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In this letter, we conducted a new method to fabricate channels inside cell structures for tissue engineering applications. A cell-embedded hydrogel is locally melted to fabricate channels inside cell structures. The fabricated channels can be used as vascular-like networks to supply nutrition and oxygen to cells, or patterning another types of cells inside the cell structures. Microelectrodes were prepared as heaters on a glass substrate by photolithography techniques. The channel patterning was conducted using the microelectrodes. The channel width and height are controlled by the heating duration when the applied voltage to the microelectrodes is constant. The multi-layered channel fabrication was achieved by repeating melting-filling hydrogel. Patterning of different cell types in hydrogel was conducted using liver cells and fibroblast cells. The proposed method can fabricate vascular-like channels in cell structures to achieve in vitro 3-D co-culture cell systems.

Constructing 3D cell module with certain shape and high cell density is a very important issue for artificial tissue engineering. In this letter, we present a novel method of fabricating compact microtissue with gear-like shape by electrodepositing of $\text{Ca}^{2+} $ induced cross-linked alginate hydrogel. The fabrication procedure is demonstrated and the fabricated gear-like tissues are characterized. Briefly, an array of gear-shaped microelectrodes are first fabricated by coating a patterned photoresist layer onto fluorine-doped tin oxide (FTO) glass slide based on photolithography technique. Then, Ca-alginate gel structures embedded with RLC-18 (rat liver) cells are electrodeposited onto the microelectrodes. These 2D cell-laden gel structures are then manually detached from the microelectrodes and treated with special reagent to be transformed into 3D alginate-Poly-L-lysine (PLL) microcapsules while maintaining the gear shape. Finally, the RLC-18 cells within the microcapsules are further promoted into compact microtissues by cultivation. The size of the fabricated gear-like tissue is about $975{\pm }{25}\;\upmu \text{m}$ in outer diameter and $320{\pm }{10}\;\upmu\text{m}$ in inner diameter based on the design of microelectrodes. 3-D images of the fabricated microtissues are obtained by a confocal microscope. The experimental results indicate the proposed method can successfully fabricate movable microtissues with predefined gear shape while maintaining the viability ( $\sim 94\%$ ). We believe the fabricated movable gear-like microtissues can be further assembled and applied to tissue engineering.

Microfluidic devices employed as “printing” head provide a mild condition to fabricate cell-laden hydrogel modules for three-dimensional (3D) assembly to create cellular constructs. However, because of the poor controllability of hydrogels and unstable microfluidic fabrication process, it remains a challenge to fabricate morphologically accurate structures to mimic in vivo tissues, which hinders the building of in vitro models of organs. In this paper, we combine a magnetic-driven strategy into a microfluidic “printing” method to handle this challenge. To enhance the controllability, we encapsulate magnetic nanoparticles (MNPs) into cell-laden alginate hydrogel microfibers and then magnetically assemble these microfibers on the surface of designed support models. To keep a continuous spinning process, we immerse the spinning orifice of microfluidic device into phosphate-buffered saline filled in a Petri dish to eliminate the influence of droplets generated during microfibers ejection. Meanwhile, a dextran flow impulse is employed to prevent the blockage of microchannels. Interestingly, this impulse can achieve to temporarily cease the spinning process. Moreover, an optimized magnetic assembly is achieved by considering both the assembling area on a ring magnet and the MNPs concentration in microfibers. After the test of cell survival, a high cell viability of 97.2 % can be confirmed in assembled structures, which indicates that our method allows a biocompatible assembly of cell-laden hydrogels to build macroscopic 3D cellular structures similar to tissues observed in vivo.

We studied bending of multilayered graphene into 3D structure. In this paper, we proposed a nanogripper folded by multilayered graphene as one of the 3D device. The nanogripper is actuated by electro static forces to open and close. The multilayered graphene was fabricated by focused ion beam (FIB) to form nano-gripper design. Reversible action is realized by controlled two different electrodes. In the experimental section, the relationship of actuation distance and applied voltage was evaluated for folded multilayered graphene.

Field-effect transistors (FETs) have been developed from silicon based to carbon nanotubes (CNTs) based, and the fabrication space became three-dimensionl (3D). Such fabrication process requires to accurately assemble a single CNT in 3D. However, most of the current assembly technologies were used for planar structures but not for 3D structures. In this study, we aim to use nanomanipulation based on a scanning electron microscopy (SEM) to realize the 3D assembly. To achieve this goal, we first proposed a novel 3D structure named Tri-gate CNT-FET. The Tri-gate CNT-FET has three cuboid micro-electrodes and it is wrapped by CNTs with front, top and back sides. After fabrication of the electrodes, a single CNT was picked up by an Au-coated probe and placed on the front side of the three micro-electrodes by suspending over a substrate to a certain height. The CNT pick-up and placement highly depended on attractive interactions at a CNT-metal contact interface by van der Waals force. Electron beam induced deposition (EBID) technique was then used to deposit Tungsten at the interface to fix CNT. Mechanical cutting was finally carried out to release the probe from the assembled structure. The whole assembly was achieved by using only one nanomanipulator. Experiment results validated our proposed 3D assembly method for the fabrication of Tri-gate CNT-FET.

