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.

Helical structure assembled by hydrogel microfibers is significant for culture of smooth muscle cells. However, the helical structure is only fabricated at the macroscale, while the fabrication of helical microstructure is still a challenge due to the lack of assembly method. In this paper, we propose a robotics-based assembly method to handle such challenge. An electromagnetic needle (EMN) is employed as end-effector to magnetically reel the microfiber encapsulating magnetic nanoparticles around a micropillar, and a dual-ring structure is designed to keep the microfiber being attracted at the EMN tip. For enhancing the stability of tip attraction, the manipulation mode of anticlockwise pushing microfiber is established. Moreover, the interaction mechanism between EMN tip and microfiber is analyzed by developing a static force model, and then the key condition of stably reeling microfiber is concluded. Furthermore, a robotics-based motion trajectory of EMN tip is planned to achieve a smooth reeling process. Based on such planning, the size of dual-ring structure is further optimized to improve the success rate of reeling. Finally, the helical microstructure with there-turn coils is successfully fabricated.

Practical education about information security is needed for system developers and administrators in Web service suppliers. We propose a hacking contest CTF as attack and defense style with a website for a multiple online game. The contest organizer prepares a game server site as a contest environment which contains several vulnerability. The contest participants are divided into an attacker team as game players and a defender team as game Website operators. While the attackers access the game site with normal actions, they find vulnerabilities and try cheating actions. While the defenders monitor server logs, they detect illegal events and prevent them for maintenance. An action of each side is calculated as a point and makes each score for victory or defeat by the contest rule. The contest organizer reviews the progress situation with all participants. In this paper, we introduce several functions of a trial system and discuss the result of a trial practice.

This paper presents a novel In situ nanomanipulation integrated with scanning electron microscope- computed tomography (SEM-CT) imaging system for 3D nanomanipulation. In our previous works, a nanorobotic manipulation system was established inside an environmental-SEM (E-SEM) for water-contained samples, including biological organism, based on a real-time high resolution SEM observation. However, the SEM image is limited in two dimensional (2D) and surficial information from the signals of secondly electrons. For nanosurgery applications, such as nanoinjection, it is needed to evaluate the sample in 3D space with its internal information after manipulation. The SEM-CT imaging system is developed for In situ nanomanipulation based on SEM observation. The CT is an effective method to obtain the internal 3D information as a non-destructive manner. The imaging resolution of our SEM-CT system is in less than 400 nm. A Caenorhabditis elegans (C. elegans) was used as a target of biological sample. To improve the contrast of SEM-CT imaging of C. elegans, the X-ray was tested by generating using brass and copper materials. Finally, the nanoinjection was demonstrated with SEM-CT imaging system to C. elegans using the nanoinjector which was fabricated by focused ion beam (FIB) process.

In this paper, we propose a new method to assemble microstructures with biological cells towards in vitro 3D cellular structures. The proposed assembly method uses self-assembly process of magnetized toroidal microstructures. Biocompatible toroidal hydrogel microstructures are prepared by electrodeposition method, and ferrite particles are put on the fabricated structures using poly-L-lysine (PLL). The microstructures are magnetized by the magnetic field at 3 T, and assembled by the magnetic self-assembly process. Biological cells were encapsulated in the microstructures and cultured to achieve high density of cells inside structures. The magnetized microstructures were assembled automatically. The magnetic force generated from the ferrite embedded microstructures was estimated and compared to the fluid resistance applied to the microstructures. The proposed method can be applied to achieve 3D in vitro cell structures with vascular networks for tissue engineering applications.

Due to the poor controllability in hydrogels, Hydrogels-based assembly to form larger 3D complex shapes is still a big challenge. In this paper, we have reported a novel “bottom-up” method to fabricate three-dimensional (3D) magnetic alginate microfibers (MAMs) assemblies with complex shapes. Specifically, Alginate microfibers encapsulating Fe3O4magnetic nanoparticles (MNs) and fibroblasts (NIH/3T3) have been spun using microfluidic method with “pinch-off” scheme. Experimental results show that the MAMs can respond quickly to the magnetic field, enabling their enhanced controllability. The magnetic assembly system is constructed by PDMS microfluidic device, 3D supporting model adhered on the bottom surface of dish filled with deionized water and magnet. The microfluidic “printing“ and magnetic deposition in magnetic assembly process are experimentally demonstrated, respectively. Because of magnetic field, the complex assembly shapes can be fabricated just by moving the microfluidic device in a plane. To match well with the shape of supporting model and to keep a stable assembly structure, the secondary cross-linking method is employed. From the LIVE/DEAD assay, cells can survive well during the magnetic assembly process.

In this paper, we conducted a new method to assemble microstructures inside a droplet named “Lab in a Droplet (LiD)”. The method can realize the assembly of micro-nano structures inside a droplet size. The surface tension is used to assemble microstructures automatically. Micro-scale or nano-scale objects are ejected from an inkjet nozzle. They are gathered at the center of the droplet and assembled by the surface tension. We conducted preliminary experiments to check whether the self-assembly of microstructures can be achieved by LiD. The experimental results show that the self-assembly of microbeads is conducted inside a droplet. The nano-scale objects can be patterned on a substrate depend on their sizes. A microstructure made of photo-crosslinkable resin was prepared, and microbeads were assembled with the microstructure inside a droplet. The method can be used for autonomous assembly of micro-nano structures in high precision.

