Two-photon Polymerization Research Articles

P-259 Intracytoplasmic sperm injection conducted within a micro 3D printed device that requires no holding pipette or vacuum reduces porcine oocyte invagination during microinjection

Abstract Study question Can the use of a custom-designed and 3D printed microICSI™ device reduce the degree of oocyte invagination during intracytoplasmic sperm injection (ICSI)? Summary answer Invagination was significantly less in porcine oocytes when ICSI was performed in microICSI™ device compared to conventional ICSI. What is known already ICSI is a difficult procedure for embryologists to master, yet until recently, technically little had changed. In particular, the holding pipette (HP) with vacuum to hold the oocyte has not been surpassed. However, a source of shear-stress during ICSI of oocytes is the invagination of the zona pellucida and cytoplasm that occurs during microinjection, due to the pressure of the injection pipette against the small surface area of the HP. A recent study demonstrated microinjection within a two-piece 3D printed device. Here we evaluated a design refinement that displaced injection pressure more evenly across the oocyte to improve embryo quality. Study design, size, duration A control (conventional ICSI) and study group (microICSI™) were compared over three replicates investigating the level of invagination (3-point score, 1 is lowest and 3 is greatest) during ICSI. Incidence of cytoplasmic lysis was also recorded 24 h following ICSI. Participants/materials, setting, methods Porcine oocytes collected by follicle aspiration of abattoir-sourced ovaries were matured for 39-44 h in NCSU-17 medium supplemented with eCG, hCG, porcine follicular fluid, EGF and insulin. Mature oocytes, stripped of cumulus, were randomly allocated to either conventional ICSI or microICSI™. ICSI was performed, with treatment order randomised, and the injected oocytes were cultured in NCSU-17 culture medium at 39 C. Main results and the role of chance The microICSI™ device was modelled as a one-piece structure with contoured surfaces within a microwell array to support the oocyte during injection without the requirement for a holding pipette. Two-photon polymerisation 3D micro printing was used to print and assess multiple prototype geometries to optimise the support of the oocyte during ICSI. Over the three replicates (n = 10-20/treatment/replicate), the degree of invagination was consistently lower in the microICSI™ group compared with conventional ICSI (Mean ± SEM): Replicate 1: 1.40 ± 0.16 vs. 2.00 ± 0.19; Replicate 2: 1.20 ± 0.11 vs. 2.00 ± 0.13; Replicate 3: 1.42 ± 0.12 vs. 1.78 ± 0.15, with a paired t-test showing a significant difference between the two groups (p = 0.035). The incidence of lysis was not significantly different between the two treatments (ranging from 15-20% across both groups and varied between replicates). Limitations, reasons for caution The use of a porcine ICSI model may not sufficiently replicate what occurs with human ICSI as human oocytes experience greater levels of invagination (typically scored out of 4 rather than 3). Wider implications of the findings Reducing the invagination due to the injection process may reflect a less stressful procedure, with improvement to fertilization rates and embryo quality. Trial registration number not applicable

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 isdeveloped. 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 candeform to the customized shape. In addition, a microcrawler printed using this method canimitate 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.

In-situ measurement of anisotropic Young’s modulus in fused deposition modeling printed cantilevers

In this study, we investigate the effect of fused deposition modeling printing direction on the effective Young’s modulus value of cantilevers. Through finite-element simulations and experiments with seven different dimensions and totaling over 100 cantilevers, we have observed the impact of printing direction on cantilever resonance. Unlike the conventional compressive and tensile stress—strain characterization, observation of the resonance allows for in-situ testing on the final device under test during operation. Initially, we observed the bulk filament modulus to be 4.5 GPa based on the optimal match between experiments and realistic finite element models expressing the internal structures of the longitudinal and transverse printed cantilevers. Then, the effective Young’s modulus of the cantilevers is inferred through sweeping the Young’s modulus that provides the best fit between the experiments, conventional cantilever formulations and finite-element simulations with solid, homogeneous, and isotropic cantilever model. Overall, we observed an average effective Young’s modulus of 3.35 GPa for the cantilevers with longitudinal (along the cantilever axis) deposited filaments and an average effective Young’s Modulus of 2.50 GPa for the transverse (perpendicular to the cantilever axis, along the width dimension) deposited Polylactic acid cantilevers. Eventually, simplified shape outline and effective Young’s modulus for the corresponding printing direction eases the subsequent theoretical and simulation analyses. The presented methodology is also applicable to micrometric and sub-micrometric scale serial manufacturing techniques (i.e. two-photon polymerization) where the laser beams steering direction causes anisotropy in the mechanical properties of the device under test.