This study presents the high-precision microinjection of fluorescent micro-gel beads into Caenorhabditis elegans trapped in a suction microchannel. In our previous works, we demonstrated survival microinjection by a conventional micromanipulation technique. However, the focal planes differed between the tip of the microinjection tool and the target axon inside the C. elegans body. To resolve this problem, we here propose a suction microchannel that traps C. elegans during the microinjection operation. The focal plane of the target nerve axon matches that of the fluorescent microbead in the microinjection tool, enabling high-precision microinjection into the interior of the C. elegans body under a microscopic view. In an experimental evaluation, the positioning accuracy of the injection into C. elegans was within the target accuracy (15 μm). The head-flrst navigation alignment of C. elegans along the microchannel was controlled by electrotaxis. Injection of the fluorescent micro-gel bead into the C. elegans body was quantitatively confirmed by confocal microscopy.

The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological change between the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversion percentage based on the OD800absorbance data. From the optical microscope observations as expected, the normal cells had an oval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is, yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl2(CaS). It was also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile water mediums. The OD800absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-like surface after the loss of its integrity when removing the cell wall.

In this paper, we propose a new method to construct microchannels inside cell structures. A local melting of cell embedded hydrogel is employed to make microchannels inside cell structures. Local melting of hydrogel was conducted using the fabricated electrodes. Electrodes were fabricated using photolithography technique on a glass substrate. Electrode designs were prepared ladder-shape. The ladder-shape is used to demonstrate fabrication of arbitrary shapes of channel. The hydrogel block was placed on the electrodes and 100 μm width electrode was used to melt the hydrogel. The hydrogel was prepared at the concentration of 5.6 % gelatin and 1.4 % agar. The results indicate that the proposed method can be used to making vascular-like networks inside cell structures to construct in vitro 3D cell systems.

In this study, the internal structure of collagen scaffold is observed by a scanning electron microscope-computed tomography (SEM-CT) technique operated inside an environmental-scanning electron microscope (E-SEM). The E-SEM is able to use maintain the aqueous condition inside the sample by controlling the temperature and pressure. We demonstrated the SEM-CT observation of collagen scaffold by culturing osteoblast cells in certain culture term, and confirmed the calcification change of osteoblast cells.

In this paper, a novel approach is presented to obtain the cell sheets with mimicking the morphology of liver lobule tissue by electrodeposition method. We would take some steps to gain the sheets. It includes: 1) fabricate microelectrode which coated pattern onto fluorine-doped tin oxide (FTO) glass based on photolithography technique. 2) electrodeposition experiment which embedded with RLC-18 (rat liver) cells. 3) detach the sheet from the microelectrode by shaking it. 4) assemble 3D tissue structure. In the result, precise pattern in the sheet could be kept and the viability of the cells could be maintained. We also checked the height of the sheet. We believe the use of cell sheets by electrodeposition method is great potential in fabricating 3D cell structures.

Carbon nanotubes (CNT) have been developed in recent decades for nanodevices such as nanoradios, nanogenerators, carbon nanotube field effect transistors (CNTFETs) and so on, indicating that the application of CNTs for nanoscale electronics may play a key role in the development of nanotechnology. Nanorobotics manipulation systems are a promising method for nanodevice construction and assembly. For the purpose of constructing three-dimensional CNTFETs, a nanorobotics manipulation system with 16 DOFs was developed for nanomanipulation of nanometer-scale objects inside the specimen chamber of a scanning electron microscope (SEM). Nanorobotics manipulators are assembled into four units with four DOFs (X-Y-Z-θ) individually. The rotational one is actuated by a picomotor. That means a manipulator has four DOFs including three linear motions in the X, Y, Z directions and a 360-degree rotational one (X-Y-Z-θ stage, θ is along the direction rotating with X or Y axis). Manipulators are actuated by picomotors with better than 30 nm linear resolution and

This paper presents a electrostatic actuation of multi-graphene which was folded by nanomanipulation for novel nano-gripper application. The development design of multi-graphene was initially fabricated by focused ion beam (FIB) process. The nanomanipulation system was used for folding and electrostatic actuation inside a FESEM (Field Emission Scanning Electron Microscope). The movement of multi-graphene was confirmed by electrostatic forces. The actuation was evaluated depending on the applied voltages.

Nanotool feeding system (NTFS) is presented for efficient nanomanipulation. We constructed a unique bio-nanomanipulation system inside an Environmental-SEM (E-SEM) using various nanotools, for example stiffness measurement nanotool, dual electrical nanoprobe, nanopicker, nanofork, nanocutter, nanoputter, and nanoinjector. For continues usage of different multiple tools, it needs to exchange the nanotools continuously. In this paper, the Rotary-table Nanotool Feeding System (R-NTFS) is proposed to realize continuous exchange of different multiple tools individually. An efficient continuous nanomanipulation was achieved by a short exchange time which was about 13 seconds. Finally, the multiple nanoinjection was demonstrated using the proposed R-NTFS.

