Agarose microfabrication technology is one of the micropatterning techniques of cells having advantages of simple and flexible real-time fabrication of three-dimensional confinement microstructures even during cell cultivation. However, the conventional photothermal etching procedure of focused infrared laser on thin agarose layer has several limitations, such as the undesired sudden change of etched width caused by the local change of absorbance of the bottom surface of cultivation plate, especially on the indium-tin-oxide (ITO) wiring on the multi-electrode array (MEA) cultivation chip. To overcome these limitations, we have developed a new agarose etching method exploiting the Joule heating of focused micro ionic current at the tip of the micrometer-sized capillary tube. When 75 V, 1 kHz AC voltage was applied to the tapered microcapillary tube, in which 1 M sodium ion buffer was filled, the formed micro ionic current at the open end of the microcapillary tube melted the thin agarose layer and formed stable 5 μm width microstructures regardless the ITO wiring, and the width was controlled by the change of applied voltage squared. We also found the importance of the higher frequency of applied AC voltage to form the stable microstructures and also minimize the fluctuation of melted width. The results indicate that the focused micro ionic current can create stable local spot heating in the medium buffer as the Joule heating of local ionic current and can perform the same quality of microfabrication as the focused infrared laser absorption procedure with a simple set-up of the system and several advantages.

The original version of this Article contained an error in Figure 1, where panel G did not display correctly. The original Figure 1 and accompanying legend appear below. The original Article has been corrected.

Agarose photothermal microfabrication technology is one of the micropatterning techniques that has the advantage of simple and flexible real-time fabrication even during the cultivation of cells. To examine the ability and limitation of the agarose microstructures, we investigated the collective epithelial cell migration behavior in two-dimensional agarose confined structures. Agarose microchannels from 10 to 211 micrometer width were fabricated with a spot heating of a focused 1480 nm wavelength infrared laser to the thin agarose layer coated on the cultivation dish after the cells occupied the reservoir. The collective cell migration velocity maintained constant regardless of their extension distance, whereas the width dependency of those velocities was maximized around 30 micrometer width and decreased both in the narrower and wider microchannels. The single-cell tracking revealed that the decrease of velocity in the narrower width was caused by the apparent increase of aspect ratio of cell shape (up to 8.9). In contrast, the decrease in the wider channels was mainly caused by the increase of the random walk-like behavior of component cells. The results confirmed the advantages of this method: (1) flexible fabrication without any pre-designing, (2) modification even during cultivation, and (3) the cells were confined in the agarose geometry.

<title>Abstract</title>Conventional neuronal network pattern formation techniques cannot control the arrangement of axons and dendrites because network structures must be fixed before neurite differentiation. To overcome this limitation, we developed a non-destructive stepwise microfabrication technique that can be used to alter microchannels within agarose to guide neurites during elongation. Micropatterns were formed in thin agarose layer coating of a cultivation dish using the tip of a 0.7 <inline-formula><alternatives><tex-math>$$\upmu \mathrm{m}$$</tex-math><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML">
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</mml:math></alternatives></inline-formula> from the edge of the etched agarose microchannel. We exploited the fast temperature decay property to guide cell-to-cell connection during neuronal network cultivation. The first neurite of a hippocampal cell from a microchamber was guided to a microchannel leading to the target neuron with stepwise etching of the micrometer resolution microchannel in the agarose layer, and the elongated neurites were not damaged by the heat of etching. The results indicate the potential of this new technique for fully direction-controlled on-chip neuronal network studies.

We investigated the dominant rule determining synchronization of beating intervals of cardiomyocytes after the clustering of mouse primary and human embryonic-stem-cell (hES)-derived cardiomyocytes. Cardiomyocyte clusters were formed in concave agarose cultivation chambers and their beating intervals were compared with those of dispersed isolated single cells. Distribution analysis revealed that the clusters’ synchronized interbeat intervals (IBIs) were longer than the majority of those of isolated single cells, which is against the conventional faster firing regulation or “overdrive suppression.” IBI distribution of the isolated individual cardiomyocytes acquired from the beating clusters also confirmed that the clusters’ IBI was longer than those of the majority of constituent cardiomyocytes. In the complementary experiment in which cell clusters were connected together and then separated again, two cardiomyocyte clusters having different IBIs were attached and synchronized to the longer IBIs than those of the two clusters’ original IBIs, and recovered to shorter IBIs after their separation. This is not only against overdrive suppression but also mathematical synchronization models, such as the Kuramoto model, in which synchronized beating becomes intermediate between the two clusters’ IBIs. These results suggest that emergent slower synchronous beating occurred in homogeneous cardiomyocyte clusters as a community effect of spontaneously beating cells.