Two is better than one: ESIPT dyes as photoinitiators in two-photon polymerization

In this work, we present the successful application of Excited-State Intramolecular Proton Transfer (ESIPT) fluorophores as photoinitiators for microfabrication. The influence of chemical structure on such application was investigated, followed by two-photon polymerization. We have determined the quality of fabricated structures and polymerization thresholds for various dye/monomer weight ratios with a staggeringly low value of 11 nJ for one of the chromophores.

4D direct laser writing of photo-triggered liquid crystal elastomer microactuators with large actuation strain

4D printed photo-triggered liquid crystal elastomers (LCEs) microactuators by direct laser writing via two-photon polymerization (DLW-TPP) have attracted increasing attention due to their manipulation flexibility, reversible and rapid actuation capabilities. However, their development is hampered by the lack of room-temperature printable liquid crystal (LC) photoresists. Here, we developed new light-responsive LC photoresists by incorporating novel conjugated polymers (CPs) as photothermal agents for the DLW-TPP technology. The CPs displayed a remarkable photothermal effect and effectively avoided the aggregation problems that always happened for inorganic nanoparticles in photoresists. Moreover, the CPs incorporation lowered the nematic-to-isotropic temperature of the LC photoresists which is beneficial for room-temperature DLW-TPP. The printing parameters, including laser power and scanning speed, were investigated using the developed LC photoresists. It was found the range of printing parameters decreased with the increase of the CPs loading fraction from 0.1 to 0.5 wt%, which was attributed to the high photothermal conversion efficiency (52.7%). A well-defined CPs/LCEs microactuator with CPs as low as 0.3 wt% was printed, which could achieve a large 25.0% actuation strain in 5 s upon near-infrared (NIR) light stimulation. It could be used for thriving soft micro-robotics and micro-membranes with controllable separation capabilities.

High-Precision and Rapid Direct Laser Writing Using a Liquid Two-Photon Polymerization Initiator.

Two-photon polymerization based direct laser writing (DLW) is an emerging micronano 3D fabrication technology wherein two-photon initiators (TPIs) are a key component in photoresists. Upon exposure to a femtosecond laser, TPIs can trigger the polymerization reaction, leading to the solidification of photoresists. In other words, TPIs directly determine the rate of polymerization, physicochemical properties of polymers, and even the photolithography feature size. However, they generally exhibit extremely poor solubility in photoresist systems, severely inhibiting their application in DLW. To break through this bottleneck, we propose a strategy to prepare TPIs as liquids via molecular design. The maximum weight fraction of the as-prepared liquid TPI in photoresist significantly increases to 2.0 wt %, which is several times higher than that of commercial 7-diethylamino-3-thenoylcoumarin (DETC). Meanwhile, this liquid TPI also exhibits an excellent absorption cross section (64 GM), allowing it to absorb femtosecond laser efficiently and generate abundant active species to initiate polymerization. Remarkably, the respective minimum feature sizes of line arrays and suspended lines are 47 and 20 nm, which are comparable to that of the-state-of-the-art electron beam lithography. Besides, the liquid TPI can be utilized to fabricate various high-quality 3D microstructures and manufacture large-area 2D devices at a considerable writing speed (1.045 m s-1). Therefore, the liquid TPI would be one of the promising initiators for micronano fabrication technology and pave the way for future development of DLW.

Water flow tuned by micro/nano hierarchical structures fabricated through two-photon polymerization

Two-photon polymerization (TPP) is a sub-classification of vat photopolymerization. It has been widely used to fabricate micro/nano hierarchical structures that have the potential to be used to tune the fluid flow in chips. The present work first studied the influence of laser power on the feature size of TPP-fabricated 2D structures. It was found that the increase of laser power from 1.0 μW to 1.4 μW leads to the feature size increasing from 203 nm to 307 nm. Then, the effect of scanning parameters on the accuracy and surface quality of 3D structures was investigated. The arrangement of the voxels at different scanning strategies explains the mechanisms of the variation of accuracy and surface roughness. Finally, the pinecone structures with micro/nano hierarchical features were built and evaluated for their water repellence. The increase in the number of rows of pinecone structures significantly enhances the water-repellent performance.

A sinterless, low-temperature route to 3D print nanoscale optical-grade glass.