In vitro tissues are significant biological substitute for drug test, cell morphogenesis exploration and organ transplantation. In this letter, a novel microrobotic bioassembly method is proposed to engineer 3-D cellular structure, which can be utilized to culture in vitro tissue with microstructural features. A microrobotic system is set up under optical microscope with 30 nm positioning resolution. To assemble cellular micromodules, the system repeats a programmed single-step contact motion with one manipulator, which effectively avoids human intervention and frequent alignment of micromanipulators. The contact force is analyzed and the system is modeled to be a cantilever handing beam for dynamic force characterization. Through the characterization, we figure out the key parameter as the stiffness of micromanipulator at contact point, which influences assembly success rate. After the calibration of stiffness, the high-speed assembly of cellular structure is performed with an assembly speed of 20 micromodules/min. Finally, the 3-D cellular structure incorporating vascular-like microtube is engineered for the fabrication of in vitro micro tissues.

Semiconductor nano-devices based on carbon nanotubes (CNTs) have attracted great interests recently such as field effect transistors (CNTFETs). One of the challenges is to reduce the contact resistance between CNT and electrodes. This paper presents a robotic system with two nano-manipulators for two-lead contact resistance measurement. Electron beam induced deposition (EBID) technique was applied to decrease the contact resistance between multi-walled CNTs (MWCNTs) and Tungsten deposition fabricated by focused ion beam (FIB). A Tungsten (W) probe fixed on one manipulator is used to pick up a CNT selectively from a CNT bundle. The picked CNT was positioned to approach a W deposition on an atomic force microscopy (AFM) cantilever. The cantilever mounted on another manipulator was coordinated by positioning for well contact between the W deposition and CNT. To strengthen this contact, four W points are deposited on the contact area of CNT for totally 9min by EBID. A resistance between W probe and cantilever was measured before and after EBID. Based on these measured resistance, contact resistance was calculated by a theoretical model. It showed the contact resistance of MWCNT/Tungsten deposition was reduced by 98.8% by the EBID deposition.

In this paper, we constructed three dimensional (3D) microchannel networks inside gelatin-based hydrogel towards 3D in vitro cell structures. To achieve functional 3D cell structures, vascular networks are important to supply oxygen and nutrients to cells, and remove waste product from cells. An alginate hydrogel fibers were employed to construct the microchannel networks like blood vessels. The pattern of the hydrogel fiber can be fixed using magnetic force and the fibers are embedded in the gelatin-based hydrogel. Cells can be embedded inside the gelatin-based hydrogel. The patterned hydrogel fiber can be melted using standard saline citrate (SSC) buffer and the melted area can be used as vascular networks. The flow inside the melted alginate hydrogel fibers were visualized using fluorescent microbeads. The results validate that the proposed method can be used to make arbitrary patterns of vascular networks inside cell structures and contribute to achieve in vitro 3D cell structures.

In situ nanomanipulation system is presented with 3D scanning electron microscope - computed tomography (SEM-CT) system integrated with inside an environmental - SEM (E-SEM). The E-SEM was used to achieve a real-time high resolution imaging for high precision nanomanipulation. The SEM-CT system was used for obtaining 3D cross-sectional image with sub-micron high resolution. In this paper, Caenorhabditis elegans (C. elegans) was used as a target of biological sample to demonstrate a nanoinjection operation. The nanoinjection was determined by the SEM-CT system with its 3D information.

In this paper, we propose a new method to construct microchannels inside cell structures. A local melting of cell embedded hydrogel is employed to make microchannels inside cell structures. The fabricated microchannels can be used as vascular-like networks to supply nutrition and oxygen to cells. Electrodes were fabricated using photolithography technique on a glass substrate. The hydrogel to embed cells were prepared by mixing gelatin and agar in culture media. A microchannel was experimentally fabricated using the fabricated electrodes to locally melt the hydrogel on the electrodes. The fabricated channel width and height by local heating was changed proportionally by the heating time when the applied voltage to the electrodes was constant. The results indicate that the proposed method can be used to making vascular-like networks inside cell structures to construct in vitro 3D cell systems.

In this study, we describe a novel method of fabricating shape-controlled calcium alginate gel microcapsules. Alginate-poly-L-lysine (PLL) hydrogel microcapsules with predefined shapes were constructed based on electrodeposition method. Firstly, electrolysis of water in alginate solutions with calcium carbonate particles induced alginate gelation on micro-patterned electrode to form 2D gel structures. Then, these structures will be detached from the electrode surface and treated with the alginate-PLL microcapsules system. By passive control based on the micro-pattern geometric confinement and electrodeposition parameters, we succeeded in producing calcium alginate-PLL microcapsules with diverse shapes (such as sphere rod and cubic). The shape and size of the calcium alginate microcapsules could be tuned by adjusting the geometric design of micro-pattern on electrode and the apply voltage of electrodepostion. The preparation conditions of size- and shape-controlled calcium alginate-PLL microcapsules and influence factors were studied. This proposed method can lead to more accurate and creative studies of fabricating biocompatibility scaffold for tissue engineering.