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.

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.

In this study, cell-laden alginate-poly-L-lysine (PLL) hydrogel structures with arbitrary shapes were constructed based on electrodeposition method. Electrolysis of water in alginate solutions with calcium carbonate particles induced alginate gelation on micro-patterned anode electrode, and cell-laden alginate gel structures were formed. The several different shapes of gel were generated at one time by predefined micro-patterns. The micro-patterned electrode was fabricated by coating photoresist on Fluorine-doped tin oxide (FTO) glass slide. Alginate-PLL encapsulation technique was introduced to this platform forming alginate-PLL hydrogel structures. This proposed method can lead to more accurate and creative studies of fabricating cell-laden scaffold for tissue engineering.

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.

3D assembly of cellular structures is important for the fabrication of biological substitute in tissue engineering. In this paper, a novel rail-guided multi-microrobotic system was proposed for the assembly of cellular structure. The cellular 2-dimensional (2D) module was fabricated by UV illumination of the crosslinkable hydrogel. The coordinated manipulation among the micromanipulators was performed with newly designed concentric movement along the rail, which realized the arbitrary change of micromanipulator posture. Through the rotation of the end-effectors around the specimen without swapping out the visual field, the manipulation flexibility was improved. The distance information between the micromanipulator and the module was acquired from vision feedback system and utilized for the automatic pick-up of the microstructure. Through the cooperation among multi-manipulators with hybrid motors, the micromanipulation to assemble the 3D structure with 30 nm operation resolution was achieved. Finally, the rail-guided DeSCom system realized the bottom-up fabrication of cellular vascular-like microtube with vision feedback.

Microfluidic devices provide efficient approaches for building cellular tubular structures for in vitro tissue models in tissue engineering. In this paper, we report a novel method of constructing three-dimensional (3D) multilayered tubular structures based on axis translation of two-dimensionally (2D) microstructures inside microfluidic devices. The on-chip fabrication of movable 2D microstructures embedding fibroblasts (NIH/3T3) based on Poly (ethylene glycol) Diacrylate (PEGDA) was reported. Novel two-layered microfluidic devices were fabricated by Polydimethylsiloxane (PDMS), for conducting the fluidic self-assembly of the 2D microstructures. The self-assembly process was experimentally demonstrated. For improving the assembly results, a funneled structure and 3 micro grooves were added inside the microfluidic channel. Improved self-assembly result of constructing a multilayered tubular microstructure with higher efficiency was demonstrated.

In this paper, we achieved three dimensional (3D) assemblies of multi-cell spheroids towards 3D in vitro cell structures. The 3D assembly of cells in vitro is one of the important techniques to reveal cell growth, differentiation and hystogenesis. The techniques can be used for applications in tissue engineering, drug screening, and cell characterization. The multi-cell spheroids were used as building blocks for 3D structure, and thermoresponsive gel was used for spheroids assembly. The mouse myoblast cells C2C12 and mouse fibroblast cells NIH 3T3 were cultured in an ultra-low atachment surface flask. The cells were naturally aggregated in the flask and cell spheroids were prepared in self-assembly process. The rat liver cell RLC-18 was also cultured to prepare spheroids. The thermoresponsive Gel probe (GeT probe) we proposed was employed for assembly of the prepared spheroids in 3D. The handled spheroids by GeT probe were cultured inside the thermoresponsive gel and the cell viability was confirmed. The cultured structure was handled by the GeT probe for further assembly. The results validate that the GeT probe can manipulate spheroids with keeping live condition of cells and achieve 3D cell assembly inside the thermoresponsive gel with arbitrary and precise patterning.

Cellular vascular-like microtubes occupy an important position in tissue engineering for building in vitro tissue models. In this paper, we report a method of constructing three-dimensional (3D) multilayered vascular-like microtubes based on fluidic axis-translation self-assembly of two-dimensional (2D) microstructures inside microfluidic devices. The on-chip fabrication of cell (fibroblasts NIH/3T3) embedded 2D microstructures based on Poly (ethylene glycol) Diacrylate (PEGDA) and biodegradable material Gelatin Methacrylate (GelMa) were reported. A multilayered Polydimethylsiloxane (PDMS) microfluidic device was fabricated for conducting the fluidic self-assembly of 2D microstructures. The fluidic axis-translation self-assembly process was experimentally demonstrated. Multiple hydrogels embedded microtube was constructed. The fabrication of GelMa microstructures was demonstrated. The degradability of cell embedded GelMa microstructures was evaluated by long-term observation, and it shows the great potential of GelMa to be used for constructing cellular vascular-like microtubes.

This paper introduces a novel catalytic mobile micro/nanorobot made only of platinum that realizes nanometer locomotion in hydrogen peroxide solution. The innovative mechanism and principle of the nanorobot are presented. A simple and effective fabrication process by focused ion beam and a stable manipulation method of the nanorobot are demonstrated. The nanorobot can steer or navigate by its finely designed geometry, and keep a stable rotational motion rather than arbitrary and uncertain movement. This paper evaluates the influence of some critical factors on the movement of the nanorobot, such as the concentration and temperature of the hydrogen peroxide solution, and the geometry of a nanorobot. The control of the nanorobot's movement can be realized based on the result of this evaluation. Compared with previous studies, this catalytic platinum nanorobot realizes bidirectional rather than unidirectional movement. The Langevin equation is used to describe the dynamic model of the platinum nanorobot.