Three-dimensional (3D) printing of silica glass is dominated by techniques that rely on traditional particle sintering. At the nanoscale, this limits their adoption within microsystem technology, which prevents technological breakthroughs. We introduce the sinterless, two-photon polymerization 3D printing of free-form fused silica nanostructures from a polyhedral oligomeric silsesquioxane (POSS) resin. Contrary to particle-loaded sacrificial binders, our POSS resin itself constitutes a continuous silicon-oxygen molecular network that forms transparent fused silica at only 650°C. This temperature is 500°C lower than the sintering temperatures for fusing discrete silica particles to a continuum, which brings silica 3D printing below the melting points of essential microsystem materials. Simultaneously, we achieve a fourfold resolution enhancement, which enables visible light nanophotonics. By demonstrating excellent optical quality, mechanical resilience, ease of processing, and coverable size scale, our material sets a benchmark for micro- and nano-3D printing of inorganic solids.

High-efficiency two-photon polymerization initiators based on coumarin derivatives for additive manufacturing with sub-wavelength resolution

Efficient photoinitiators are very important for two-photon polymerization additive manufacturing. In this paper, a series of new initiators based on coumarin groups have been designed and synthesized, and their optical properties and two-photon polymerization properties have been studied and discussed in detail, and the relationship between their structures and properties has been analyzed. The synthesized initiators can realize a two-photon polymerization process with a speed of 100,000 μm/s under a laser power of 35–50 mW, and successfully obtain a series of clear three-dimensional structures with sub-wavelength resolution. Starting from the principle of two-photon polymerization, the relationship between laser power and writing speed and resolution was discussed, two simple mathematical models were established, and a fitting formula with physical meaning was obtained, which provides a way for the prediction and control of resolution. Finally, under the conditions of 100,000 μm/s and 50 mW laser power, a low-density ring with an outer diameter of 2.4 mm and an inner diameter of 2.0 mm with sub-wavelength resolution was manufactured in just over 10 min for the fusion target experiment. This proves the possibility of using the resulting initiator to manufacture precision micro-nano devices.

Single-step fabrication of superhydrophobic surfaces by two-photon polymerization micro 3D printing

Single-step fabrication of superhydrophobic surfaces by two-photon polymerization micro 3D printing

A Directly Moldable, Highly Compact, and Easy-for-Integration 3D Micromixer with Extraordinary Mixing Performance.

Micromixers are a critical component in microfluidics. However, most 2D passive micromixers produce optimal mixing at a high flow rate range and 3D micromixers require mm-scale channels or a complex assembly that is unsuitable for microfluidic applications. Here, we reported a 3D PDMS micromixer based on the splitting-stretching-recombination (SSR) of streams to facilitate molecular diffusion, which can effectively and rapidly mix solutions with low Reynolds numbers (0.01-10). The fabrication of our micromixer is convenient with only two steps─two-photon polymerization (2PP) 3D printing and soft lithography, with high resolution, reproducibility, and ease for integration. We investigated the mixing performance of the micromixer by CFD simulations and experimental studies under a confocal microscope; the results confirmed its better performance and higher chip miniaturization than others. It can achieve a mixing efficiency above 0.90 (which is generally regarded as complete mixing) for low-Re solutions (flow rates ≤60 μL/min) with a mixing volume smaller than 20 nL. The time for complete mixing is in the range of milliseconds (e.g., 21 ms for Re = 10, 194 ms for Re = 0.88). The device shows negligible degradation in mixing performance for highly viscous solutions (∼50 times more viscous than water), macromolecule solutions, and colloidal solutions of nanoparticles.

Suppression of auto-fluorescence from high-resolution 3D polymeric architectures fabricated via two-photon polymerization for cell biology applications

Suppression of auto-fluorescence from high-resolution 3D polymeric architectures fabricated via two-photon polymerization for cell biology applications

Two-Photon Excitation Photoredox Catalysis Enabled Atom Transfer Radical Polymerization.

In recent years, metal-free photoredox-catalyzed atom transfer radical polymerization (O-ATRP) has gained wide attention because of its advantages (e.g., no metal contamination and mild reaction conditions). However, this traditional one-photon excitation catalysis has thermodynamic limits. Most photocatalysts cannot effectively reduce the initiators and drive the polymerization under visible light. Herein, we investigate the two-photon excitation-catalyzed O-ATRP, in which the catalyst can absorb two photons to accumulate energy. Compared to one-photon excitation catalysis, this method not only has distinct advantages in the controllability, reaction rate, and catalyst loading but also can chemically reduce the various initiators (e.g., aryl halides) to initiate the polymerization. Density functional theory (DFT) calculation reveals that the two-photon excitation process reached a higher energy end state with stronger reduced ability via a thermodynamically more stable intermediate. We believe that this work will provide a new strategy for photoredox-catalyzed O-ATRP.

