Mie-Resonant Nanophotonic-Enhancement of Asymmetry in Sodium Chlorate Chiral Crystallization.

Studies on chiral spectroscopy have recently demonstrated strong enhancement of chiral light-matter interaction in the chiral near-field of Mie resonance in high-refractive-index dielectric nanostructures by studies on chiral spectroscopy. This situation has motivated researchers to demonstrate effective chiral photosynthesis under a chiral near-field beyond circularly polarized light (CPL) as a chiral source. However, the effectivity of the chiral near-field of Mie resonance for chiral photosynthesis has not been clearly demonstrated. One major challenge is the experimental difficulty in evaluating enantiomeric excess of a trace amount of chiral products synthesized in the near-field. Here, by adopting sodium chlorate chiral crystallization as a phenomenon that includes both synthesis and the amplification of chiral products, we show that crystallization on a Mie-resonant silicon metasurface excited by CPL yields a statistically significant large crystal enantiomeric excess of ∼18%, which cannot be achieved merely by CPL. This result provides implications for efficient chiral photosynthesis in a chiral near-field.

Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Spatiotemporal Control of Polymorphic Phase Transition of Glycine Crystals by Three-Dimensional Femtosecond Laser Ablation Processing.

Spatiotemporal control of the polymorphic phase transition of glycine crystals was demonstrated by three-dimensional (3D) processing with a focused femtosecond laser pulse as an external stimulus. We found that the transition from a metastable form (ß-form) to more stable ones (α- or γ-form) could be triggered from the irradiated area of not only the surface but also inside of glycine crystals. This 3D processing with a focused femtosecond laser pulse enabled us to precisely monitor the transition dynamics from a targeted position to the entire part of crystals. The systematic study with the space-selective phase transition method revealed that the phase transition inside of glycine crystals was significantly slower (e.g., ∼50 times) than that at the crystal surface, which indicates the crucial role of water molecules in air on the phase transition dynamics. We foresee that this laser method can be a practical tool for monitoring spatiotemporal dynamics of phase transition.

Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells.

Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).1,2,3.

Surface-limited reactions for spatial control of kinesin-microtubule motility assays using indirect irradiation of an electron beam.

Gliding of microtubules (MTs) on kinesins has been applied to lab-on-a-chip devices, which enable autonomous transportation and detection of biomolecules in the field of bioengineering. For rapid fabrication and evaluation of the kinesin-MT based devices, optical control techniques have been developed for control of kinesin activity and density; however, use of caged molecules lacks spatial controllability for long-term experiments, and direct irradiations of UV light onto kinesin-coated surfaces are inherently damaging to MTs due to their depth limit since the heights of the kinesin-MT systems are at the tens of a nanometer scale. Considering surface electric fields in electrolytic solutions are shielded at the nanometer scale due to Debye shielding, in this study, we show that fine spatial control of kinesin density and activity is enabled using surface-limited electrochemical reactions induced by indirect irradiations of an electron beam (EB). An EB is indirectly irradiated onto the kinesins through a 100-nm-thick silicon nitride membrane, and the electrons scattered in the membrane can cause localized electrochemical effects to the kinesins. We show that these localized electrochemical effects cause both ablation of kinesins and motility control of kinesin activity by changing the EB acceleration voltage. In particular, the latter is achieved without complete ablation of MTs, though the MTs are indirectly irradiated by the EB. As a demonstration of on-demand control of gliding MTs, we show the accumulation of the MTs on a target area by scanning the EB. The proposed accumulation technique will lead to rapid prototyping of microdevices based on MT-kinesin motility assay systems.

Mechanical guidance of self-condensation patterns of differentiating progeny.

Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG > 0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.

Quantitative evaluation of the impact of artificial cell adhesion via DNA hybridization on E-cadherin-mediated cell adhesion.

Programmable cell adhesion with DNA hybridization is a promising approach for fabricating various tissue architectures without sophisticated instrumentation. However, little is known about how this artificial interaction influences the binding of cell adhesion proteins, E-cadherin. In this work, we designed a planar and fluid lipid membrane displaying E-cadherin and/or single-strand DNA with well-defined densities. Visualization of cells on membranes by fluorescence and interference microscopy revealed cell adhesion to be a two-step process: artificial adhesion by DNA hybridization within a few minutes followed by biological adhesion via cadherin-cadherin binding within hours. Furthermore, we discovered that DNA hybridization can substantially facilitate E-cadherin-mediated cell adhesion. The promotive effect is probably due to the enforced binding between E-cadherin molecules in geometrical confinement between two membranes. Our in vitro model of cell adhesion can potentially be used to design functional synthetic molecules that can regulate cell adhesion via cell adhesion proteins for tissue engineering.

