In this study, we propose the manipulation and cell injection of a fluorescent microsensor using multiple wavelengths of light. The fluorescent microsensor is made of a 1-μm polystyrene particle containing infrared (IR: 808 nm) absorbing dye and Rhodamine B. The polystyrene particle can be manipulated in water using a 1064-nm laser because the refractive index of the polystyrene is 1.6 (refractive index of water: 1.3). The IR absorbing dye absorbs 808-nm light but does not absorb the 1064-nm laser. Rhodamine B is a temperature-sensitive fluorescent dye (excitation wavelength: 488 nm, emission wavelength: 560 nm). The functions of manipulation, heating for injection, and temperature measurement are achieved by different wavelengths of 1064 nm, 808 nm, and 488 nm, respectively. The temperature increase of fluorescent microsensor with 808-nm (40 mW, 10 s) laser was approximately 15°C, and enough for injection of fluorescent microsensor. We demonstrated manipulation and injection of the microsensor into Madin-Darby canine kidney cell using 1064-nm and 808-nm lasers. These results confirmed the effectiveness of our proposed cell injection of a fluorescent microsensor using multiple wavelengths of light.

Quality evaluation of oocyte and embryo has become one of the most important issues for many biomedical applications such as domestic animals and fertilization treatment. We developed measurement of oxygen consumption rate of individual oocyte by indirect fluorescence measurement using fluorescence oxygen sensor array having stripe-shape on a microfluidic chip. The fluorescence oxygen sensor was made of the mixture of photo-crosslinkable resin, polyethylene glycol diacrylate (PEG-DA) and Tris (2, 2'-bipyridyl) dichloro ruthenium (II) hexahydrate (Ru(bpy)3Cl2). The sensor was patterned to stripe-shape in the polydimethylsiloxane using photofabrication and molding technique. Oxygen concentration around the oocyte was distributed according to spherical diffusion theory because of similar oxygen permeability of polydimethylsiloxane and that of medium. The sensor array detects two-dimensional distribution of oxygen concentration and oxygen consumption ratio of the oocyte is calculated by image processing and Fick's low. We demonstrated non-contact measurement of oxygen consumption ratio of single mouse oocyte on a microfluidic chip (0.59 ± 0.03 fmol/s).

We developed selective and rapid injection of fluorescence sensor into the cell for intracellular measurement. The sensor encapsulated in the liposome contains photochromic chemical, 1, 3, 3-Trimethylindolino-6'- nitrobenzopyrylospiran, for optical control of zeta potential. The molecular structure of spiropyran changes between spiropyran type and merocyanine type by UV/VIS illumination. Zeta potential of merocyanine type is higher than that of spiropyran type. The charge of the liposomes are normally negative. Zeta potential of the liposome is switched from negative to positive by photoisomerization of spiropyran. Positive-charged liposome manipulated by optical tweezers adheres to the cell membrane by electrostatic force since the charge of the cell membrane is negative. The local vibration stimulus using optical tweezers is applied to the cell membrane for rapid injection of the sensor by membrane fusion and endocytosis. We demonstrated selective adhesion of the liposome to the cell membrane by optical control of zeta potential (adhesion rate: 71%). Moreover, we also demonstrated rapid injection of 1 μmφ fluorescence sensor (approximately 30 minutes, injection rate: 100%) into a cell using local vibration stimulus by optical tweezers.

We developed fabrication and manipulation of hybrid nanorobots by 3D nano exposure and optical tweezers. Hybrid nanorobot is composed of photoresist and silicon nanowire. The robot is fabricated by femtosecond laser exposure and is connected to the glass substrate by a micropillar. The processing resolutions of the femtosecond laser exposure are 270 nm (line width) and 600 nm (thickness), respectively. The robot is released by cutting of the pillar by ablation with femtosecond laser. Release time is within four minutes. The released robot is manipulated by holographic optical tweezers (HOT). We succeeded in high-speed manipulation of the robot using HOT (transport speed: 100 μm/s, rotation speed: 1140 deg/s). The robot can be fabricated by incorporating temperature indicator to the robot body and inserting a silicon nanowire to the probe. The robot can be used as a thermal sensor by measuring temperature change from the probe to the robot body because silicon nanowire has high thermal conductivity (168 W/m·K). In this paper, we demonstrated fabrication, on-demand release, and manipulation of the robot in a solution. We also demonstrated temperature calibration and measurement to confirm the effectiveness of the temperature sensor.