This paper presents a new in-vitro simulator that can simulate the new technology of three-dimensional digital subtraction angiography (DSA) to perform a cerebral aneurysm surgery. Then, propose a new technique for the numerical evaluation of the surgical tools applied within this interventional surgery to aid in the design and selection of surgical instruments. Initially, the in-vitro intravascular simulator was constructed, followed by cerebral aneurysm models that were fabricated with highly sensitive photoelastic materials of epoxy resin. Then reconstruct virtual reality modeling of the blood vessel and catheter to record the position information and realize virtual guidance for training. Meanwhile, conduct image processing of photoelastic effects for numerical evaluations of surgical instruments such as catheter insertion and stent expansion. During the stent expansion experiment, the average stress in the selected region of interest caused by the expansion was calculated and compared with the corresponding value obtained before the expansion. The presented simulator and measurement technique can assist in improving a surgeons skills, quantifying the performance of medical mechanisms, and contribute to 3D analysis of stress distribution. This approach can also provide feedback control for robot assisted endovascular systems.

In this paper, we propose a new method to assemble 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 blocks can be maintained the gel condition by the hysteresis character and the microstructures can be formed by assembling the gel blocks. The temperature distribution around a microheater was analyzed to generate the thermoresponsive gel and avoid thermal damages to cells. The generation of thermoresponsive gel was conducted using the microheater which was embedded in a probe tip and manipulated by a micromanipulator. The fabrication of a gel block was achieved using the hysteresis character and the fabricated gel block was picked and placed by the probe. Cells were embedded in the gel by controlling the position of microheater to avoid the influence of thermal convection flow. 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.

3D tailor-made biodegradable scaffold integrated with biological cells or molecules is of great importance for tissue engineering. This paper addresses an improved method for exploring magnetic tweezers in patterning and aligning magnetic hydrogel fiber to fabricate large-scale engineered cell-hydrogel constructs. Magnetic hydrogel fibers were fabricated based on microfluidic device. The fabricated hydrogel fiber is made of alginic acid sodium and with a diameter of 34 μm. Magnetic nanoparticles is added into the alginic acid sodium solution to append magnetic material inside the fibers. The magnetic material inside the hydrogel fiber is regulated by the microfluidic device. Magnetic tweezers system based on solenoid electromagnet is utilized to evaluate the magnetic response of the magnetic hydrogel fiber. Evaluation results show the hydrogel fiber can be maneuvered by the proposed system with a positioning resolution of sub-micro level. The cultivation results of hydrogel fiber with C2C12 cells shows the potential for real applications of the proposed method in tissue engineering.

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.

Recently the research about constructing 3 dimensional cell structures is very important for its great potential applications in tissue engineering. In this paper, we report a novel method of constructing multi-layered microstructures embedding cells via microfluidic devices. The on-chip fabrication of movable microstructures embedding fibroblasts (NIH/3T3) based on Poly (ethylene glycol) Diacrylate (PEGDA) was reported. Two approaches for assembling these movable microstructures were presented. One was a manual assembly method based on micromanipulation system and the other one was a self-assembly method based on microfluidic channel. Several manual assembly ways were demonstrated and a tube-shaped microstructure with 17 layers was assembled by an efficient assembly method. A novel microfluidic channel was presented for conducting self-assembly method and a 2-layered experimental microfluidic device was fabricated by Polydimethylsiloxane (PDMS). The self-assembly process of fabricated microstructures via this device was preliminarily demonstrated.

In this paper, we try to establish new technique that the components of the biological clock are reconstituted into the liposome. In other words, we try to produce a nano size clock, capsulated into the liposome, made by protein molecules. The circadian clock is a basic cellular system found in almost all organisms. This clock generates self-sustained oscillations under constant conditions with a ≈ 24-hour (circadian) period. In cyanobacteria, circadian clock could be reconstituted in vitro only by mixing the three clock proteins, KaiA, KaiB, KaiC, with adenosine triphosphate (ATP). So we reconstitute these proteins and adenosine triphosphate (ATP) into phospholipid-coated microdroplet and confirmed the clock function. The clocks in phospholipids-coated microdroplet indicate long period more than 24 hours. In this case, period length became 35 hour, self-sustaining oscillation was reaming with little dumping. To reveal why the time cycle became long period, we observed localization of Kai proteins in phospholipid-coated microdroplets by using fluorescents labeled Kai proteins under fluorescent (confocal) microscopy. From the observation of localization of Kai proteins, we found KaiB protein was distributed equivalently, on the other hand, KaiC protein was located near membrane of phospholipid-coated microdroplet. These results indicate that different localization between Kai proteins cause long period oscillation and we could control period length depend on calculation data from localization.

Recently, micro-nano robots intended for application to various fields are developed. However, motors which are robot's power are not yet practical. In this paper, to realize the motor which can be applied to micro-nano robot's power source, we established bio-motor by using surface swarming of Vibrio alginolyticus. First, we succeeded in driving the micro gear in the closed micro channel. Next, to transmit power of rotational movement, we fabricated the open micro channel, and succeeded in driving the gear in the open micro channel similarly. In addition, we revealed that ratchet type gear rotated faster than other type. Finally, we assembled the micro gear with shaft. Therefore, it became possible transmitting the power from rotational movement to outside. Thus, we achieved to construct base of bio-motor.