This paper presents a fabrication method of multi-graphene box structure by a nanomanipulation in a Field Emission Scanning Electron Microscope (FESEM). To bend the multi-graphene in plastic deformation region, the bending stress were measured using a silicon cantilever based on the nanomanipulation. In this study, defects were introduced inside the multi-graphene on the folding line of the multi-graphene by irradiating an electron beam assisted with oxygen gas. From experimental bending stress measurement, the bending stress was dropped with electron beam irradiation in oxygen gas environment. The fabrication of 3D box structure of multi-graphene was demonstrated from a cubic net of multi-graphene which was fabricated by a Focused Ion Beam (FIB) etching process. For bending the one side of the cubic net graphene, a tungsten probe was used after reforming by FIB etching as end-effector of nanomanipulator. The multi graphene was bent to form a box structure in 3D space.

A nanolaboratory is proposed to realize the capabilities of nanofabrication, nanoinstrumentation and nanoassembly to make three-dimensional structures, devices and systems in the nanoworld. A nanorobotic manipulation system is introduced to realize these capabilities inside a scanning electron microscope or a transmission electron microscope. Some precursors are introduced into the small working space in the nanoworld, so that cutting, bending, peeling and fixing operations in the nanoworld could be realized using nanomanipulators, electron-bean-induced deposition and other methodologies as in the macroworld. After making the three-dimensional structures, the nanodevices such as nanosensors and nanoactuators are fabricated using carbon nanotubes and a focused ion beam chemical vapor deposition process. Some nanodevices for bioapplications are also presented.

This paper presents the hybrid nanowire growth via field emission based on nanorobotic manipulations inside field emission scanning microscope (FESEM). The hybrid nanowires are made of different kinds of materials. A multi-walled carbon nanotube (MWNT) was used as an emitter. A tungsten probe was positioned as anode at counter position by nanomanipulation. By changing the trimethyl cyclopentadienyl platinum (CpPtMe 3 ) and tungsten hexacarbonyl (W(CO) 6 ) as precursors, the metallic nanowires were grown via field emission for several minutes. The growth processes was repeated to control the length of nanowires. Finally, the hybrid nanowire was fabricated by growing the tungsten nanowires at the tip of platinum nanowire. The components of hybrid nanowires were evaluated by the energy-dispersive X-ray spectroscopy (EDS) of transmission electron microscope (TEM). The hybrid nanowire has various applications in nanoelectronics, nanosensor device, nanomechanical system and so on.

A nanolaboratory is one of the systems to realize various nanoscale fabrications and assemblies to develop novel nanodevices to integrate borderless technologies based on a nanorobotic manipulation system. We have presented the nanolaboratory inside electron microscopes including a transmission electron microscope (TEM), scanning electron microscope (SEM), and environmental-SEM (E-SEM) for three-dimensional (3D) and real-time nanomanipulation, nanoinstrumentation, and nanoassembly. The following is a presentation of our current work of nanomanipulation and nanoassembly based on the hybrid nanorobotic manipulation inside a TEM and an SEM toward carbon nanotube (CNT) applications. Single cell stiffness measurement has been also presented based on the nanorobotic manipulation system inside an E-SEM.

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galbeta1-->3GlcNAc, LNB) and galacto-N-biose (Galbeta1-->3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBPBl). In the presence of alpha-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a kcat/Km value for GNB that was approximately 50 times higher than that for LNB, whereas LNBPBl showed similar kcat/Km values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-alpha-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.

Carbon nanotubes (CNTs) are potentially useful for developing functional nanodevices such as thermal probes, flow sensor arrays, and AFM probes. Controlling the growth position of CNTs grown by chemical vapor deposition (CVD) is crucial to fabrication of these nanodevices. We propose an easy, non-lithographic process of making CNT sensor probes using CVD. Growth positions of CNTs were precisely controlled by depositing a patterned barrier layer above the catalyst layer. Then, the CNTs were grown from the exposed catalyst layer using CVD. This method is easily applied to assembling bridged CNTs. We used focused ion beam etching to remove the barrier layer and expose the catalyst membrane. Through the proposed method, we made a bridge of CNTs at the tip of a Si edge. This type of bridge can be used as the nanoprobes for local sensing of physical properties.

We report nanomanipulation and nanoassembly of carbon nanotubes (CNTs) through nanorobotic manipulation inside electron microscopes. A hybrid nanorobotic manipulation system, which is integrated with a nanorobotic manipulator inside a transmission electron microscope (TEM) and nanorobotic manipulators inside a scanning electron microscope (SEM), is used. The elasticity of a multi-walled CNT (MWNT) is measured inside a TEM. The telescoping MWNT is fabricated by peeling off outer layers through destructive fabrication process. The electrostatic actuation of telescoping MWNT is directly observed by a TEM. A cutting technique for CNTs assisted by the presence of oxygen gas is also presented. The cutting procedure was conducted in less than 1 minute using a low-energy electron beam inside a scanning electron microscope. A bending technique of a CNT assisted by the presence of oxygen gas is also applied for the 3-D fabrication of nanostructure. We expect that these techniques will be applied for the rapid prototyping nanoassembly of various CNT nanodevices.