We developed selective injection of a single particle sensor into a specific cell by optically-regulated cell adhesion and membrane fusion of liposome for intracellular measurement. Fluorescence sensor encapsulated in fusogenic liposome is fabricated by spontaneous transfer method. The liposome is consists of neural lipids and spiropyran (SP). SP is a photochromic chemical and used to control the surface potential of the liposome. The surface potential of the liposome is neutral under VIS illumination. On the other hand, the surface potential becomes positive by UV-induced photoisolation of the SP. Charged liposome adheres to the cell membrane by coulomb's force since the surface potential of the membrane is negative. The estimated coulomb's force is approximately several hundred pN. The particle sensor can contain various fluorescence indicators such as Rhodmaine B as temperature sensor. The sensor is manipulated by optical tweezers and contacted to the cell membrane. We evaluated encapsulation efficiency of the single sensor into the liposome and adhesion efficiency to cell membrane under UV illumination. We demonstrated the optically-controlled adhesion of the sensor to MDCK cell and observed the injected sensor by laser confocal fluorescence method. As results, we succeeded in injection of particle sensor into the cell cytoplasm and nucleus.

In this paper, we proposed long-lifetime and high-sensitive measurement using fluorescent intensity by compensation of photo-bleaching with the difference compensation for cell measurement. Photo-bleaching of fluorescence is one of the most crucial factors to cause measurement error in physiological measurement. In our approach, difference of fluorescent intensity between imaging time is compensated using single exponential function to make the difference amount uniform in each imaging time. We evaluated the dependencies of measurable time and the sensitivity by fluorescent lifetime and the decay coefficient of fluorescent lifetime using numerical simulation. From these results, the proposed method has much higher sensitivity and longer measurable time against conventional compensation method of photo-bleaching. By using the proposed method, we demonstrate the photosynthesis activity of single Synechocystis sp. PCC 6803 in the micro chamber with gas barrier layer.

We developed Optical pH regulation using functional nanotool impregnated with photo-responsive chemical for selective cell injection of nanotool. The nanotool was modified by fluorescent dye for intracellular measurement. The nanotool was included in the fusogenic liposome. Membrane fusion of the liposome to the cell membrane was used for invasive cell injection of the nanotool. The liposome fuses to the cell in weak acidic condition. Local pH regulation inside the liposome was developed using photochromic chemical for selective cell injection of the nanotool. The nanotool was modified by Leuco crystal violet (LCV). LCV emits the proton by ultraviolet (UV) illumination. The emitted proton decreases the pH value in the liposome. This pH regulation is reversible by UV/VIS illumination. The liposome was manipulated by optical tweezers. After contact of the liposome to the cell, the liposome was adhered to the cell by UV induced membrane fusion. Injected nanotool was manipulated by optical tweezers. Intracellular temperature was detected by measuring the fluorescence intensity from the nanotool. We demonstrated optical pH regulation, selective cell injection of the nanotool, and manipulation of the nanotool in the cell.

In this paper, we proposed cell investigation tool for measurement of physiological conditions of single cell. We developed a novel temperature measurement method using temperature dependence of the quantum dot. The cell investigation tool encapsulates the quantum dot and adheres to the cell membrane. These tools can be manipulated in a solution by optical tweezers. We proposed image processing of the tools using the color information for temperature measurement. We calibrated the temperature using the tool impregnated with quantum dot by several color spaces such as HSV and YCrCb (H: hue, S: saturation, V: values (brightness). Y: brightness, Cr: color difference of red, Cb: color difference of blue). Y and Cr represent the monotone decrease according to the temperature increase, while H and Cb do not represent monotone variation. Measurement value by Cr is stable rather than the values by Y and V information because the color difference is robust against brightness fluctuation. From these results, we confirmed temperature measurement using Cr information suitable for cell analysis. Sensitivity is −1.3 %/K and accuracy is 0.3 K.