A novel nanotool exchanger system is proposed based on Environmental Scanning Electron Microscope (E-SEM) nanorobotic manipulation system. We proposed to use the E-SEM nanomanipulation system for the analysis of biological specimen using various “nanotools” to realize flexible and complex nano-scale stiffness measurement, adhesion force measurement, cutting, and injection. The E-SEM can use to observe the biological samples in nano-scale and real-time without any drying or dyeing processes. As previous works, we applied the system to manipulate biological specimens, such as Caenorhabditis elegans (C. elegans) and yeast cells. To maintain the livable condition of biological cells, it is important to reduce the exchange time of the nanotools. This is also important to improve the efficiency of biological specimen analysis using various nanotools without break the chamber pressure. This paper presents a novel nanotool exchanger system for exchanging different nanotools within the ESEM chamber. Through the nanotool exchanger system, the following advantages are mainly obtained, 1) it is not needed to open the sample chamber to exchange the nanotools and to evacuate the sample chamber pressure again, 2) it is not needed to operate manually to exchange nanotools, 3) it is possible to recover the nanotools by exchanging new one, 4) it is possible to use different tools continuously. Firstly, the design and fabrication are presented for the proposed nanotool adaptor, nanotool attachment and nanotool holder. Finally, we demonstrate to exchange the different nanotools based on E-SEM nanomanipulation system.

In this paper, an automatic system for single cell analysis inside an environmental scanning electron microscopy (ESEM) was proposed. Single yeast cell was put on an tungsten probe substrate inside ESEM. The endeffector for single cell analysis was fixed to an nanorobotic manipulator, which has three degrees of freedom, i.e. X, Y and Z translation. The real time images during the experiment can be observed by ESEM system in realtime. Therefore, the position of the endeffector and the single cell can be recognized by imaging processing. These position information were used as the feedback signal to control the movement of the nanorobotic manipulator. Finally, a single cell cutting experiment was performed to demonstrate the working mechanism of this system. Two types of cell pattern substrates were also designed and fabricated as the cell analysis chips for the automation single cell analysis in the future.

Constructing different patterns of cells and immobilizing these cells inside certain structures are very important issues for artificial tissue engineering. In this paper, we present methods of forming line pattern of yeast cells by dielectrophoresis (DEP) and immobilizing patterned cells by photo-crosslinkable resin. High speed cell pattering by DEP and on-chip fabrication of microstructure which contains patterned yeast cells is demonstrated. In order to applying DEP force for forming cell pattern, several novel microelectrodes are fabricated by Indium Tin Oxides (ITO) which are coated on the glass. The two kinds of DEP responses of yeast cell (W303) and the precise experimental parameters of them are confirmed. Based on negative DEP phenomenon, cell traps generated by microelectrode are demonstrated. Position control and transportation of yeast cells is performed by using cell traps. Besides, a cell trap matrix is fabricated and high speed cell pattering is performed. The experimental results show that the cell line patterns which contain hundreds of yeast cells can be formed by DEP within 1 second. The on-chip fabrication for arbitrary shapes of microstructures based on Poly Ethylene Glycol Diacrylate (PEG-DA) is reported. With the cell patterning by DEP and immobilizing by on-chip fabrication, microstructure which contains 3 lines of yeast cells is fabricated in the microfluidic channel, inside PEG-DA and NaCl solution.

This paper proposes PRDC-SSIS, a new compressibility-feature based semantic-sensitive image segmentation method using PRDC. One of the drawbacks of traditional signal (pixel-color) based image segmentation is the poor capability to capture the semantical information contained in the images. Because the semantic information tends to be carried by a set of neighboring pixels, rather than an individual pixel, we divide the image into patches and classify the patches based on their semantical contents. The crucial problem is classifying the patches into groups of similar patches according to their contents, and so we exploit the compressibility feature vector space of PRDC to accomplish this. An application of this method to an EO-image confirmed the proposed scheme can be carried out without any of the human-tailored target object models required by almost all traditional methods.

Magnetic field has been used for manipulation of micro-robots; in this research we use it for scaffold fabrication. Magnetic sugar particle (MSP) was used as porogen to control pore size, pore structure and pore density in the scaffold. We studied the influence of the strength of magnetic fields for controlling the coating thickness of unmagnetized MSPs during the fabrication of sheet-like scaffolds. The experimental relationship between magnetic flux density and the thickness of MSP layer was illustrated. Furthermore we investigated the infiltration capacity of poly(L-lactide-co-ε-caprolactone) (PLCL) which was used as scaffold material on the MSP clusters. 5% and 10% PLCL solutions were employed in the experiments. After polymer casting and removal of the sugar template, spherical pores were generated inside scaffold, the thickness evaluation of a single layer scaffold was carried out and the cytocompatibility experiment of NdFeB powder used in fabrication of MSP was confirmed with human umbilical vein endothelial cells.

We proposed a micro fluidic device to control the position of Caenorhabditis elegans (C. elegans) and to analyze the condition of C. elegans as a bioindicator during its lifespan. In this device, we can filter C. elegans depending on size and control the position of C. elegans to culture and test C. elegans and to measure the capacitance change. The capacitance change depends on the body volume of C. elegans. Through the correlation with the capacitance change and the body volume of C. elegans, the body volume of C. elegans is assessed without imaging system. The proposed micro fluidic device was implemented using a PDMS layer and a glass substrate that were bonded together. The PDMS layer had a valve system and pattern rows to filter, culture and test C. elegans. A pair of electrodes is used to measure the capacitance change. In this study, we filtered C. elegans into the same size by filter and one C. elegans was introduced into a chamber row to be cultured and tested. And it was confirmed the correlation between the capacitance change and the body volume of C. elegans. And the proposed micro fluidic device will be applied to observe the effect of chemical or toxicant like heavy metal (Cd) on C. elegans as bioindicator.