The electrostatic actuation of a telescoping multiwalled carbon nanotube (MWNT) is presented. Its inner core was actuated by applying electric voltages between the telescoping nanotube and a counter-electrode inside a transmission electron microscope (TEM). The telescoping nanotube was fabricated by peeling off its outer layers through a destructive fabrication process inside the TEM. Its inner core extended (max: ~179 nm) and retracted with applied DC voltage (0–65 V). Estimations of electrostatic forces by finite element method (FEM) and van der Waals interactions indicated that the actuation force is mainly caused by electrostatic forces. These experimental processes were carried out through a hybrid nanorobotic manipulation system, which can be used inside both a scanning electron microscope (SEM) and a TEM. The present result shows the possibility of nanoscale actuator applications using a telescoping MWNT with precision.

Carbon nanotubes have been used as building blocks for assembly of nanostructures and nanodevices. Therefore, the interaction between the carbon nanotube and the environment is important. Interaction forces which were mainly consisted of van der Waals forces between a single multi-walled carbon nanotube tip and a gold surface were investigated and measured with an atomic force microscope cantilever. We also found that the electron beam irradiation can increase the interaction forces. The Hamaker constant for the CNT and the gold surface was obtained from the theoretical calculation and the experimental results. The results of the experiment provide good agreement with theoretical calculation.

Fabricated vessel-mimetic microtubes are essential for delivering sufficient nutrient to engineered composite tissues. In this paper, vascular-like microtubes are engineered by automated assembly of donut-shaped micromodules that embed fibroblast cells. A microrobotic system is set up with dual manipulators of 30-nm positioning resolution under an optical microscope. The system assembles the micromodules by repeated single-step pick-up motions. This process is specifically designed to avoid human interference and ensure high reproducibility for automation. We optimized the single-step motion by calibrating the key parameters (the micromodule dimensions) in a force analysis. The optimal motion achieved a 98% pick-up success rate. The automated repetitive single-step assembly is achieved by an algorithm that acquires the 3-D location and tracks the micromanipulator without being affected by low contrast. The accuracy of the acquired 3-D location was experimentally determined as approximately 1 pixel (2 μm under 4× magnification), and the tracking under different observation conditions is proved effective. Finally, we automatically assembled microtubes at 6 micromodules/min, sufficiently fast for fabricating macroscopic vessel-mimetic substitutes in biological applications.

This study presents the survival microinjection into Caenorhabditis elegans (C. elegans) with in vivo observation based on micromanipulation. The microinjections were achieved with micro-gel beads which are enable to encapsulate chemicals for injection. In this study, a fluorescent material was used to evaluate the injection positional precision inside the C. elegans. The fluorescent microbead was picked up at the tip of a micropipette injection tool and injected by a piezo actuated microinjector. The distance between the injected micro-gel bead and closest nerve axon was measured as 20.3 µm and 16.5 µm by in vivo observation of a confocal microscopy. The two types of pipette tools were used to evaluate the success and survival rates of microinjection, and the smaller pipette (pipette A, 0.8 µm in diameter) showed higher rates as 50 % and 67 % respectively.

The fabrication of vessel-mimetic microchannel provides critical access to enable sufficient nutrient delivery for engineered composite tissues. This paper presents a novel method to engineer vascular-like microchannel by automated assembly of donut-shaped micromodules embedding fibroblast cells. A railed microrobotic system is set up with multi manipulators of 30 nm positioning resolution under an optical microscope. The coordinated manipulation among the micromanipulators is performed with newly designed concentric movement along the rail, which realized the arbitrary change of micromanipulator posture. With the novel developed image processing algorithm for the tracking of micromanipulator and micromodules, the automated bio-assembly of vascular-like microchannel is achieved which is free from the influence of the occlusion during contact manipulation. Experimental results showed that the pick-up success rate is around 98% and tracking algorithm is robust. Finally, we achieved automated assembly of microchannel at a speed of 6 micromodules/min, which is efficient enough to fabricate vessel-mimetic substitute with macro-significance dimension for biological application.

This paper presents (he aqueous observation of three-dimensional internal information using scanning election microscope- computed tomography (SEM-CT) inside an environ mental-SEM (E-SEM). For this purpose, it Is required to control the sample temperature by controlling sampk' duimhiT pressure of the E-SEM. In this paper, a cooling system was installed into the SEM-CT system to decrease the sample temperature. 1 he aqueous observation was confirmed by the E-SEM and SEM-CT observations using sodium poh/acrylate particles as a sample.

