrocarbons might not be sufficiently obvious. The effective application range of the alkylnaphthalene-related maturity parameters (2-/1-methylnaphthalenes, (2,6- + 2,7-)/1,5-dimethylnaphthalenes, 2,3,6-/(1,4,6- + 1,3,5-) trimethylnaphthalenes, and (2,3,6- + 1,3,7-)/(1,4,6- + 1,3,5- + 1,3,6-) trimethylnaphthalenes) and the alkyldibenzothiophene maturity parameters (4-/1-methyldibenzothiophenes, 4,6-/(1,4- + 1,6-) dimethyldibenzothiophenes, and (2,6- + 3,6-)/(1,4- + 1,6-) dimethyldibenzothiophenes) was 0.84-2.06% Ro. The alkylphenanthrene-related maturity parameters had a wide application range for lacustrine source rocks with an Ro < 2.06%. These parameters included 1.5 × (2- + 3-)/(phenanthrene +1- + 9-) methylphenanthrenes, 3 × 2-/(phenanthrene + 1- + 9-) methylphenanthrenes, (2- + 3-)/(1- + 9-) methylphenanthrenes, 2-/1-methylphenanthrenes, (3- + 2-)/(1- + 2- + 3- + 9-) methylphenanthrenes, 2-/(1- + 2- + 3- + 9-) methylphenanthrenes, and 2,7-/1,8-dimethylphenanthrenes. In addition, the effective applicable range of the methylnaphthalene-related maturity parameter 3-/1-methylchrysenes was an Ro value less than 1.79%. The results clarified the validity scope of some aromatics' maturity parameters and provided a theoretical basis for the scientific application of these parameters.

A 4D-Printing Inverse Design Strategy for Micromachines with Customized Shape-Morphing.

An active heterostructure with smart-response material used as "muscle" and inactive material as "skeleton" can deform over time to respond to external stimuli. 4D printing integrated with two-photon polymerization technology and smart material allows the material or characteristic distribution of active heterostructures to be defined directly at the microscale, providing a huge programmable space. However, the high degree of design freedom and the microscale pose a challenge to the construction of micromachines with customized shape morphing. Here, a reverse design strategy based on multi-material stepwise 4D printing is proposed to guide the structural design of biomimetic micromachines. Inspired by the piecewise constant curvature model of soft robot, a reverse design algorithm based on the Timoshenko model is developed. The algorithm can approximate 2D features to a constant-curvature model and determine an acceptable material distribution within the explored printing range. Three Chinese "Long" (Chinese dragon heralds of good fortune) designed by the strategy can deform to the customized shape. In addition, a microcrawler printed using this method can imitate a real inchworm gait. These results demonstrate that this method can be an efficient tool for the action or shape design of bionic soft microrobots or micromachines with predetermined functions.

Self-Sensing Scanning Superlens for Three-Dimensional Noninvasive Visible-Light Nanoscale Imaging on Complex Surfaces.

Microsphere-assisted super-resolution imaging technology offers label-free, real-time dynamic imaging via white light, which has potential applications in living systems and the nanoscale detection of semiconductor chips. Scanning can aid in overcoming the limitations of the imaging area of a single microsphere superlens. However, the current scanning imaging method based on the microsphere superlens cannot achieve super-resolution optical imaging of complex curved surfaces. Unfortunately, most natural surfaces are composed of complex curved surfaces at the microscale. In this study, we developed a method to overcome this limitation through a microsphere superlens with a feedback capability. By maintaining a constant force between the microspheres and the sample, noninvasive super-resolution optical imaging of complex abiotic and biological surfaces was achieved, and the three-dimensional information on the sample was simultaneously obtained. The proposed method significantly expands the universality of scanning microsphere superlenses for samples and promotes their widespread use.

Upconversion optogenetics-driven biohybrid sensor for infrared sensing and imaging.

Living organisms are far superior to state-of-the-art devices in visual perception as they have evolved a wide number of capabilities that encompass our most advanced technologies. By leveraging the performance of living organisms and directly interfacing them with artificial components, it can use the intricacy and metabolic efficiency of biological visual sensing within artificial machines. Inspired by the molecular basis (transient receptor potential, TRP) for infrared detection of pit-bearing organisms, we propose a TRP-like biohybrid sensor by integrating upconversion nanoparticles (UCNP) and optogenetically engineered cells on a graphene transistor for infrared sensing and imaging. The UCNP converts infrared light irradiation into blue light, the blue light activates the cells expressed with channelrhodopsin-2 (ChR2) and induces transmembrane photocurrent, and the photocurrent is detected by a biocompatible graphene transistor. Stepwise and overall experimental results show that, upon infrared light irradiation, the UCNP can rapidly mediate cellular photocurrents, which further translates into the extra output current of the graphene transistor. More notably, the response speed of the biohybrid sensor is 1∼3 orders of magnitude faster than those of TRPs heterologously expressed in cell lines in the literature, which confirms the response time advantage of the combination of UCNP and ChR2 within the sensor in place of TRPs. The biohybrid sensor can successfully image infrared targets, proving the feasibility of developing bionic infrared sensing devices by biohybrid integration of nonliving nanomaterials and biological components. This work opens up an avenue for biohybrid sensors to develop the bionic infrared vision that promisingly reproduces the functional superiority of natural organisms. STATEMENT OF SIGNIFICANCE: Infrared sensing and imaging have a wide range of military and civilian applications. Organisms have evolved excellent infrared vision with the molecular basis, transient receptor potential (TRP), and the performance is superior to existing state-of-the-art infrared devices. Inspired by this, a TRP-like biohybrid sensor based on upconversion optogenetics and a 2D material-based device is developed for infrared sensing and imaging. The biohybrid sensor has a relatively fast response speed that is 1∼3 orders of magnitude faster than that of the heterologously expressed TRPs, which enables its capability of infrared imaging with a single pixel-based method. This work broadens the spectrum of biohybrid sensing based on engineered cells to infrared, advancing the process of reproducing the excellent infrared detection of organisms.

Customized construction of microscale multi-component biostructures for cellular applications.

In vitro biofabrication is employed in fields such as biomedicine and those using biomimetic materials. However, it suffers from drawbacks such as low resolution, applicability on a limited range of components, and difficulty in purposefully depositing specific cells in three-dimensional space. Hence, this paper proposes a digital micromirror device-based optical projection lithography (DOPL) system for producing multi-component microstructures with resolutions of tens of microns and explores the behavior of cells with these structures. The printability and mechanical properties of these microstructures were investigated to assess their reproduction quality and the ability to control their structural characteristics. The results show that when DOPL is used with polyethylene glycol dimethacrylate (PEGDMA) hydrogel, an array of micropits can be fabricated within a few minutes. Furthermore, uniform cell spheroids form rapidly with high throughput when they are seeded into the micropits. Additionally, PEGDMA and gelatin methacryloyl (GelMA) were used to construct multi-component microstructures, and it was demonstrated that cells with various morphologies selectively adhere to the heterogeneous interface. In addition, DOPL could enable deposition of various cells for constructing microenvironments and for drug screening. Finally, a biomimetic peritoneal model was constructed. Overall, this work demonstrates the versatility of this system and its potential in cellular applications such as cell behavior research, drug screening, and tissue engineering.

Correlative AFM and Scanning Microlens Microscopy for Time-Efficient Multiscale Imaging.

With the rapid evolution of microelectronics and nanofabrication technologies, the feature sizes of large-scale integrated circuits continue to move toward the nanoscale. There is a strong need to improve the quality and efficiency of integrated circuit inspection, but it remains a great challenge to provide both rapid imaging and circuit node-level high-resolution images simultaneously using a conventional microscope. This paper proposes a nondestructive, high-throughput, multiscale correlation imaging method that combines atomic force microscopy (AFM) with microlens-based scanning optical microscopy. In this method, a microlens is coupled to the end of the AFM cantilever and the sample-facing side of the microlens contains a focused ion beam deposited tip which serves as the AFM scanning probe. The introduction of a microlens improves the imaging resolution of the AFM optical system, providing a 3-4× increase in optical imaging magnification while the scanning imaging throughput is improved ≈8×. The proposed method bridges the resolution gap between traditional optical imaging and AFM, achieves cross-scale rapid imaging with micrometer to nanometer resolution, and improves the efficiency of AFM-based large-scale imaging and detection. Simultaneously, nanoscale-level correlation between the acquired optical image and structure information is enabled by the method, providing a powerful tool for semiconductor device inspection.

Characterization of interconnectivity of gelatin methacrylate hydrogels using photoacoustic imaging.