Micro-nano robots intended for application to various fields are developed. However, motors, which are robot's power, are not yet practical. In this paper, to realize the motor, which can be applied to micro-nano robot's power source, we established bio-motor by using surface swarming of Vibrio alginolyticus. We succeeded in controlling movement of surface swarming by ratchet shape micro channel and then driving the micro gear in the closed micro channel. As a next step, to transmit power of rotational movement, we fabricated the open micro channel and micro object (floating pillar, micro gear etc.) and succeeded in driving the object in the open micro channel. In addition, we revealed that rotational power of floating pillar by deformation of nano probe. Finally, we assembled the micro gear with shaft like crank. Therefore, it became possible transmitting the power from rotational movement to outside. Thus, we achieved to construct base of bio-motor.

This paper presents a novel selective nano-injection method using fluorescent nanobeads based on nanomanipulation for in-vivo single cell analysis. The nanomanipulation system was constructed under hybrid microscope. The hybrid microscope consists of the optical microscope (OM) and environmental-scanning electron microscope (E-SEM) to realize biological specimen analysis by optical imaging including fluorescent imaging, and nano-scale manipulation by E-SEM imaging. Based on the bio-nano manipulation system, we propose a nanoinjection method by nanoprobe insertion and nanobead embeddedness. The nanoprobe was designed to have four tips to fix single nanobead. It is fabricated by focused ion beam (FIB) process. Its nano-scale size is considered to be important for minimal damage to a biological specimen. In this work, as biological specimen, the Caenorhabditis elegans (C. elegans) was used. It is one of the important model organisms for various diseases analysis. The nanoinjection technique is applied to transport fluorescent materials or specific biological organism into a specific cell for in-vivo experiment of C. elegans. The proposed system is considered to be important as future nano-surgery system for life innovation using model organism.

Single cell's mechanical property characterization is important for the understanding of the cell's condition and activity. In this paper, an automatic nanomanipulation system was proposed for single cell mechanical property characterization inside an environmental scanning electron microscopy (ESEM). A nanoneedle was fabricated from AFM (atomic force microscopy) cantilever by FIB (focused ion beam) etching technique. The nanoneedle was fixed to a nanorobotic manipulator, which has three degrees of freedom, i.e. X, Y and Z translation. Single yeast cell was put on an tungsten probe substrate inside ESEM. The position information of the nanoneedle and single cell were used as the feedback signals to control the movement of the nanorobotic manipulator. Finally, the stiffness of the single cell was measured using the nanoneedle driven by the automatic nanomanipulation system.

For tissue engineering, it is very important to construct cell patterns and immobilize patterned cells inside certain structures. In this paper, we present methods of forming cell pattern by dielectrophoresis (DEP) and immobilizing cells by photo-crosslinkable resin inside microfluidic chips. High speed cell manipulation, including patterning and concentration control by DEP is demonstrated. Movable microstructure embedding cells is on-chip fabricated. Several microelectrodes are fabricated by Indium Tin Oxides (ITO) and Cr/Au. The two kinds of DEP responses of yeast cell (W303) and other particles are experimentally confirmed. Based on negative DEP phenomenon, line and circle patterns of cells and microbeads are high speed performed. Cell manipulation for patterning is completed within 1 second. The on-chip fabrication of movable microstructures embedding cells based on Poly Ethylene Glycol Diacrylate (PEG-DA) is reported. The microfluidic chip with separated patterning and fabrication areas is fabricated. With the cell concentration control by DEP and on-chip fabrication method, movable microstructures embedding microbeads of which the concentration is controllable are fabricated in the microfluidic channel, inside PEG-DA and NaCl solution.

Toward to realizing a novel drug delivery system, a simulation system is considered to be important for a future analysis of it. In this paper, a novel, miniature, and energy-efficient bio-mimetic propulsion concept was proposed. The antibody binding technique is developed and used to attach bacteria to liposome's surface for enhancing liposome mobility. Bacteria and liposome are strongly combined through antibody. Consequently, the effect of antibody when bacteria attached to liposome is studied experimentally. The stochastic nature of bacterial propulsion of liposome is investigated experimentally and analytically. It is shown that antibody plays an important role in attaching bacteria to liposome and the liposome with bacteria moved broader compared to the liposome without bacteria. Statistical calculation matches well with experimental data.

The microstructures are expected to be used as the components of on-chip single cell analysis system such as cell manipulation and measurement tools. We previously proposed a method to fabricate and assemble the microstructures within a microfluidic channel. Micro objects with arbitrary shape are fabricated by illumination of patterned UV-ray through the mask. The arbitrary shape objects are made of the photo-crosslinkable resin on the microfluidic device under a microscope and these objects are manipulated by optical tweezers. In this paper, we report a method of hybrid-microstructures fabrication which improves the trapping efficiency by optical tweezers. The hybrid-microstructure is composed of the photo-crosslinkable resin and polystyrene beads. The manipulation speed of the hybrid-microstructure increases when the optical tweezers trap the polystyrene beads. This is because the trapping efficiency of spherical configuration is higher than that of planar shape. We fabricate this hybrid-microstructure and evaluate the trapping efficiency compared with normal microstructure.