Muhi-graphene cubic structure was fabricated by a nanomanipulation system. Two nanomanipulators were coordinately used with three degrees of freedoms to handle two end-effectors respectively. A cubic net was designed and fabricated using a multi graphene by a FIB (Focused Ion Beam) etching. A tungsten probe was also reformed by the FIB etching to adjust the size of cubic net design for bending manipulation. 1 he each surface of cubic net was bent to form a cubic 3D structure. In this study, the bending stress of multi graphene were also measured using a silicon cantilever to reveal the elastic or plastic deformation regions by bending manipulation.

Although a variety of carbon nanotube field-effect transistors (CNTFETs) have been successfully fabricated, the contact property between multi-walled carbon nanotubes (MWCNTs) and electrodes is still elusive. In this study, we aims at further characterizing the contact properties among MWCNTs, Au electrode (gold coated glass substrate) and tungsten deposit. First, an atomic force microscope (AFM) cantilever actuated by a nanorobotic manipulation system was used to move the MWCNTs to directly contact Au electrode or tungsten deposit. With the adjustment of contact length, the contact resistance could be derived by a theoretical model using two-lead method. We measured the resistance between the AFM cantilever and Au electrode or tungsten deposit before and after fixing MWCNTs with electron beam induced deposition (EBID).The results imply that the contact resistance decreased ∼11%after being fixed with EBID. Moreover, EBID is confirmed to be able to strengthen the contact between MWCNT and tungsten deposit.

A hybrid nanorobotic manipulation system inside a scanning electron microscope (SEM) and a transmission electron microscope (TEM) is presented. The SEM manipulators have been constructed with 8 degrees of freedom (DOFs) with three units for effective TEM sample preparation. The TEM manipulator consists of a 3-DOF manipulator actuated with four multilayer piezoelectric actuators and a 3-DOF passively driven sample stage. High resolution and transmission image of TEM is readily used for measurement and evaluation of samples. The stage is premanipulated by the SEM manipulator for sample preparations inside the SEM. This methodology is called the hybrid nanorobotic manipulation so as to differentiate it from those with only an exchangeable specimen holder. To show the effectiveness of the system, the Young's modulus of a carbon nanotube (CNT) was measured to be 1.23 TPa inside a TEM after being premanipulated inside the SEM. With this system, we can measure the inner diameter of a CNT and improve the accuracy in measuring the Young's modulus of a CNT.

We propose a nanofixation method with melting/solidifications of a low melting metal based on nanomanipulation inside an electron microscope. Indium micro particles are experimentally used as the low melting metal. Formation changes of their melting/solidification are observed by scanning electron microscope (SEM) images. To control the position, temperature and pressure of samples, the nano-manipulation system is constructed inside an environmental-SEM (E-SEM). The nanomanipulators are newly constructed with 6 degrees of freedom (DOFs) and 3 units inside the E-SEM. Bio-cells are experimentally observed on water-containing conditions with non-drying treatments. The individual carbon nanotubes (CNTs) are experimentally fixed by melting/solidification of indium micro particles.

This paper presents a local microinjection system using hydro-gel beads based on micromanipulation near nerve axon of Caenorhabditis elegans (C. elegans), which is one of the important model organism for disease analysis and nerve analysis. The hydro-gel beads were prepared using Polyethylene Glycol Diacrylate (PEG-DA) to encapsulate biomaterials. The tip of micro-pipette tool was used to attach the hydro-gel beads by a negative pressure with a syringe. The local-nano-injection technique was demonstrated to inject fluorescent materials into C. elegans using the micro-pipette tool by a piezo actuator. The microinjection was evaluated in a three dimensional space by a confocal microscope. The proposed microinjection method is considered to be important as future micro-nano surgery system for life innovation using model organism.

In this paper, we presented a lab-on-chip microfluidic system for single cell mass measurement. Single cell mass has been related with drag force exerted on cell and Newton law of motion. Drag force has been generated using pressure driven syringe micropump. Motions of the cells were measured using optical observation under an inverted microscope. We have calibrated the approach using known mass of polystyrene microbeads with a diameter of 5.2 µm. Our experimental results showed that, mass of single microbead is 88.9 fg, which is very close to the theoretical mass (77.3 fg) of the particle. From which it is believed, our approach is suitable for single particle's mass measurement. We used Saccharomyces cerevisiae baker's yeast cell as a cell sample. Yeast cells have been cultured in our laboratory using YPD medium. For the diameter of 4 µm cell, mass of single cell was measured as 1.9 pg which is very consistent with previously reported single yeast cell mass (1–3 pg).

We present the fabrication of catalytic driven micro-nano robot by direct laser writing. The micro-nanorobot has a spiral design of 3,5, and 7 μm named D1, D2 and D3 respectively and a flat top design D4. The micro-nanorobot is catalytically driven in 6% hydrogen peroxide (H 2 O 2 ) from platinum catalytic area. The result shows that the spiral micro-nanorobot increased in speed except when the micro-nanorobot exhibits the drilling motion. It showed spinning motion, self-propulsion and drilling motion for micro-nanorobot D1, D2 and D3 respectively. The flat top micro-nanorobot has the highest speed and spinning motion was displayed. Future studies should include the actual pollutants in the environment and the effects of the drilling motion on its final content should be observed.