Hydrogels can provide a three-dimensional microenvironment for cells and thus serve as an extracellular matrix in a biofabrication process. The properties of hydrogels, such as their porosity and mechanical properties, significantly influence the cell growth. However, there is still a lack of effective methods for characterizing the hydrogel structure noninvasively. Herein, a photoacoustic (PA) imaging-based method is proposed for the characterization of gelatin methacrylate (GelMA) hydrogels. Owing to their high PA contrast, red blood cells (RBCs) are included as mediators in the GelMA hydrogel to analyze its pore distribution. The interconnectivity of the pores is further analyzed through the lysis of RBCs. The diffusion of the RBC lysis buffer in the GelMA is consistent with the trend observed in simulations. The analyzed vitality of HEK293 cells in different GelMA hydrogels reveals that understanding the diffusion of solutes (i.e., nutrients) is a potential strategy to optimize the hydrogel parameters during biofabrication.

Imaging properties of microsphere superlenses with varying background refractive indices under inclined illumination.

Microsphere lenses can overcome the optical diffraction limit and can be used to observe features smaller than 200 nm under white light. Inclined illumination benefits from the second refraction of evanescent waves in the microsphere cavity, prohibiting the influence of background noise and improving the imaging resolution and quality of the microsphere superlens. Currently, there is a consensus that microspheres immersed in a liquid environment can improve imaging quality. Microsphere imaging under inclined illumination is performed using barium titanate microspheres immersed in an aqueous environment. However, the background medium of a microlens varies depending on its diverse applications. In this study, the effects of continuously changing background media on the imaging properties of microsphere lens under inclined illumination are investigated. The experimental results demonstrate that the axial position of the microsphere photonic nanojet changes with respect to the background medium. Consequently, owing to the refractive index of the background medium, the imaging magnification and the position of the virtual image change. Using a sucrose solution and polydimethylsiloxane with the same refractive index, we demonstrate that the imaging performance of microspheres is related to the refractive index rather than the background medium type. This study helps associate microsphere superlenses with a more universal application spectrum.

Density Regulation and Localization of Cell Clusters by Self-Assembled Femtosecond-Laser-Fabricated Micropillar Arrays.

Tumor cell clusters of varying sizes and densities have different metastatic potentials. Three-dimensional (3D) patterned structures with rational topographical and mechanical properties are capable of guiding the 3D clustering of tumor cells. In this study, single femtosecond laser pulses were used to fabricate individual high-aspect-ratio micropillars via two-photon polymerization (TPP). By combining this approach with capillary-force self-assembly, complex 3D microstructure patterns were constructed with a high efficiency. The microstructures were able to regulate the formation of cell clusters at different cell seeding densities and direct self-guided 3D assembly of cell clusters of various sizes and densities. Localization of cell clusters was achieved using grid-indexed samples to address individual cell clusters, which holds great promise for in situ cell cluster culture and monitoring and for applications such as RNA sequencing of cell clusters.

Real-time red blood cell counting and osmolarity analysis using a photoacoustic-based microfluidic system.

Counting the number of red blood cells (RBCs) in blood samples is a common clinical diagnostic procedure, but conventional methods are unable to provide the size and other physical properties of RBCs at the same time. In this work, we explore photoacoustic (PA) detection as a rapid label-free and noninvasive analysis technique that can potentially be used for single RBC characterization based on their photoabsorption properties. We have demonstrated an on-chip PA flow cytometry system using a simple microfluidic chip combined with a PA imaging system to count and characterize up to ∼60 RBCs per second. Compared with existing microfluidic-based RBC analysis methods, which typically use camera-captured image sequences to characterize cell morphology and deformation, the PA method discussed here requires only the processing of one-dimensional time-series data instead of two- or three-dimensional time-series data acquired by computer vision methods. Therefore, the PA method will have significantly lower computational requirements when large numbers of RBCs are to be analyzed. Moreover, we have demonstrated that the PA signals of RBCs flowing in a microfluidic device could be directly used to acquire the osmolarity conditions (in the range of 124 to 497 mOsm L-1) of the medium surrounding the RBCs. This finding suggests a potential extension of applicability to blood tests via PA-based biomedical detection.

Self-assembled microcage fabrication for manipulating and selectively capturing microparticles and cells.

Single-cell-scale selective manipulation and targeted capture play a vital role in cell behavior analysis. However, selective microcapture has primarily been performed in specific circumstances to maintain the trapping state, making the subsequent in situ characterization and analysis of specific particles or cells difficult and imprecise. Herein, we propose a novel method that combines femtosecond laser two-photon polymerization (TPP) micromachining technology with the operation of optical tweezers (OTs) to achieve selective and targeted capture of single particles and cells. Diverse ordered microcages with different shapes and dimensions were self-assembled by micropillars fabricated via TPP. The micropillars with high aspect ratios were processed by single exposure, and the parameters of the micropillar arrays were investigated to optimize the capillary-force-driven self-assembly process of the anisotropic microcages. Finally, single microparticles and cells were selectively transported to the desired microcages by manipulating the flexibly of the OTs in a few minutes. The captured microparticles and cells were kept trapped without additional forces.