In this paper, we established the transport systems for micro object by using surface swarming of Vibrio alginolyticus. First, we confirmed that V. alginolyticus were able to transport the micro beads. In this experiment, we used YM19 and YM19ΔcheY that were a kind of the variant strain of V. alginolyticus appropriate for surface swarming. Next, we examined relationship between the transport speed and the diameter of the micro bead. The micro channel was applied to control the cell movement. And then we succeeded in causing the rotational transport of object through this method.

This paper presents a novel nano-injection method by nanoprobe insertion based on environmental-scanning electron microscope (E-SEM) under hybrid microscope. The hybrid microscope is designed to combine the optical microscope (OM) and E-SEM to realize biological specimen analysis by optical imaging including fluorescent imaging, and nano-scale manipulation by E-SEM imaging. Based on the bio-nano manipulation system, we propose a nano-injection method by nanoprobe insertion. The nanoprobe is designed to have a sharp tip to insert the probe into biological specimen. It is fabricated by focused ion beam (FIB) process. The nano-scale size is important for minimal damage to biological specimen. The Caenorhabditis elegans (C. elegans) is used as a target biological specimen, which is one of the important model organism for diseases analysis. The 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-surgery system for life innovation using model organism.

In this paper, a novel oscillating nano knife for single cell cutting at its native state was proposed. The nano knife with a small edge angle 5° was fabricated from a commercial AFM cantilever by focused ion beam (FIB) etching. The spring constant of the nano knife was calibrated by nanorobotic manipulation method. A piezo actuator was employed to generate the vibration of the nano knife. The oscillating amplitude response to the applied voltage was calculated based on the realtime image observation. Single yeast cell (W303) cutting using oscillating nano knife was performed based on the nanorobotic manipulation system inside an environment-SEM. The maximum cutting forces and the slices angle were measured from the deformation of the nano knife beam and the single cell. The result showed the oscillating nano knife was capable and effective for single cell cutting task.

In this paper, we propose new probe device which can realize soft handling of single cells using thermal gel. In general, probe devices are one of the important devices for single cell manipulation. However, conventional probe devices have some problems: damage to the manipulated cells and difficulty to release the manipulated objects by the effect of surface force. As one solution of these problems, we propose "soft handling probe" in this paper. The probe has an electrode as a microheater. The thermoresponsive polymer can be gelled when a current is applied to the electrode in the thermoresponsive polymer solution. The electrode was fabricated by cupper wire and sputtering of gold on the probe. Thermal gelation of the thermoresponsive polymer was realized at the fabricated probe tip. The manipulation of micro objects and assembly of three dimensional structures were demonstrated by the probe. The handling of yeast cell and liposome were also realized by the probe. It is expected that the probe can realize the soft handling by the generated thermal gel and realize the precise positioning by reducing the effect of surface force.

In this paper, we try to establish newly technique that the components of the biological clock are reconstituted into the liposome. In other words, we try to produce a nano size clock, capsulated into the liposome, made by protein molecules. The circadian clock is a basic cellular system found in almost all organisms. This clock generates self-sustained oscillations under constant conditions with a ≈ 24-hour (circadian) period. In cyanobacteria, circadian clock could be reconstituted in vitro only by mixing the three clock proteins, KaiA, KaiB, KaiC, with adenosine triphosphate (ATP). So we reconstitute these proteins and adenosine triphosphate (ATP) into phospholipid-coated microdroplet and confirmed the clock function. The clocks in phospholipids-coated microdroplet indicate long period more than 24 hour. In this case, period length became 35 hour, self-sustaining oscillation was reaming with little dumping. To reveal why the time cycle became long period, we observed localization of Kai proteins in phospholipid-coated microdroplets by using fluorescents labeled Kai proteins under fluorescent (conforcal) microscopy. From the observation of localization of Kai proteins, we found KaiB protein was distributed equivalently, on the other hand, KaiC protein was located near membrane of phospholipid-coated microdroplet. These results indicate that deferent localization between Kai proteins cause long period oscillation.

Micro/Nano robots have attracted scientific attention to develop novel technologies such as drug delivery systems. Recently, flagellated bacteria have been used as the driving force of microobjects to develop bacteria-driven microrobots. In this paper, we propose a method of smart manipulation of multiple bacteria-driven microobjects with single manipulated guiderobot based on bacterial autonomous movement, named ¿SMARTBOT¿, for enabling arbitrary and low cost manipulation of multiple bacteria-driven microrobots. We verify the capability of smart manipulation of multiple bacteria-driven microobjects based on bacterial autonomous movement by using micro/nano pipettes instead of a guiderobot. For the fabrication of the guiderobot and the cargo driven by the bacteria, the fabrication of the arbitrarily-shaped microobjects using photo lithography technique is demonstrated. Additionally, we demonstrate the remote control using optical tweezers and driving using flagellated bacteria of the fabricated arbitrarily-shaped microobjects.