Pause of the target gliding microtuble on the virtual cathode.

We report the transient response of gliding microtubules on a virtual cathode. In vivo activities, microtubule-kinesin systems are known to act as motor proteins with respect to cell motility cytokinesis and cellular transport by hydrolyzing ATP molecules. With development of in vitro assays, motor proteins have been attracting much attention as a key component for highly efficient nano-transportation systems. The molecular functions based on structural states are affected by changing the ionic condition of the molecular functions and by changing the electrical field in solution because of electrical charges of the molecules. The virtual cathode, which was generated on the SiN display surface by a low energy electron beam, locally induced electrochemical reactions and electric field around the targeted molecules on the display surface, and then the gliding motions of the targeted microtubules were regulated. In this study, we demonstrated that the virtual cathode display temporally stops a selected gliding microtubule by only applying the virtual cathode to the microtubule. The pause mode of the microtubule was easily canceled by simply turning the virtual cathode off, and then the gliding motion was restarted.

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

Design of Polymer Networks Involving a Photoinduced Electronic Transmission Circuit toward Artificial Photosynthesis.

Many strategies have been explored to achieve artificial photosynthesis utilizing mediums such as liposomes and supramolecules. Because the photochemical reaction is composed of multiple functional molecules, the surrounding microenvironment is expected to be rationally integrated as observed during photosynthesis in chloroplasts. In this study, photoinduced electronic transmission surrounding the microenvironment of Ru(bpy)3(2+) in a polymer network was investigated using poly(N-isopropylacrylamide-co-Ru(bpy)3), poly(acrylamide-co-Ru(bpy)3), and Ru(bpy)3-conjugated microtubules. Photoinduced energy conversion was evaluated by investigating the effects of (i) Ru(bpy)3(2+) immobilization, (ii) polymer type, (iii) thermal energy, and (iv) cross-linking. The microenvironment surrounding copolymerized Ru(bpy)3(2+) in poly(N-isopropylacrylamide) suppressed quenching and had a higher radiative process energy than others. This finding is related to the nonradiative process, i.e., photoinduced H2 generation with significantly higher overall quantum efficiency (13%) than for the bulk solution. We envision that useful molecules will be generated by photoinduced electronic transmission in polymer networks, resulting in the development of a wide range of biomimetic functions with applications for a sustainable society.

Kinesin-Driven Active Substrate Giving Stochastic Mechanical Stimuli to Cells for Characterization.

We present a new platform to give stochastic mechanical stimuli to cells for their characterization. There nano- and micrometer scaled fluctuations are generated by an engineered motor protein system of kinesin-microtubules (MTs) on a solid surface. Cells have abilities to deform in many ways during homeostatic metabolism, tissue forming processes, cancer developments, and so on. Namely, cells in biological tissues are exposed to noise-like stochastic movements at nano- and micrometer-scales, which mainly come from the mechanical environment surrounding the cells. Although cells seem to have the potential to respond to such types of mechanical stimuli, the influences on cellular behaviors are poorly understood. As a first attempt to verify an effect of noise-like mechanical stimuli in vitro, we prepared a system to give stochastic mechanical stimuli to cells using a technique of in vitro motility assay for a kinesin-MT system. An active substrate was obtained by integrating movements of MTs on a kinesin-coated glass surface via cross-linkage, and stochastic mechanical stimuli at the cell-scale were successfully applied to the seeded cells. There, traveling distances of the cells over one cell length were observed until they started to adhere. When metastatic melanoma cells were exposed to the stochastic mechanical stimuli, unusually long protrusions or extensions of cell bodies were observed. Cellular aggregations were also promoted through the movements on this active substrate which could disturb the landing and enhance the collisions of the cells. This approach giving mechanical stimuli to cells in a stochastic manner at nano- and micrometer-scales might allow us to uncover unknown behaviors of cells, which might contribute to research fields requiring our understanding on the mechanical nature of cells, such as cancer diagnosis and regenerative medicine.

Oscillating high-aspect-ratio monolithic silicon nanoneedle array enables efficient delivery of functional bio-macromolecules into living cells.

Delivery of biomolecules with use of nanostructures has been previously reported. However, both efficient and high-throughput intracellular delivery has proved difficult to achieve. Here, we report a novel material and device for the delivery of biomacromolecules into live cells. We attribute the successful results to the unique features of the system, which include high-aspect-ratio, uniform nanoneedles laid across a 2D array, combined with an oscillatory feature, which together allow rapid, forcible and efficient insertion and protein release into thousands of cells simultaneously.

Effect of microtubule polymerization on photoinduced hydrogen generation.