22 nm Resolution Achieved by Femtosecond Laser Two-Photon Polymerization of a Hyaluronic Acid Vinyl Ester Hydrogel.

Three-dimensional (3D) bioinspired hydrogels have played an important role in tissue engineering, owing to their advantage of excellent biocompatibility. Here, the two-photon polymerization (TPP) of a 3D hydrogel with high precision has been investigated, using the precursor with hyaluronic acid vinyl ester (HAVE) as the biocompatibility hydrogel monomer, 3,3'-((((1E,1'E)-(2-oxocyclopentane-1,3-diylidene) bis(methanylylidene)) bis(4,1-phenylene)) bis(methylazanediyl))dipropanoate as the water-soluble initiator, and dl-dithiothreitol (DTT) as the click-chemistry cross-linker. The TPP properties of the HAVE precursors have been comprehensively investigated by adjusting the solubility and the formulation of the photoresist. The feature line width of 22 nm has been obtained at a processing laser threshold of 3.67 mW, and the 3D hydrogel scaffold structures have been fabricated. Furthermore, the average value of Young's modulus is 94 kPa for the 3D hydrogel, and cell biocompatibility has been demonstrated. This study would provide high potential for achieving a 3D hydrogel scaffold with highly precise configuration in tissue engineering and biomedicine.

Four-Dimensional Printing of a Fiber-Tip Multimaterial Microcantilever as a Magnetic Field Sensor

“Lab on Fiber” technology integrates different micro- and nanoscale structures or materials onto optical fibers to create additional functionality. With the advanced femtosecond (Fs) laser-induced two-photon polymerization (TPP) technology, polymer microstructures printed on a fiber tip have great potential as micro-optical sensors. However, additional functionalization is usually required, which is costly and complex. Based on the advanced four-dimensional (4D) printing theory, for the first time, a successive multimaterial on-fiber TPP strategy is proposed here, and a fiber-tip polymer microcantilever-based magnetic sensor is prepared in one step. First, the cantilever and the supporting base on the fiber tip are printed by TPP, and then the magnetically responsive cube on the cantilever tip is printed with a self-made magnetic fluid photoresist. The whole process is completed in one step without additional treatment. The prepared sensor shows a minuscule size and a high magnetic sensitivity of 119 pm mT–1 in the range of 0–90 mT, suitable for weak or microspace magnetic field measurement at high spatial resolution. Especially, the proposed 4D successive on-fiber TPP strategy can be extended to other stimulus-responsive materials, providing new guidance for manufacturing various stimulus-responsive microsensors and microactuators on the fiber tip.

Biodegradable Microrobots for DNA Vaccine Delivery.

The delivery of nucleic acid vaccine to stimulate host immune responses against Coronavirus disease 2019 shows promise. However, nucleic acid vaccines have drawbacks, including rapid clearance and poor cellular uptake, that limit their therapeutic potential. Microrobots can be engineered to sustain vaccine release and further control the interactions with immune cells that are vital for robust vaccination. Here, the 3D fabrication of biocompatible and biodegradable microrobots via the two-photon polymerization of gelatin methacryloyl (GelMA) and their proof-of-concept application for DNA vaccine delivery is reported. Programmed degradation and drug release by varying the local exposure dose in 3D laser lithography and further functionalized the GelMA microspheres with polyethyleneimine for DNA vaccine delivery to dendritic cell and primary cells is demonstrated. In mice, the DNA vaccine delivered by functionalized microspheres elicited fast, enhanced, and durable antigen expression, which may lead to prolonged protection. Furthermore, we demonstrate the maneuverability of microrobots by fabricating GelMA microspheres on magnetic skeletons. In conclusion, GelMA microrobots may provide an efficient vaccination strategy by controlling the expression duration of DNA vaccines.

