Instantaneous extracellular solution exchange for concurrent evaluation of membrane permeability of single cells.

The osmotic stress imposed on microorganisms by hypotonic conditions is perceived to regulate water and solute flux via cell membranes, which are crucial for survival. Some cells that fail to perceive osmotic stress die because this results in the rupture of the cell membrane. The flux through the membrane is characterized by the membrane permeability, which is measured using a stopped-flow apparatus in response to a millisecond-order osmolarity change. However, the obtained data are an ensemble average of each cell response. Additionally, the measurement of permeability, considering cellular viability, contributes to a more accurate evaluation of osmoadaptation. Here, we present a novel on-chip instantaneous extracellular solution exchange method using an air-liquid interface. The presented method provides a concurrent evaluation at the single-cell level in response to a millisecond-order osmotic shock, considering cellular viability by solution exchange. This method utilizes a liquid bridge with a locally formed droplet on the surface of a micropillar fabricated inside a microchannel. We evaluated a solution exchange time of 3.6 ms and applied this method to Synechocystis PCC 6803 under two different osmolarity conditions. The live/dead ratio of 1 M to 0.5 M osmotic down shock condition was 78.8/21.2% while that of 1 M to 0.25 M osmotic down shock condition was 40.0/60.0%. We evaluated the water permeability of two groups: cells that were still live before and after osmotic shock (hereafter named cell type 1), and cells that were live before but were dead 10 minutes after osmotic shock (hereafter named cell type 2). The results indicated that the water permeability of cell type 2 was higher than that of cell type 1. The results obtained using the presented methods confirmed that the effect of osmotic stress can be accurately evaluated using single-cell analysis.

Integration of Microfluidic Chip and Probe with a Dual Pump System for Measurement of Single Cells Transient Response.

The integration of liquid exchange and microfluidic chips plays a critical role in the biomedical and biophysical fields as it enables the control of the extracellular environment and allows for the simultaneous stimulation and detection of single cells. In this study, we present a novel approach for measuring the transient response of single cells using a system integrated with a microfluidic chip and a probe with a dual pump. The system was composed of a probe with a dual pump system, a microfluidic chip, optical tweezers, an external manipulator, an external piezo actuator, etc. Particularly, we incorporated the probe with the dual pump to allow for high-speed liquid change, and the localized flow control enabled a low disturbance contact force detection of single cells on the chip. Using this system, we measured the transient response of the cell swelling against the osmotic shock with a very fine time resolution. To demonstrate the concept, we first designed the double-barreled pipette, which was assembled with two piezo pumps to achieve a probe with the dual pump system, allowing for simultaneous liquid injection and suction. The microfluidic chip with on-chip probes was fabricated, and the integrated force sensor was calibrated. Second, we characterized the performance of the probe with the dual pump system, and the effect of the analysis position and area of the liquid exchange time was investigated. In addition, we optimized the applied injection voltage to achieve a complete concentration change, and the average liquid exchange time was achieved at approximately 3.33 ms. Finally, we demonstrated that the force sensor was only subjected to minor disturbances during the liquid exchange. This system was utilized to measure the deformation and the reactive force of Synechocystis sp. strain PCC 6803 in osmotic shock, with an average response time of approximately 16.33 ms. This system reveals the transient response of compressed single cells under millisecond osmotic shock which has the potential to characterize the accurate physiological function of ion channels.

Microfluidic Bioreactor Made of Cyclo-Olefin Polymer for Observing On-Chip Platelet Production.

We previously proposed a microfluidic bioreactor with glass-Si-glass layers to evaluate the effect of the fluid force on platelet (PLT) production and fabricated a three-dimensional (3D) microchannel by combining grayscale photolithography and deep reactive ion etching. However, a challenge remains in observing the detailed process of PLT production owing to the low visibility of the microfluidic bioreactor. In this paper, we present a transparent microfluidic bioreactor made of cyclo-olefin polymer (COP) with which to observe the process of platelet-like particle (PLP) production under a bright-field, which allows us to obtain image data at a high sampling rate. We succeeded in fabricating the COP microfluidic bioreactor with a 3D microchannel. We investigated the bonding strength of COP-COP layers and confirmed the effectiveness of the microfluidic bioreactor. Results of on-chip PLP production using immortalized megakaryocyte cell lines (imMKCLs) derived from human-induced pluripotent stem cells show that the average total number of produced PLPs per imMKCL was 17.6 PLPs/imMKCL, which is comparable to that of our previous glass-Si-glass microfluidic bioreactor (17.4 PLPs/imMKCL). We succeeded in observing PLP production under a bright-field using the presented microfluidic bioreactor and confirmed that PLP fragmented in a narrow area of proplatelet-like protrusions.

