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.

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.

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.

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 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.

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.

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.