A method is presented for pico-Newton (pN) order force measurement using a carbon nanotube (CNT) probe, which is calibrated by electromechanical resonance. A CNT probe is constructed by attaching a CNT to the end of a tungsten needle or an atomic force microscope (AFM) cantilever using nanorobotic manipulators inside a field-emission scanning electron microscope (FE-SEM). Conductive electron-beam-induced deposition (EBID) is used for the fixation of CNTs with an internal vaporized precursor W(CO)6. For manipulating them easily and quickly, CNTs are dispersed in ethanol by ultrasonic waves and oriented on copper electrodes by electrophoresis. The elastic moduli of CNT probes are calibrated for use as a force measurement probe by electrically exciting at fundamental frequency. We analyzed the resolution of force measurement using a CNT probe. This force measurement can be used to characterize the mechanical properties of nanostructures and to measure friction or exfoliation forces in nanometer order.

Rat eosinophils contain eosinophil-associated ribonucleases (Ears) in their granules. Ears are thought to be synthesized as pre-forms and stored in the granules as mature forms. However, the N-terminal amino acid of mature Ear-1 and Ear-2 is still controversial. Therefore, we prepared two recombinant mature forms of Ear-1 and Ear-2 in which the N-terminal amino acids are Ser24 (S) [Ear-1 (S) and Ear-2 (S)] and Gln26 (Q) [Ear-1 (Q) and Ear-2 (Q)], and analyzed their biological activities by comparing them with those of pre-form Ear-1 and pre-form Ear-2. The four mature Ears showed RNase A activity as well as bovine pancreatic RNase A activity, but pre-Ear-1 and pre-Ear-2 showed no RNase A activity. Mature Ear-1 (Q) and mature Ear-2 (Q) showed more potent RNase A activity than mature Ear-1 (S) and mature Ear-2 (S), respectively. The RNase A activities of mature Ear-1 (Q) and mature Ear-2 (Q) were reduced by treatment at 96 degrees C for 20 min or with RNase inhibitor. The growth of Escherichia coli was inhibited by both pre-Ears and mature Ears in a concentration-dependent manner, and was almost completely suppressed at 1.0 microM. The bactericidal activities of mature Ear-1 (Q) and mature Ear-2 (Q) were not inhibited by RNase inhibitor, but was increased by treatment at 96 degrees C for 20 min.

Three-dimensional carbon nanotube field-effect transistors (3D CNTFETs) possess predictable characteristics that rival those of planar CNTFETs and Si-based MOSFETs. However, due to the lack of a reliable assembly technology, they are rarely reported on, despite the amount of attention they receive. To address this problem, we propose the novel concept of a 3D CNTFET and develop its assembly strategy based on nanomanipulation and the electron-beam-induced deposition (EBID) technique inside a scanning electron microscope (SEM). In particular, the electrodes in our transistor design are three metallic cuboids of the same size, and their front, top and back surfaces are all wrapped up in CNTs. The assembly strategy is employed to build the structure through a repeated basic process of pick-up, placement, fixing and cutting of CNTs. The pick-up and placement is performed through one nanomanipulator with four degrees of freedom. Fixing is carried out through the EBID technique so as to improve the mechanical and electrical characteristics of the CNT/electrodes connection. CNT cutting is undertaken using the typical method of electrical breakdown. Experimental results showed that two CNTs were successfully assembled on the front sides of the cubic electrodes. This validates our assembly method for the 3D CNTFET. Also, when contact resistance was measured, tens of kilohms of resistance was observed at the CNT-EBID deposition-FET electrodes junction.. This manifests the electrical reliability of our assembly strategy.

In this paper, we propose and demonstrate a new method for local cell stiffness measurement using multiple probes system. Thin glass pipettes were employed to measure the stiffness of cells on a general culture dish. The thin micropipettes were prepared and their spring constant was measured using the Atomic Force Microscopy (AFM) cantilever before stiffness measurement. The deformation of the pipette tips and the cell were observed under an optical microscope. The cell stiffness was calculated from the deformations based on the Hooke's low. The measurement results show that the myoblast C2C12 cell on a glass substrate has the stiffness around 0.1 N/m. The experimental results indicate that the proposed method can conduct the time-lapse cell stiffness measurement at multiple points simultaneously.

Lin1840 is a putative β-glucosidase that is predicted to be involved in 1,2-β-glucan metabolism since thelin1839gene encoding a 1,2-β-oligoglucan phosphorylase and thelin1840gene are located in the same gene cluster. Here, Lin1840 was crystallized. The crystals of Lin1840 diffracted to beyond 1.8 Å resolution. The crystal belonged to space groupI121, with unit-cell parametersa= 89.75,b= 95.10,c= 215.00 Å, α = 90.00, β = 96.34, γ = 90.00°.