This paper presents a novel homogeneous power management system for heterogeneous self-reconfigurable multi-module systems consisting of active power suppliers, diverse power consumers and hybrids. To optimize functionality of each module, a concept separating functionality from power supply is proposed and applied in immobile payload items which can be combined with mobile modules in varying configurations autonomously. By allowing consumers and power sources to connect to common power bus, the concept enhances flexibility, reusability and performance of modular robotic systems. The power management system based on the concept is implemented and evaluated. The experimental results show that power is efficiently transferred in power bus among individual modules, consumer modules without battery packs can be kept alive during switching, and the power management system can protect individual modules from hardware faults reliably.

The on-chip fabrication and manipulation of microstructures are expected to be applied for single cell analysis system such as cell manipulation and measurement tools. In this paper, we previously present a methodology for fabricating and assembling microstructures inside a microfluidic channel. By the illumination of patterned UV-ray through the mask under a microscope, microstructures with arbitrary shape are made of the photo-crosslinkable resin inside microfluidic device. The microstructures are fabricated at the desired place inside microfluidic channel and manipulated by optical tweezers. Based on the technique which can manipulate multiple points simultaneously by high-speed scanning of a single laser with galvanometer mirror, a rotational microstructure made of a microgear and a rotation axis is assembled and rotated. We also report two methods of solution replacement inside microfluidic channel which reduces viscosity of solvent in order to improve manipulation performance. By adjusting the concentration of photo-crosslinkable resin and replacing solution components, the viscosity of solvent inside channel can be changed. The manipulation speed of the rotational microstructure increases when the viscosity of solvent decreases, because the viscosity resistance for the movement of microstructure is weaker inside lower viscosity solvent. We fabricate rotational microstructures inside lower viscosity solvent and evaluate the movement efficiency compared with microstructures inside former high viscosity solvent.

Micro/nano robots have been actively studied toward the realization. Now, the micro living organisms have been used as the driving forces for the micro objects. To achieve the fine manipulation of bio driven micro objects, the manipulation methods have to be established. Therefore, the local environmental control technique is desired to manipulate a single cell and to analyze the detailed properties. We have developed the local environmental control system with nano/micro dual pipettes, and achieved the quick-response and iterative rotational speed manipulation of Na+-driven flagellar motor, which is a rotary molecular machine, by switching the local spout between Na+-containing and Na+-free solutions with dual pipettes. In this paper, we upgrade our local environmental control system with nano/micro dual pipettes to achieve the quantitative and long-time-stable manipulation of the rotational speed of the Na+-driven flagellar motor. We demonstrate fine and long-time-stable rotational speed manipulation of Na+-driven flagellar motor by simultaneous local spouts of Na+-containing and Na+-free solutions using dual pipettes with controlling the spouting velocities independently. And, as the driving force, the rotational torque generated by the flagellar motor is estimated at the range from ~2.3×103to ~2.8×103pN·nm.

In this paper, we propose a semi-closed microchip which can realize the probe type manipulation in the microchip with the internal environment sealed off from the external environment. Long-term experiments can be conducted for in-situ culture and analysis of cells by the semi-closed microchip. The semi-closed microchip has a bath in the middle of microchannel to allow the insertion of micropipettes into the microchip. The bath is sealed off by thin oil film to prevent the evaporation of the solution in the bath. The seal by the oil film can maintain even if micropipettes are inserted into the bath and pulled out from the bath because the oil film can fill the hole by its surface tension. The seal by the oil film was evaluated by measuring the weight of the bath after 12/24 hours. To use the semi-closed microchip for cell culture and cell analysis, the exchange of the solution in the bath was demonstrated. It was also demonstrated that the cell fixation with the thermo sensitive gel in the proposed semi-closed microchip by fabricating the ITO electrodes as heaters in the bath. Cell culture and the target cell harvesting were conducted by the semi-closed microchip. The proposed semi-closed microchip can be used for cell culture, cell analysis and cell harvesting under a microscope as the biological applications for single cell analysis.

The microstructures are expected to be used as the components of on-chip single cell analysis system such as cell manipulation and measurement tools. In this paper, we propose the on-chip fabrication method of microstructures with minimum dimensions of 5 ¿m. The micro objects with arbitrary shape are fabricated by illumination of patterned UV-ray through the mask. The arbitrary shape objects are made of the photo-crosslinkable resin on the microchip under a microscope. The polymerized micro-objects, which we call as ¿Microtools¿, can be used for the single cell analysis. The microtools are fabricated at the desired place on the microchip in less than 1 second. Additionally, the microtools are manipulated by optical tweezers. By high-speed scanning of a single laser with galvanometer mirror, it is able to manipulate multiple points simultaneously. Using this technique, the rotational microstructure composed of the rotation axis and the microgear is assembled and rotated. We propose the two method of microstructure assembly. The assembled rotational microstructure is evaluated about the rotation speed depending on the laser power.

This paper presents the local stiffness measurements of Caenorhabditis Elegans (C. elegans) by buckling nanoprobes through the nanorobotic manipulation system inside an environmental-scanning electron microscope (E-SEM). C. elegans has complex outer and inner structures constructed by approximately one thousand cells. For example, the lateral alae are the surface fine structure by seam cell body. In this paper, their fine structures were observed by E-SEM directly; without any drying or dyeing processes. The observation environments are controlled under different E-SEM chamber pressures for clear observation of C. elegans. The local stiffness of C. elegans was measured by buckling measurement method of the nanoprobe fabricated by focus ion beam (FIB) etching at the tip of atomic force microscope (AFM) cantilever. The measurement position can arbitrarily be controlled by the nanorobotic manipulator inside the E-SEM. In this work, from experimental results, the measured elasticity on lateral alae was 1.6 times larger than it around lateral alae. This local stiffness measurement technique can readily be applied to reveal unknown biological local stiffness, cell health conditions and novel cell diagnosis.