Scanning Super-Resolution Imaging in Enclosed Environment by Laser Tweezer Controlled Superlens.

Super-resolution imaging using microspheres has attracted tremendous scientific attention recently because it has managed to overcome the diffraction limit and allowed direct optical imaging of structures below 100 nm without the aid of fluorescent microscopy. To allow imaging of specific areas on the surface of samples, the migration of the microspheres to specific locations on two-dimensional planes should be controlled to be as precise as possible. The common approach involves the attachment of microspheres on the tip of a probe. However, this technology requires additional space for the probe and could not work in an enclosed environment, e.g., in a microfluidic enclosure, thereby reducing the range of potential applications for microlens-based super-resolution imaging. Herein, we explore the use of laser trapping to manipulate microspheres to achieve super-resolution imaging in an enclosed microfluidic environment. We have demonstrated that polystyrene microsphere lenses could be manipulated to move along designated routes to image features that are smaller than the optical diffraction limit. For example, a silver nanowire with a diameter of 90 nm could be identified and imaged. In addition, a mosaic image could be constructed by fusing a sequence of images of a sample in an enclosed environment. Moreover, we have shown that it is possible to image Escherichia coli bacteria attached on the surface of an enclosed microfluidic device with this method. This technology is expected to provide additional super-resolution imaging opportunities in enclosed environments, including microfluidic, lab-on-a-chip, and organ-on-a-chip devices.

Microsphere-Based Super-Resolution Imaging for Visualized Nanomanipulation.

Nanomanipulation provides high operating accuracy and has been successfully applied in many fields such as nanoparticle assembly, nanowire alignment, and semiconductor device manufacturing. However, because of the limits of optical diffraction, the use of nanomanipulation is challenged by a lack of visual feedback at the nanoscale, and thus, its efficiency is difficult to be improved. In this study, we developed a novel method of microlens-enhanced nanomanipulation capable of real-time super-resolution imaging. Nanomanipulation was performed using the atomic force microscopy (AFM) mechanism by coupling a microlens to an AFM probe, and optical imaging with a minimum characteristic size of 80 nm is realized by combining the microlens with the optical imaging system. Under the conditions of fluorescent illumination and white light illumination, nanomanipulations were achieved under real-time visual guidance for fluorescent nanoparticles with a diameter of 100 nm and silver nanowires with a diameter of 80 nm, respectively. This method enables the possibility of in situ observation and manipulation, which can potentially be used for biological samples.

Cross-scale additive direct-writing fabrication of micro/nano lens arrays by electrohydrodynamic jet printing.

High-quality micro/nanolens arrays (M/NLAs) are becoming irreplaceable components of various compact and miniaturized optical systems and functional devices. There is urgent requirement for a low-cost, high-efficiency, and high-precision technique to manufacture high-quality M/NLAs to meet their diverse and personalized applications. In this paper, we report the one-step maskless fabrication of M/NLAs via electrohydrodynamic jet (E-jet) printing. In order to get the best morphological parameters of M/NLAs, we adopted the stable cone-jet printing mode with optimized parameters instead of the micro dripping mode. The optical parameters of M/NLAs were analyzed and optimized, and they were influenced by the E-jet printing parameters, the wettability of the substrate, and the viscosity of the UV-curable adhesive. Thus, diverse and customized M/NLAs were obtained. Herein, we realized the fabrication of nanolens with a minimum diameter of 120 nm, and NLAs with different parameters were printed on a silicon substrate, a cantilever of atomic force microscopy probe, and single-layer graphene.

Facile Method for Fabricating Microfluidic Chip Integrated with Microwell Arrays for Cell Trapping.

With the development of biomedical technology, personalized diagnosis and treatment at the single-cell level are becoming more important in the medical field. As one of the most powerful tools, microfluidic chips have shown significant potential for various applications related to cell separation, cell proliferation, and cell behavior analysis. However, fabricating microfluidic devices requires complicated procedures and high-cost equipment. In this study, an optofluidic maskless lithography method was proposed for rapid fabrication of microfluidic devices integrated with microwells. Through the use of this approach, microwells can be on-line designed and the exposure patterns can be modulated. Single or multi polystyrene microspheres were successfully trapped by using the designed microwells. The capture of MCF-7 cells and cell arrays indicated that the microfluidic devices fabricated in the present study can be applied for cell research.