Weakening of resistance force by cell-ECM interactions regulate cell migration directionality and pattern formation.

Collective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell-cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, we analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, we showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Our results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.

Evaluating Young's Modulus of Single Yeast Cells Based on Compression Using an Atomic Force Microscope with a Flat Tip.

In this research, atomic force microscopy (AFM) with a flat tip cantilever is utilized to measure Young's modulus of a whole yeast cell (Saccharomyces cerevisiae BY4741). The results acquired from AFM are similar to those obtained using a microfluidic chip compression system. The mechanical properties of single yeast cells are important parameters which can be examined using AFM. Conventional studies apply AFM with a sharp cantilever tip to indent the cell and measure the force-indentation curve, from which Young's modulus can be calculated. However, sharp tips introduce problems because the shape variation can lead to a different result and cannot represent the stiffness of the whole cell. It can lead to a lack of broader meaning when evaluating Young's modulus of yeast cells. In this report, we confirm the differences in results obtained when measuring the compression of a poly(dimethylsiloxane) bead using a commercial sharp tip versus a unique flat tip. The flat tip effectively avoids tip-derived errors, so we use this method to compress whole yeast cells and generate a force­deformation curve. We believe our proposed method is effective for evaluating Young's modulus of whole yeast cells.

Hydrogel Fluorescence Microsensor with Fluorescence Recovery for Prolonged Stable Temperature Measurements.

This work describes a hydrogel fluorescence microsensor for prolonged stable temperature measurements. Temperature measurement using microsensors has the potential to provide information about cells, tissues, and the culture environment, with optical measurement using a fluorescent dye being a promising microsensing approach. However, it is challenging to achieve stable measurements over prolonged periods with conventional measurement methods based on the fluorescence intensity of fluorescent dye because the excited fluorescent dye molecules are bleached by the exposure to light. The decrease in fluorescence intensity induced by photobleaching causes measurement errors. In this work, a photobleaching compensation method based on the diffusion of fluorescent dye inside a hydrogel microsensor is proposed. The factors that influence compensation in the hydrogel microsensor system are the interval time between measurements, material, concentration of photo initiator, and the composition of the fluorescence microsensor. These factors were evaluated by comparing a polystyrene fluorescence microsensor and a hydrogel fluorescence microsensor, both with diameters of 20 µm. The hydrogel fluorescence microsensor made from 9% poly (ethylene glycol) diacrylate (PEGDA) 575 and 2% photo initiator showed excellent fluorescence intensity stability after exposure (standard deviation of difference from initial fluorescence after 100 measurement repetitions: within 1%). The effect of microsensor size on the stability of the fluorescence intensity was also evaluated. The hydrogel fluorescence microsensors, with sizes greater than the measurement area determined by the axial resolution of the confocal microscope, showed a small decrease in fluorescence intensity, within 3%, after 900 measurement repetitions. The temperature of deionized water in a microchamber was measured for 5400 s using both a thermopile and the hydrogel fluorescence microsensor. The results showed that the maximum error and standard deviation of error between these two sensors were 0.5 °C and 0.3 °C, respectively, confirming the effectiveness of the proposed method.

Hydrogel Heart Model with Temperature Memory Properties for Surgical Simulation.

The continual development of surgical technology has led to a demand for surgical simulators for evaluating and improving the surgical technique of surgeons. To meet these needs, simulators must incorporate a sensing function into the organ model for evaluating the surgical techniques. However, it is difficult to incorporate a temperature sensor into the conventional cardiac training model. In this study, we propose a heart model for surgical training of cardiac catheter ablation made from hydrogel, which has temperature memory properties. The heart model consists of a photo-crosslinkable hydrogel mixed with an irreversible temperature indicator that exhibits a color change from magenta to colorless at 55 °C. The Young's modulus, electrical resistivity, thermal conductivity, and specific heat capacity of the hydrogel material were evaluated and compared with those of human heart. Furthermore, temperature calibration based on the color of the hydrogel material confirmed that the temperature measurement accuracy of the material is ± 0.18 °C (at 56 °C). A heart model for catheter ablation was fabricated using the hydrogel material and a molding method, and the color change due to temperature change was evaluated.