Herein we report a novel reaction field for photoinduced H2 generation by using microtubules as a medium. By controlling the tubulin/microtubule hierarchical structure, synergistic effects by which the Ru(bpy)3(2+)-conjugated microtubule network causes suppression of energy loss by collision are clarified.

Microtubule teardrop patterns.

Several strategies for controlling microtubule patterns are developed because of the rigidity determined from the molecular structure and the geometrical structure. In contrast to the patterns in co-operation with motor proteins or associated proteins, microtubules have a huge potential for patterns via their intrinsic flexural rigidity. We discover that a microtubule teardrop pattern emerges via self-assembly under hydrodynamic flow from the parallel bundles without motor proteins. In the growth process, the bundles ultimately bend according to the critical bending curvature. Such protein pattern formation utilizing the intrinsic flexural rigidity will provide broad understandings of self-assembly of rigid rods, not only in biomolecules, but also in supramolecules.

Controlling the bias of rotational motion of ring-shaped microtubule assembly.

Biomolecular motor system microtubule (MT)-kinesin is considered a building block for developing artificial microdevices. Recently, an active self-organization method has been established to integrate MT filaments into ring-shaped assembly that can produce rotational motion both in the clockwise and in the counterclockwise directions. In this work, we have investigated the effect of parameters such as MT and kinesin concentration, length, and rigidity of MT and type of kinesin (structure of tail region) on the preferential rotation of the ring-shaped MT assembly produced in an active self-organization. We elucidated that these factors can significantly affect the bias of rotation of the ring-shaped MT assembly, which seems to be related to the fluctuation of leading tip of moving MT filaments. This new finding might be important for designing handedness regulated artificial biomachine using the ring-shaped MT assembly in future.

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy.

The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered-integrin bindings link cells mechanically to the ECM. In order to quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin-substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.

Detection of microtubules in vivo using antibody-immobilized nanoneedles.

We present here an alternative, force-based measurement method for the detection of intracellular cytoskeletal proteins in the live cell. High aspect ratio nanoneedles of 200 nm in diameter were functionalized with anti-tubulin antibodies and inserted, using an atomic force microscope (AFM), into live NIH3T3 cells, without affecting cell viability. Force curves were recorded during insertion and evacuation of nanoneedles from the cells, and used to analyse intracellular interactions of the nanoneedles with the microtubule cytoskeleton during evacuation from the cell. Disruption of microtubules led to a correlated time-dependent decrease in the measured intracellular binding forces, pointing to the high-sensitivity and high-specificity of this detection method. This analytical technique allows for real-time evaluation of the microtubule network in the live cell, without the need to use potentially harmful molecular markers as do conventional detection methods, and may prove beneficial in the diagnosis and investigation of cytoskeleton-associated diseases.

Thermo- and photo-enhanced microtubule formation from Ru(bpy)3 2+-conjugated tubulin.

Ru(bpy)3 2+-conjugated tubulin is able to substantially enhance polymerization to form microtubules with increased rate at lower temperatures. Additionally, the polymerization is enhanced by photo-irradiation and the possible mechanism is discussed focusing on the photo-thermal energy conversion.

Controlled cell adhesion using a biocompatible anchor for membrane-conjugated bovine serum albumin/bovine serum albumin mixed layer.

We report here a method for controlling cell adhesion, allowing simple yet accurate cell detachment from the substrate, which is required for the establishment of new cytometry-based cell processing and analyzing methods. A biocompatible anchor for membrane (BAM) was conjugated with bovine serum albumin (BSA) to produce a cell-anchoring agent (BAM-BSA). By coating polystyrene substrates with a mixture of BAM-BSA and BSA, controlled suppression of the substrate's adhesive properties was achieved. Hook-shaped nanoneedles were used to pick up cells from the substrate, while recording the cell-substrate adhesion force, using an atomic force microscope (AFM). Due to the lipid bilayer targeting property of BAM, the coated surface showed constant adhesion forces for various cell lines, and controlling the BAM-BSA/BSA ratio enabled tuning of the adhesion force, ranging from several tens of nano-Newtons down to several nano-Newtons. Optimized tuning of the adhesion force also enabled the detachment of cells from BAM-BSA/BSA-coated dishes, using a shear flow. Moreover, the method was shown to be noncell type specific and similar results were observed using four different cell types, including nonadherent cells. The attenuation of cell adhesion was also used to enable the collection of single cells by capillary aspiration. Thus, this versatile and relatively simple method can be used to control the adhesion of various cell types to substrates.

Nanoneedle insertion into the cell nucleus does not induce double-strand breaks in chromosomal DNA.

An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs.