Hisataka Maruyama

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.

Pinpoint injection of microtools for minimally invasive micromanipulation of microbe by laser trap

This paper reports transportation of the target microbe by the laser trapped microtools with minimum laser irradiation to the target. The size of a microtool (MT) is around micrometer. The MTs are manipulated by the focused laser under the microscope to manipulate the target microbe. Here we propose a pinpoint injection method of MTs at the desired location in the microchamber, which is filled with liquid. At first, we classified the injection method of the MTs in four categories. Here we employed a new method to install the MTs inside the microchamber. We developed a MT holding chip to install the MTs. The MTs were injected in the microchamber, and were manipulated successfully by the laser scanning micromanipulator to transport the target microbe for separation. The proposed method is useful for the pinpoint injection of MTs and separation by the indirect micromanipulation.

Multi-beam laser micromanipulation of microtool by integrated optical tweezers

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.

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.

Ultra-small site temperature sensing by carbon nanotube thermal probes

We have assembled thermal probes with single carbon nanotubes (CNTs) for sensing temperature distribution in local area. The temperature coefficient of resistance of the thermal probes shows the positive characteristics. The feasibility of the CNT thermal probes for scanning local area temperature is investigated in nanometer order. We have implemented the assembled CNT thermal probes for temperature measurement and calibration of CNT nanoheater, and demonstrated them to be novel temperature sensors.

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 cells 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 cells 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 cells state by measuring thermal characteristics.

Minimally invasive micromanipulation of microbe by laser trapped micro tools

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.

Photoprocessible Hydrogel Microsensor for Local Environment Measurement on a Microfluidic Chip

On-chip environment measurement is an important technique for control ambient conditions of cells and monitoring cell activities. However, integration of many sensors in the chip has difficulties such as position and interconnection of sensors. We developed hydrogel microsensors made of photocrosslinkable resin impregnating with indicators for on-chip measurement. This sensor can be used for both area measurement and 3-D sensing by shaping the hydrogel by UV ray patterning. This sensor requires no interconnections since environmental information is detected as optical information such as brightness and color. The YCrCb color space was suitable than the HSV color space in terms of the linearity of the color calibration (H: hue, S: saturation, V: values (brightness). Y : brightness, Cr: color difference of red, Cb: color difference of blue). In this paper, we demonstrated measurement of pH distribution in the microchannel using a color indicator and measurement of oxygen consumption of brown fat cell (BFC) using a fluorescent oxygen indicator. On pH measurement, we used YCrCb color space because Cr and Cb represented monotone variation and robust against brightness fluctuation. The hydrogel was patterned by photolithography into the stripe shape and modified with the indicator. Color change was detected by a color charge-coupled device (CCD). On oxygen measurement, we formed the hydrogel impregnating with the fluorescent indicator into microsphere. Fluorescence change was detected by the spectrophotometer. We succeeded in measuring local pH distribution and oxygen consumption of BFC inside the microfluidic chip.

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.

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.