In this paper, we present the fabrication and assembly of microstructures inside a microfluidic device based on a photocrosslinkable resin and optical tweezers. We also report a method of solution replacement inside the microfluidic channel in order to improve the manipulation performance and apply the assembled microstructures for single cell cultivation. By the illumination of patterned ultraviolet (UV) through a microscope, microstructures of arbitrary shape were fabricated by the photocrosslinkable resin inside a microfluidic channel. Based on the microfluidic channel with both glass and polydimethylsiloxane (PDMS) surfaces, immovable and movable microstructures were fabricated and manipulated. The microstructures were fabricated at the desired places and manipulated by the optical tweezers. A rotational microstructure including a microgear and a rotation axis was assembled and rotated in demonstrating this technique. The improved laser manipulation of microstructures was achieved based on the on-chip solution replacement method. The manipulation speed of the microstructures increased when the viscosity of the solvent decreased. The movement efficiency of the fabricated microstructures inside the lower viscosity solvent was evaluated and compared with those microstructures inside the former high viscosity solvent. A novel cell cage was fabricated and the cultivation of a single yeast cell ( w303) was demonstrated in the cell cage, inside the microfluidic device.

Background: Human eosinophils contain two eosinophil ribonucleases, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). In rats, 8 homologues of human ECP and EDN have been identified. To clarify the biological activity of rat eosinophil ribonucleases, we cloned rat eosinophil-associated ribonuclease (EAR)-1/rat ribonuclease 7 and rat EAR-2/rat ribonuclease 4, and produced recombinant rat pre-EAR-1 and pre-EAR-2 in a bacterial expression system. Methods: As we have already cloned the complete nucleotide sequence for rat EAR-1, we determined that for rat EAR-2 cDNA by the rapid amplification of cDNA ends procedure. Recombinant rat pre-EAR-1 and pre-EAR-2 were expressed in Escherichia coli as N-terminal 6 × histidine-tagged proteins, isolated from the insoluble fraction of the cell lysate and purified by a single-step method using an Ni-NTA resin column after solubilization with a 6 M guanidine solution. Results: The deduced amino acid sequence revealed that the molecular weight of EAR-2 containing the signal peptide is 17.3 kD and the isoelectric point is 8.59. The homology in amino acid sequence between rat pre-EAR-2, and human pre-ECP and human pre-EDN is 51 and 53%, respectively. The purified and refolded recombinant rat pre-EAR-1 and pre-EAR-2 showed bactericidal activity against E. coli and Staphylococcus aureus. Conclusions: These findings suggest that rat EAR-1 and EAR-2 act as host defense factors against bacterial infection in rats.

In this paper, we conducted an assembly of microstructures made of a thermoresponsive gel using hysteresis character of the thermoresponsive polymer solution. This method can be used for 3 dimensional cell assembly by embedding cells in the thermoresponsive gel structures. Gel sheets can be fabricated by micrheaters on a substrate and maintained in the gel condition by the hysteresis character. The microstructures can be formed by assembling the gel sheets using a probe device. The generation of a thermoresponsive gel was conducted using the microheaters made of ITO on a substrate and the generated gel sheets were manipulated by a micromanipulator. The fabrication of a gel sheets was achieved using the hysteresis character and the fabricated gel sheets were picked and placed by the probe. The positioning of the gel blocks can be precisely controlled by the micromanipulator. The results indicate the method we propose has a great possibility to achieve 3D cell assembly without large stress to cells during the assembly and cell culture.

A gyroscope based on Coriolis effect and fabricated by Carbon Nanotube (CNT) was presented. The gyroscope was designed A CNT is vibration at x direction, when there a rotation around z direction, acceleration happens to the y direction. The natural frequency of MWCNT is depending on the inner diameter, outer diameter and length. The CNT was driven by the electrostatic force to its nature frequency. A CNT with a length of 1 μm was used for emitter and an anode with a bias angle is etched by Focused Ion Beam (FIB) which is set to the opposite to the CNT. An electrode is set near the CNT tip, which is employed to pulling the CNT for mechanical resonances by an AC voltage. The CNT with a resonate frequency of 1 MHz and mechanical quality factor Q of ~400 was tested at rotation speed of 20 rad.s.

This paper presents a local drug micro-injection method by fluorescence micro-gel-bead with micro-pipette tool based on nano-manipulation under optical micro scope (OM) and environment scanning electron micro scope (E-SEM). Micro-gel-bead is fabricated from Polyethylene Glycol Diacrylate (PEG-DA). The drug is leaked after injection. The tip of micro-pipette tool is used to attach micro-gel-beads by applying a negative pressure with syringe pump. The Caenorhabditi selegans (C.elegans) is used as a target biological specimen, which is one of the important model organism for disease analysis and nerve analysis. The local-nano-injection technique is needed to transport fluorescent materials or specific biological organism into specific cell for in-vivo experiment. The proposed system is considered to be important as future nano-sugery system for life innovation using model organism.