Influenza virus replication raises the temperature of cells.

Influenza virus invades the cell by binding sialic acid on the cell membrane through haemagglutinin (HA), and then genome replication and transcription are carried out in the nucleus to produce progeny virus. Multiplication of influenza virus requires metabolites, such as nucleotides and amino acids, as well as cellular machinery to synthesize its genome and proteins, thereby producing viral particles. Influenza virus infection forces the start of several metabolic systems in the cell, which consume or generate large amounts of energy. Thus, the viral multiplication processes involved in both genome replication and transcription are considered to require large numbers of nucleotides. The high-level consumption of nucleotides generates large amounts of energy, some of which is converted into heat, and this heat may increase the temperature of cells. To address this question, we prepared a tool based on rhodamine B fluorescence, which we used to measure the temperatures of influenza virus-infected and uninfected cells. The results indicated that influenza virus multiplication increased the temperature of cells by approximately 4 °C - 5 °C, ATP levels in the cells decreased at 3 h after infection, and mitochondrial membrane potential decreased with multiplication level. Thus, the increase in cellular temperature during influenza virus infection appears to be due to the massive consumption of ATP over a short period.

Three-Dimensional Blood Vessel Model with Temperature-Indicating Function for Evaluation of Thermal Damage during Surgery.

Surgical simulators have recently attracted attention because they enable the evaluation of the surgical skills of medical doctors and the performance of medical devices. However, thermal damage to the human body during surgery is difficult to evaluate using conventional surgical simulators. In this study, we propose a functional surgical model with a temperature-indicating function for the evaluation of thermal damage during surgery. The simulator is made of a composite material of polydimethylsiloxane and a thermochromic dye, which produces an irreversible color change as the temperature increases. Using this material, we fabricated a three-dimensional blood vessel model using the lost-wax process. We succeeded in fabricating a renal vessel model for simulation of catheter ablation. Increases in the temperature of the materials can be measured by image analysis of their color change. The maximum measurement error of the temperature was approximately -1.6 °C/+2.4 °C within the range of 60 °C to 100 °C.

Application of an indicator-immobilized-gel-sheet for measuring the pH surrounding a calcium phosphate-based biomaterial.

The present study was designed to investigate the local microenvironment of octacalcium phosphate in a granule form upon biomolecules adsorption utilizing an indicator-immobilized-gel-sheet for measuring pH. We previously showed that octacalcium phosphate enhances bone regeneration during its progressive hydrolysis into hydroxyapatite if implanted in bone defects. The gel-sheet was made from a photocrosslinkable prepolymer solution, which can easily immobilize a pH indicator (bromothymol blue; BTB) in the hydrogel. The indicator-immobilized-gel-sheet was mounted on a biochip which was made of polydimethylsiloxane (PDMS) with a flow channel. The pH value was calculated by detecting the color changes in the gel-sheet and displayed as the pH distribution. After pre-adsorption of bovine albumin, ß-lactoglobuline or cytochrome C onto octacalcium phosphate granules, the granules with the gel-sheet were further incubated in Tris-HCl buffer solution in the absence or presence of fluoride, known as an accelerator of octacalcium phosphate hydrolysis. pH values of the gel-sheet surrounding octacalcium phosphate granules showed a decrease from pH 7.4 to 6.6 in relation to the proteins adsorbed. Overall, the proposed pH-sensitive gel can be used to detect the pH around octacalcium phosphate granules with a high spatial resolution.

Manipulation and Immobilization of a Single Fluorescence Nanosensor for Selective Injection into Cells.

Manipulation and injection of single nanosensors with high cell viability is an emerging field in cell analysis. We propose a new method using fluorescence nanosensors with a glass nanoprobe and optical control of the zeta potential. The nanosensor is fabricated by encapsulating a fluorescence polystyrene nanobead into a lipid layer with 1,3,3-trimethylindolino-6'-nitrobenzopyrylospiran (SP), which is a photochromic material. The nanobead contains iron oxide nanoparticles and a temperature-sensitive fluorescent dye, Rhodamine B. The zeta potential of the nanosensor switches between negative and positive by photo-isomerization of SP with ultraviolet irradiation. The positively-charged nanosensor easily adheres to a negatively-charged glass nanoprobe, is transported to a target cell, and then adheres to the negatively-charged cell membrane. The nanosensor is then injected into the cytoplasm by heating with a near-infrared (NIR) laser. As a demonstration, a single 750 nm nanosensor was picked-up using a glass nanoprobe with optical control of the zeta potential. Then, the nanosensor was transported and immobilized onto a target cell membrane. Finally, it was injected into the cytoplasm using a NIR laser. The success rates of pick-up and cell immobilization of the nanosensor were 75% and 64%, respectively. Cell injection and cell survival rates were 80% and 100%, respectively.