We performed in situ measurements of mechanical properties of individual W303 wild-type yeast cells by using an integrated environmental scanning electron microscope (ESEM) - nanomanipulator system. Compression experiments to penetrate a cell wall of an individual cell under two difference conditions, i.e. ESEM (600 Pa) and high vacuum (HV) (about 3 mPa) modes have been conducted. Data from the experiments clearly show a significant increase of about twenty five times of penetration force values from ESEM mode to HV mode; i.e. 152 nN and 4 muN for 5 mum cell size with sharp tip indenter. This can be further confirmed from quantitative estimation of average cell rigidity through the Hertz model, i.e. ESEM mode (2.16 plusmn 1.21 MPa) and HV (20.58 plusmn 2.09 MPa) for all cell sizes with sharp tip indenter. Further analysis also revealed that the size of the cells can influence its mechanical strength. As the size of the cell increases, the compression force needed to penetrate its wall also increases. From these experimental results, we can conclude that the force needed to penetrate a single cell does not only depend on the internal factor, i.e. cell size but also being influenced by the external factors, i.e. the environmental conditions.

In situ fabrication and electrostatic actuation of a telescoping multi-walled carbon nanotube are presented. The inner core of the telescoping nanotube is actuated by applied bias voltage with a certain gap inside a vacuum chamber. From modeling analysis, the electrostatic forces are mainly driven force for actuation. At the experimental section, one end opened telescoping multi-walled carbon nanotube (MWCNT) is directly observed inside a transmission electron microscope (TEM). The telescoping MWNT is fabricated by peeling off outer layers through destructive fabrication process. Its inner core is extended and retracted with applied DC bias voltage. These processes have been done through a hybrid nanorobotic manipulation system, which are integrated nanomanipulators inside a scanning electron microscope (SEM) and a TEM. From experimental and analytical results, the possibility of nano-scale actuator applications using telescoping MWCNT with precision has been presented

Nanoscale linear servomotors with integrated position sensing are investigated from experimental, theoretical, and design perspectives. Prismatic motion is realized using the interlayer motion of telescoping multi-walled carbon nanotubes (MWNTs). Position sensing can be achieved by monitoring field emission or by measuring resistance change between a MWNT and a gold substrate during sliding movement. Experimental results demonstrate resolution in the nanometer range. Actuation experiments demonstrate the feasibility of a linear nano servomotor with integrated position sensing based on field emission. A local “kink”-like fluctuation of emission current is observed, which is caused by the change of the protruding length of the nanotube core, thus demonstrating the potential of using emission as a “linear encoder”. Complete extension of the inner core is observed and the electrostatic force is calibrated to be tens of nano-Newtons for individual nanotubes— 13.3 to 23.3 nN for voltages from 20 to 30V. These results demonstrate the possibility of fabricating linear servomotors at the nanometer scale with integrated position sensing.

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 presented. The new type of nanomanipulation system has high enough resolution to identify nano-scale objects due to the adoption of a TEM, and efficient and rapid nanomanipulations are realized with wide enough working space of the SEM nanomanipulators. The TEM nanomanipulator has been constructed with 4 multi-layer piezoelectric actuators for driving in 3 translational degrees of freedom (DOFs) and a passively driven 3-DOF sample stage through SEM nanorobotic manipulators to perform relatively complex manipulations whereas to keep compact volume to be installed inside the narrow vacuum chamber of a TEM. To show the effectiveness of the system, the interlayer resistance of a destructively fabricated multi-walled carbon nanotube has been measured inside a SEM whereas the telescoping structure formed by pulling out inner layers from outer layers of the tube is observed inside a TEM.

Force measurement with pico-Newton (pN) order resolution using a carbon nanotube (CNT) probe, which is calibrated by the electromechanical resonance is presented. Based on the theoretical analysis, a CNT is suitable material for the sensitive force measurement. A CNT probe is constructed by attaching a CNT to the tip of the commercially available atomic force microscope (AFM) cantilever or tungsten needle probe by the electron-beam-induced deposition (EBID) though the nanorobotic manipulators inside a field-emission scanning electron microscope (FE-SEM). In order to attach a CNT quickly and correctly, CNTs are dispersed in ethanol by ultrasonic waves for several hours and oriented by electrophoresis. The elastic moduli of CNTs are calibrated from electromechanical resonance frequency by applied electrostatic forces. We measured pico-Newton order contact forces with a CNT probe, which is constructed with nanorobotic manipulators, by measuring deformation of a CNT probe from FE-SEM images.

A nano laboratory-a prototype nano manufacturing system-is presented, which is composed with a nanorobotic manipulation system with 4 units and 16 degrees-of-freedom (DOFs), a nano fabrication system based on electron-beam-induced deposition (EBID) with an internal or external precursor evaporation reservoir equipped with a thermal field emission electron source or a nanotube cold cathode, and a real-time observation and measurement system based on a field emission scanning electron microscope (FESEM) equipped with 3-4 conventional atomic force microscope (AFM) cantilevers, piezoresistive levers or nanotube probes. Nanotube devices including a mass flow sensor, a linear bearing, and nanotube scissors are fabricated in the nano laboratory.