Production of functional microtools having an arbitrary shape by self-assembly of microparticles and heat treatment above the glass transition temperature of the microparticles was developed. Polystyrene microbeads were used as a material of the microtool. A solution including microparticles was dispersed onto the silicon substrate having microtool patterns fabricated by photolithography and etching. Dispersed particles were introduced to the pattern by gravity force. Microparticles in the pattern aggregate autonomously by surface tension through evaporation of the solution. Aggregated microparticles were fused by heating above the glass transition temperature (100 Celsius degrees). Fused microparticles were detached from the pattern by ultrasonic treatment and used as microtools. Produced microtool has spherical part since the microtool is made of microparticles. Spherical part is suitable for trapping point of optical tweezers. We demonstrated production of microtools using self-assembly and manipulation of the fabricated microtool on a chip. Position and attitude accuracy of the fabricated microtool controlled by TSS were evaluated by image processing. Finally, the transport speed of the fabricated microtool was compared with that of the photofabricated microtool. We confirmed the improvement of the transport speed and the effectiveness of our proposed microtool.

In noncontact cell manipulation with optical tweezers, we need to use microtools to avoid damages to the cells by direct laser irradiation. When we manipulate complex structures such as microtools, a multi-beam optical tweezers which can manipulate multiple objects is suitable; however manipulation speed or trapping number are not so good. Therefore, we developed an integrated optical tweezers by using time-shared scanning method for high speed manipulation and generalized phase contrast method for a lot of trapping. Then, we proposed cell manipulation method which suits for the integrated optical tweezers and then we designed the shape of microtools made of SU-8. A new fabrication method of SU-8 microtools was proposed to improve productivity. At last, we made the experiment system and the effectiveness of proposed methods was confirmed.

In this study, we successfully produced a functional microtool made of gel microbeads using size-dependent microparticle classification. Gel microbeads are made by salting-out hydrophilic photo-crosslinkable resin ENT-3400. These gel microbeads were separated according to their size using microfilters made of polydimethylsiloxane (PDMS). The first filter is a row of fluidic microchannels that block microbeads with a size greater than the channel's width. Another filtration method has been examined using a magnetically driven microtool (MMT) to separate the beads using the centrifugal force created by this MMT actuated with a DC motor. Separated gel microbeads were recovered after filtration and used to fabricate functional microtools, for example, a tether-shaped gel tool, by contact with other gel microbeads under UV illumination. The produced gel tool is manipulated using optical tweezers in a microchip. We successfully achieved size-dependent separation of gel microbeads and production of a tether-shaped gel tool.

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.

Optical adhesion control of a functional gel microtool, made of hydrophilic photo-crosslinkable resin, was developed for on-chip cell manipulation. The functional gel microtool used can be manipulated by optical tweezers and functionalized by spiropyran chromospheres, which is a photochromic polymer. Spiropyran chromospheres were used for temporal cell adhesion of the gel microtool. The adhesiveness of the gel microtools was controlled by photo irradiation. The gel microtools adhere to glass and other gel microtools, in an electrolyte solution of adjusted concentration, after ultraviolet (UV) illumination. By controlling the electrolyte concentration, the adhesion to cells can be selected as either permanent or temporal cell adhesions. UV illumination caused the cell to adhere to the gel microtool, while VIS illumination detached it from the gel-microtool. We also produced a pH sensing gel microtool by modifying the gel microtool with a pH indicator, bromothymol blue (BTB). The pH value was measured by detecting the color of the gel microtool with calibrated color information. Local measurements of the ambient pH value of a single yeast cell were performed by a ring shaped gel microtool and by immobilizing the cell on the surface of the pH sensing gel microtool.

A novel on-chip environment measurement with functional gel-microtool was developed. Environment measurement gel-microtool was fabricated by connecting the gel-microbeads impregnated with indicators in a microchip. In this paper, Bromothymol blue (BTB) and Bromocresol green (BCG) were employed as pH indicators. BTB and BCG have the different indicator range. Rhodamine B is temperature sensitive fluorescent dye and is used for temperature measurement. Gel-microbead is made by salting-out of hydrophilic photo-crosslinkable resin and is manipulated by optical tweezers. Moreover, gel-microbead is polymerized by UV illumination and connected to other gel-microbead under an electrolyte solution. The connection of gel-microbeads is performed by contact of gel-microbeads under UV illumination. Environment measurement gel-microtool with an arbitrary shape is fabricated by connection of the gel-microtool impregnated with arbitrary indicator. Multiple environments measurement gel-microtool included with several indicators is realized by assembly of the gel-microbeads impregnated with different indicators. Environment measurement is performed by detecting the color and the fluorescence intensity of each gel-microbead. We succeeded in the on-chip fabrication of the environment measurement gel-microtools such as circular pH measurement gel-microtool and wide range pH measurement gel-microtool in a microchip.