Quantum Dot Transfer from the Organic Phase to Acrylic Monomers for the Controlled Integration of Single-Photon Sources by Photopolymerization

This paper reports on a new strategy for obtaining homogeneous dispersion of grafted quantum dots (QDs) in a photopolymer matrix and their use for the integration of single-photon sources by two-photon polymerization (TPP) with nanoscale precision. The method is based on phase transfer of QDs from organic solvents to an acrylic matrix. The detailed protocol is described, and the corresponding mechanism is investigated and revealed. The phase transfer is done by ligand exchange through the introduction of mono-2-(methacryloyloxy) ethyl succinate (MES) that replaces oleic acid (OA). Infrared (IR) measurements show the replacement of OA on the QD surface by MES after ligand exchange. This allows QDs to move from the hexane phase to the pentaerythritol triacrylate (PETA) phase. The QDs that are homogeneously dispersed in the photopolymer without any clusterization do not show any significant broadening in their photoluminescence spectra even after more than 3 years. The ability of the hybrid photopolymer to create micro- and nanostructures by two-photon polymerization is demonstrated. The homogeneity of emission from 2D and 3D microstructures is confirmed by confocal photoluminescence microscopy. The fabrication and integration of a single-photon source in a spatially controlled manner by TPP is achieved and confirmed by auto-correlation measurements.

Synthesis of biocompatible BSA-GMA and two-photon polymerization of 3D hydrogels with free radical type I photoinitiator.

Although the development of three-dimensional (3D) printing technology is growing rapidly in the biomedical field, it remains a challenge to achieve arbitrary 3D structures with high resolution and high efficiency. Protein hydrogels fabricated by two- photon polymerization (TPP) have excellent mechanical properties, high precision, and 3D architecture. However, a large number of the amino acid group in bovine serum albumin (BSA) would be consumed when the protein-based hydrogels use dyes of free radical type II photoinitiators. In this study, we use glycidyl methacrylate (GMA) to modify BSA molecules to obtain a series of BSA-GMA materials, allowing the protein material to be two-photon polymerized with a water-soluble free radical type I photoinitiator. The precisely controllable 3D structure of the BSA-GMA hydrogel was fabricated by adjusting the concentration of the precursor solution, the degree of methacrylation, and the processing parameters of the TPP technique. Importantly, BSA-GMA materials are free of acidic hazardous substances. Meanwhile, the water-soluble initiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphite (LAP) allows TPP on the vinyl group of the GMA chain and thus without consuming its amino acid group. The as-prepared BSA-GMA hydrogel structure exhibits excellent autofluorescence imaging, pH responsiveness, and biocompatibility, which would provide new avenues for potential applications in tissue engineering and biomedical fields to meet specific biological requirements.

Direct laser writing structural color for reversible encryption and decryption in different mediums.

Structural color (SC) has enormous potential for improving the visualization and identification of functional micro/nano structures for information encryption and intelligent sensing. Nevertheless, achieving the direct writing of SCs at the micro/nano scale and the change of color in response to external stimuli simultaneously is rather challenging. To this end, we directly printed woodpile structures (WSs) utilizing femtosecond laser two-photon polymerization (fs-TPP), which demonstrated obvious SCs under an optical microscope. After that, we achieved the change of SCs by transferring WSs between different mediums. Furthermore, the influence of laser power, structural parameters, and mediums on the SCs was systematically investigated, and the mechanism of SCs using the finite-difference time-domain (FDTD) method was further explored. Finally, we realized the reversible encryption and decryption of certain information. This finding holds broad application prospects in smart sensing, anti-counterfeiting tags, and advanced photonic devices.

Aberration-compensated supercritical lens for sub-diffractive focusing within 20° field of view.

The supercritical lens has shown a remarkable capability of achieving far-field sub-diffraction limited focusing through elaborating a modulated interference effect. Benefiting from the relative high energy utilization efficiency and weak sidelobe properties, the supercritical lens holds significant advantage in a series of application scenarios. However, all of the demonstrated supercritical lenses mainly work in the on-axis illumination condition, so the off-axis aberration effect will severely deteriorate its sub-diffraction limit focusing capability for the illuminating beam with an oblique angle. In this work, an aberration-compensated supercritical lens with single-layer configuration is proposed and experimentally demonstrated. Such a single-layer supercritical lens consists of multilevel phase configurations patterned with the two-photon polymerization lithography technique. The simulation and experimental recorded results show that the aberration-compensated supercritical lens with a numerical aperture value of 0.63 could achieve a far-field sub-diffraction limited focusing property within 20° field of view at a wavelength of λ = 633 nm. This monochromatic aberration-compensated supercritical lens with single-layer configuration indicates excellent potential in the development of laser scanning ultrahigh optical storage and label free super-resolution imaging.