The Influence of Virus Infection on the Extracellular pH of the Host Cell Detected on Cell Membrane.

Influenza virus infection can result in changes in the cellular ion levels at 2-3 h post-infection. More H(+) is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H(+) during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H(+) from the intracellular compartment. Increased H(+) export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (ϕ1 µm) containing Rhodamine B and Fluorescein isothiocyanate (FITC). The fluorescence intensity of FITC can respond to both pH and temperature. So Rhodamine B was also introduced in the sensor for temperature compensation. Then the pH can be measured after temperature compensation. The sensor was adhered to cell membrane for extracellular pH measurement. The results showed that the multiplication of influenza virus in host cell decreased extracellular pH of the host cell by 0.5-0.6 in 4 h after the virus bound to the cell membrane, compared to that in uninfected cells. Immunostaining revealed the presence of viral PB1 protein in the nucleus of virus-bound cells that exhibited extracellular pH changes, but no PB1 protein are detected in virus-unbound cells where the extracellular pH remained constant.

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH.

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells.

Comparative analysis of kdp and ktr mutants reveals distinct roles of the potassium transporters in the model cyanobacterium Synechocystis sp. strain PCC 6803.

Photoautotrophic bacteria have developed mechanisms to maintain K(+) homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K(+) uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K(+) (Kdp). The contributions of each of these K(+) transport systems to cellular K(+) homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K(+) uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K(+) uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K(+) depletion. Correspondingly, Kdp-mediated K(+) uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K(+) required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K(+) concentration under conditions of limited K(+) in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K(+) availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K(+) levels under fluctuating environmental conditions.

Virus enrichment for single virus infection by using 3D insulator based dielectrophoresis.

We developed an active virus filter (AVF) that enables virus enrichment for single virus infection, by using insulator-based dielectrophoresis (iDEP). A 3D-constricted flow channel design enabled the production of an iDEP force in the microfluidic chip. iDEP using a chip with multiple active virus filters (AVFs) was more accurate and faster than using a chip with a single AVF, and improved the efficiency of virus trapping. We utilized maskless photolithography to achieve the precise 3D gray-scale exposure required for fabrication of constricted flow channel. Influenza virus (A PR/8) was enriched by a negative DEP force when sinusoidal wave was applied to the electrodes within an amplitude range of 20 Vp-p and a frequency of 10 MHz. AVF-mediated virus enrichment can be repeated simply by turning the current ON or OFF. Furthermore, the negative AVF can inhibit virus adhesion onto the glass substrate. We then trapped and transported one of the enriched viruses by using optical tweezers. This microfluidic chip facilitated the effective transport of a single virus from AVFs towards the cell-containing chamber without crossing an electrode. We successfully transported the virus to the cell chamber (v = 10 µm/s) and brought it infected with a selected single H292 cell.

Temperature changes in brown adipocytes detected with a bimaterial microcantilever.

Mammalian cells must produce heat to maintain body temperature and support other biological activities. Methods to measure a cell's thermogenic ability by inserting a thermometer into the cell or measuring the rate of oxygen consumption in a closed vessel can disturb its natural state. Here, we developed a noninvasive system for measuring a cell's heat production with a bimaterial microcantilever. This method is suitable for investigating the heat-generating properties of cells in their native state, because changes in cell temperature can be measured from the bending of the microcantilever, without damaging the cell and restricting its supply of dissolved oxygen. Thus, we were able to measure increases in cell temperature of <1 K in a small number of murine brown adipocytes (n = 4-7 cells) stimulated with norepinephrine, and observed a slow increase in temperature over several hours. This long-term heat production suggests that, in addition to converting fatty acids into heat energy, brown adipocytes may also adjust protein expression to raise their own temperature, to generate more heat. We expect this bimaterial microcantilever system to prove useful for determining a cell's state by measuring thermal characteristics.

Aquaporin AqpZ is involved in cell volume regulation and sensitivity to osmotic stress in Synechocystis sp. strain PCC 6803.

The moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 contains a plasma membrane aquaporin, AqpZ. We previously reported that AqpZ plays a role in glucose metabolism under photomixotrophic growth conditions, suggesting involvement of AqpZ in cytosolic osmolarity homeostasis. To further elucidate the physiological role of AqpZ, we have studied its gene expression profile and its function in Synechocystis. The expression level of aqpZ was regulated by the circadian clock. AqpZ activity was insensitive to mercury in Xenopus oocytes and in Synechocystis, indicating that the AqpZ can be categorized as a mercury-insensitive aquaporin. Stopped-flow light-scattering spectrophotometry showed that addition of sorbitol and NaCl led to a slower decrease in cell volume of the Synechocystis ΔaqpZ strain than the wild type. The ΔaqpZ cells were more tolerant to hyperosmotic shock by sorbitol than the wild type. Consistent with this, recovery of oxygen evolution after a hyperosmotic shock by sorbitol was faster in the ΔaqpZ strain than in the wild type. In contrast, NaCl stress had only a small effect on oxygen evolution. The amount of AqpZ protein remained unchanged by the addition of sorbitol but decreased after addition of NaCl. This decrease is likely to be a mechanism to alleviate the effects of high salinity on the cells. Our results indicate that Synechocystis AqpZ functions as a water transport system that responds to daily oscillations of intracellular osmolarity.

On-chip pH measurement using functionalized gel-microbeads positioned by optical tweezers.

This paper demonstrates local pH measurement in a microchip using a pH-sensing gel-microbead. To achieve this, the gel-microbead made of a hydrophilic photo-crosslinkable resin was functionalized with the pH indicator bromothymol blue (BTB). The primary constituent of this photo-crosslinkable resin is poly(ethylene glycol). Gel-microbeads impregnated with BTB were obtained by stirring the mixture solution, which was composed of the resin, BTB, and an electrolyte solution. The gel-microbead is polymerized by UV illumination. The polymerized gel-microbead can be manipulated by optical tweezers and made to adhere to a glass surface. The local pH was measured from the color of the gel-microbead impregnated with BTB by calibrated color information in the YCrCb color space. We succeeded in measuring the local pH value using the pH-sensing gel-microbead by manipulating and positioning it at the desired point in the microchip.

On chip single-cell separation and immobilization using optical tweezers and thermosensitive hydrogel.

A novel approach appropriate for rapid separation and immobilization of a single cell by concomitantly utilizing laser manipulation and locally thermosensitive hydrogelation is proposed in this paper. We employed a single laser beam as optical tweezers for separating a target cell and locating it adjacent to a fabricated, transparent micro heater. Simultaneously, the target cell is immobilized or partially entrapped by heating the thermosensitive hydrogel with the micro heater. The state of the thermosensitive hydrogel can be switched from sol to gel and gel to sol by controlling the temperature through heating and cooling by the micro heater. After other unwanted cells are removed by the high-speed cleaning flow in the microchannel, the entrapped cell is successfully isolated. It is possible to collect the immobilized target cell for analysis or culture by switching off the micro heater and releasing the cell from the entrapment. We demonstrated that the proposed approach is feasible for rapid manipulation, immobilization, cleaning, isolation and extraction of a single cell. The experimental results are shown here.

Immobilization of individual cells by local photo-polymerization on a chip.

A novel separation method for random screening of target cells from a large heterogeneous population by using a local photo-polymerization is developed. A photo-crosslinkable resin solution is mixed with the sample liquid and we controlled the state from sol to gel by irradiating the near ultraviolet (UV) light with the mercury lamp and He-Cd laser near the target cell. We applied three types of immobilization methods such as direct immobilization method, caging method, and direct immobilization with position control method. The selected cell is immobilized in the cured resin directly or inside the cage of the cured resin. In the position control method, laser tweezers are employed to manipulate the target cell indirectly by using the droplet of the resin as a microtool. The cell is positioned properly by the laser manipulation system and is immobilized in the polymerized resin. After the selected cells are immobilized we can easily remove the other objects by the cleaning flow in the microchannel since the polymerized resin strongly binds with the cover glass and resists more than 466 mm s(-1) flow speed in the microchannel (microchannel size: width is 500 micron and depth is 100 micron). We tested the mercury lamp as well as the He-Cd laser for UV-light irradiation at the local area and confirmed improvement of resolution of the cured area by using the He-Cd laser (from 7 micron to 5 micron). Based on this method, we succeeded in single cell immobilization and basic experiments such as culture and fluorescent dyeing of immobilized yeast cells.