A local pH measurement method in a microchip using a functional gel-tool was developed. We used salting-out gel-tools impregnated with Bromothymol Blue (BTB), which is a pH indicator. The gel-tool is made of hydrophilic photo-crosslinkable resin. The primary constituent of this photo-crosslinkable resin is polyethyleneglycol. A solution mixed with photo-crosslinkable resin, BTB, high concentrations of an electrolytic solution are stirred, and gel beads impregnated with BTB are obtained. Gel-tools are cured by UV-ray and adhere to the glass plate, but we can manipulate them by the optical tweezers. We can measure pH value locally from the color of gel-tool using calibrated color information in YCrCb color space. We succeeded in measuring local pH value with the pH sensing gel-tool by manipulating and locating it at the desired point in the microchip.

We developed a novel technique to manipulate lipid nanotubes in 3D by laser tweezers in water solutions. Laser tweezers are well known as one of the noncontact manipulation methods used in a closed space. We reported on 3D 6DOF manipulation of microbeads using 3D Synchronized Laser Micromanipulation (SLM), however, the blur is generated in the microscope image when multiple points at different height are trapped by scanning the focal point of laser. Moreover, it is difficult to manipulate a nano scaled rod-like object stably in 3D, since optical trap force is weak. To manipulate a lipid nanotube for example, firstly, we newly developed an observation system to improve blur of image by synchronization of shutter timing of CCD camera and laser scanning. As a result, we can obtain a clear image of the targets at different height while manipulating them. Secondly, we developed functional gel microtools which can adhere to the lipid nanotubes. We succeeded in controlling the position and orientation of the lipid nanotubes by using 3D SLM with shutter timing control method and novel functional gel microtools.

In recent years, single cell experiment has become very important for investigating unknown cell properties. We developed a novel technique to investigate individual cell properties on a chip using newly developed cell manipulation method by laser tweezers with the photo - crosslinkable resin. We propose new fabrication method of functional microtools, such as colored microtool, fluorescent microtool, cell binding microtool, rotation free microtool, rope shaped microtool, on a chip. These microtools are made of photo - crosslinkable resin. Colored microtool and fluorescent microtool are for cell manipulation using Image processing with low cost. Cell binding microtool is made for high - speed transportation of target cell. Rotation free microtool is made for position control and precise force measurement of cell and DNA. Rope shaped microtool is for versatile manipulation. Laser tweezers is employed to position control of microtools. We used mercury lamp for UV - ray illumination at the local area for combination of microtools. We succeeded in fabrication of our functional microtools and manipulation of cell in the microchip.

Handling of microorganisms is of great interest in biology and biotechnology. Single cell capture technology has been demonstrated in our previous work with thermal gelation material and micro fluidic devise. This paper extends the system by local flow rate control to stabilize the trap with in-situ fabrication by photo-crosslinkable resin. Target cells are captured by gel and enclosed with the micro-structure. The cells, therefore, are protected from high-speed-channel flow or disturbances. This system succeeds in the separation and continuous culture of yeast cells, reaction and monitoring of the cultured cells stably by fluorescence staining in situ on a chip.

A laser micromanipulation is suitable for non contact manipulation of a microscopic object, such as a bacteria and microbe, in the closed space. Here we propose the Synchronized Laser Micromanipulation (SLM) for the trajectory control of the multiple targets by the single laser. We used the laser scanning system for high speed transportation of multiple objects. Condition for secure transportation was derived and verified through the experiment. Using the SLM, we can manipulate multiple microbeads with each designed trajectories. We assembled a band of 4 adhesive microbeads. Both ends are trapped, and it is manipulated by the SLM. It is possible to transport the microbe by the indirect manipulation of the assembled microstructure by the SLM. Experiment results are shown here.

Reports separation of the target microbe by the laser trapped micro tools for minimum invasion of laser irradiation. The size of a micro tool (MT) is around micrometer. The micro tools are manipulated by the focused lasers under the microscope to manipulate the target microbe. We propose a pinpoint injection method of micro tools at the desired location in the micro chamber, which is filled with liquid. First, we classified the injection method of the micro tools in 4 categories. We employed a new method to install the micro tools inside the micro chamber. We developed a micro tool holding chip to install the micro tools. The micro tools were injected in the micro chamber, and were manipulated successfully by the laser scanning micromanipulator to separate the target microbe. The proposed method is useful for the pinpoint injection of MTs and separation by the indirect micromanipulation.