We examined characteristics of the propagation of conduction in width-controlled cardiomyocyte cell networks for understanding the contribution of the geometrical arrangement of cardiomyocytes for their local fluctuation distribution. We tracked a series of extracellular field potentials of linearly lined-up human embryonic stem (ES) cell-derived cardiomyocytes and mouse primary cardiomyocytes with 100 kHz sampling intervals of multi-electrodes signal acquisitions and an agarose microfabrication technology to localize the cardiomyocyte geometries in the lined-up cell networks with 100–300 µm wide agarose microstructures. Conduction time between two neighbor microelectrodes (300 µm) showed Gaussian distribution. However, the distributions maintained their form regardless of its propagation distances up to 1.5 mm, meaning propagation diffusion did not occur. In contrast, when Quinidine was applied, the propagation time distributions were increased as the faster firing regulation simulation predicted. The results indicate the “faster firing regulation” is not sufficient to explain the conservation of the propagation time distribution in cardiomyocyte networks but should be expanded with a kind of community effect of cell networks, such as the lower fluctuation regulation.

Precise and quick measurement of samples’ flow velocities is essential for cell sorting timing control and reconstruction of acquired image-analyzed data. We developed a simple technique for the single-shot measurement of flow velocities of particles simultaneously in a microfluidic pathway. The speed was calculated from the difference in the particles’ elongation in an acquired image that appeared when two wavelengths of light with different irradiation times were applied. We ran microparticles through an imaging flow cytometer and irradiated two wavelengths of light with different irradiation times simultaneously to those particles. The mixture of the two wavelength transmitted lights was divided into two wavelengths, and the images of the same microparticles for each wavelength were acquired in a single shot. We estimated the velocity from the difference of its elongation divided by the difference of irradiation time by comparing these two images. The distribution of polystyrene beads’ velocity was parabolic and highest at the center of the flow channel, consistent with the expected velocity distribution of the laminar flow. Applying the calculated velocity, we also restored the accurate shapes and cross-sectional areas of particles in the images, indicating this simple method for improving of imaging flow cytometry and cell sorter for diagnostic screening of circulating tumor cells.

Biophysics in Waseda University was started in 1965 as one of the three key research areas that constitute the Physics Department. In the biophysics group, one theoretical lab and two experimental labs are now working on the cutting-edge themes on biophysics, disseminating the ideas and knowledge of biophysics to undergraduate and graduate students from the viewpoint of physics.

Exploiting the combination of latest microfabrication technologies and single cell measurement technologies, we can measure the interactions of single cells, and cell networks from “algebraic” and “geometric” perspectives under the full control of their environments and interactions. However, the experimental constructive single cell-based approach still remains the limitations regarding the quality and condition control of those cells. To overcome these limitations, mathematical modeling is one of the most powerful complementary approaches. In this review, we first explain our on-chip experimental methods for constructive approach, and we introduce the results of the “community effect” of beating cardiomyocyte networks as an example of this approach. On-chip analysis revealed that (1) synchronized interbeat intervals (IBIs) of cell networks were followed to the more stable beating cells even their IBIs were slower than the other cells, which is against the conventional faster firing regulation or “overdrive suppression,” and (2) fluctuation of IBIs of cardiomyocyte networks decreased according to the increase of the number of connected cells regardless of their geometry. The mathematical simulation of this synchronous behavior of cardiomyocyte networks also fitted well with the experimental results after incorporating the fluctuation-dissipation theorem into the oscillating stochastic phase model, in which the concept of spatially arranged cardiomyocyte networks was involved. The constructive experiments and mathematical modeling indicated the dominant rule of synchronization behavior of beating cardiomyocyte networks is a kind of stability-oriented synchronization phenomenon as the “community effect” or a fluctuation-dissipation phenomenon. Finally, as a practical application of this approach, the predictive cardiotoxicity is introduced.

<jats:p>We report a change of the imaging biomarker distribution of circulating tumor cell (CTC) clusters in blood over time using an on-chip multi-imaging flow cytometry system, which can obtain morphometric parameters of cells and those clusters, such as cell number, perimeter, total cross-sectional area, aspect ratio, number of nuclei, and size of nuclei, as “imaging biomarkers”. Both bright-field (BF) and fluorescent (FL) images were acquired at 200 frames per second and analyzed within the intervals for real-time cell sorting. A green fluorescent protein-transfected prostate cancer cell line (MAT-LyLu-GFP) was implanted into Copenhagen rats, and the blood samples of these rats were collected 2 to 11 days later and measured using the system. The results showed that cells having BF area of 90 μm2 or larger increased in number seven days after the cancer cell implantation, which was specifically detected as a shift of the cell size distribution for blood samples of implanted rats, in comparison with that for control blood. All cells with BF area of 150 μm2 or larger were arranged in cell clusters composed of at least two cells, as confirmed by FL nucleus number and area measurements, and they constituted more than 1% of all white blood cells. These results indicate that the mapping of cell size distribution is useful for identifying an increase of irregular cells such as cell clusters in blood, and show that CTC clusters become more abundant in blood over time after malignant tumor formation. The results also reveal that a blood sample of only 50 μL is sufficient to acquire a stable size distribution map of all blood cells to predict the presence of CTC clusters.</jats:p>

Formation of stable micro-droplets in multiphase flow is an important step to perform numerous microfluidic applications such as sorting experiments. We herein investigate the conditions of formation of stable micro-droplets using a flow focusing microfluidic device. Two single phases and four different multiphase flow regimes were observed depending on the pressures of fluids. By tuning sample stream pressure against fixed lower oil stream pressure, stable droplet regime can create microenvironment with a diameter ranged from 30 μm to 140 μm. Results obtained show that the formation of strictly size controlled droplets can encapsulate single cell-sized bead into droplet. Moreover, the limit between unstable and stable droplet regimes was the most suitable to efficiently encapsulate cell-sized bead in droplet sorting application. This limit can be precisely monitored by using the change of the droplet speed found at the threshold between these two regimes.

Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs’ actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. Cav1.2 or Nav1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.

We examined a simultaneous combined spatiotemporal field potential duration (FPD) and cell-to-cell conduction time (CT) in lined-up shaped human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using an on-chip multielectrode array (MEA) system to evaluate two origins of lethal arrhythmia, repolarization and depolarization. The repolarization index, FPD, was prolonged by E-4031 and astemizole, and shortened by verapamil, flecainide and terfenadine at 10 times higher than therapeutic plasma concentrations of each drug, but it did not change after lidocaine treatment up to 100 μM. CT was increased by astemizol, flecainide, terfenadine, and lidocaine at equivalent concentrations of Nav1.5 IC50, suggesting that CT may be an index of cardiac depolarization because the increase in CT (i.e., decrease in cell-to-cell conduction speed) was relevant to Nav1.5 inhibition. Fluctuations (short-term variability; STV) of FPD and CT, STVFPD and STVCT also discriminated between torsadogenic and non-torsadogenic compounds with significant increases in their fluctuation values, enabling precise prediction of arrhythmogenic risk as potential new indices.

Cells encapsuled by polymer microdroplets are an effective platform for the identification and separation of individual cells for single-cell-based analysis. However, a key challenge is to maintain and release the captured cells in the microdroplets selectively, nondestructively, and noninvasively. We developed a simple method of encapsulating cells in alginate microdroplets having different digestion characteristics. Cells were diluted with an alginate polymer of sol state and encapsulated into microdroplets with Ba2+ and Ca2+ by a spray method. When a chelating buffer was applied, alginate gel microdroplets were digested according to the difference in chelating efficiency of linkage-divalent cations
hence, two types of alginate microdroplets were formed. Moreover, we examined the capability of the alginate gel to exchange linkage-divalent cations and found that both Ca2+ exchange in Ba-alginate microdroplets and Ba2+ exchange in Ca-alginate microdroplets occurred. These results indicate that the potential applications of a mixture of alginate microdroplets with different divalent cations control the selective digestion of microdroplets to improve the high-throughput, high-content microdroplet-based separation, analysis, or storage of single cells.

We investigate an integrate and fire model for two cardiomyocytes interacting with each other. A feature of the model is to incorporate the refractory periods of the cardiomyocytes as well as the influence of firing of adjacent cells. The present model predicts that, if refractory periods of the two cells are nearly equal, the beating rhythms of the two cells always synchronize and their beating rate is tuned to the faster rate between the two cells. On the other hand, if their refractory periods significantly differ, they exhibit various kinds of harmonious beating rhythms. These results successfully explain the well known characteristics of synchronized beating of cultured cardiomyocytes. We also discuss effects of a delay time of cell-to-cell interaction, that gives further complicated phase diagrams for the beating rhythms. (C) 2017 Elsevier Ltd. All rights reserved.

The community effect of cardiomyocytes was investigated in silico by the change in number and features of cells, as well as configurations of networks. The theoretical model was based on experimental data and accurately reproduced recently published experimental results regarding coupled cultured cardiomyocytes. We showed that the synchronised beating of two coupled cells was tuned not to the cell with a faster beating rate, but to the cell with a more stable rhythm. In a network of cardiomyocytes, a cell with low fluctuation, but not a hight frequency, became a pacemaker and stabilised the beating rhythm. Fluctuation in beating rapidly decreased with an increase in the number of cells (N), almost irrespective of the configuration of the network, and a cell comes to have natural and stable beating rhythms, even for N of approximately 10. The universality of this community effect lies in the fluctuation-dissipation theorem in statistical mechanics.

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 +/- 4.5% and 90 +/- 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

Recent advances in imaging flow cytometry and microfluidic applications have led to the development of suitable mathematical algorithms capable of detecting and identifying targeted cells in images. In contrast to currently existing algorithms, we herein proposed the identification and reconstruction of cell edges based on original approaches that overcome frequent detection limitations such as halos, noise, and droplet boundaries in microfluidic applications. Reconstructed cells are then discriminated between single cells and clusters of round-shaped cells, and cell information such as the area and location of a cell in an image is output. Using this method, 76% of cells detected in an image had an error <5% of the cell area size and 41% of the image had an error <1% of the cell area size (n=1,000). The method developed in the present study is the first image processing algorithm designed to be flexible in use (i.e. independent of the size of an image, using a microfluidic droplet system or not, and able to recognize cell clusters in an image) and provides the scientific community with a very accurate imaging algorithm in the field of microfluidic applications. (C) 2016 International Society for Advancement of Cytometry

For the prediction of lethal arrhythmia occurrence caused by abnormality of cell-to-cell conduction, we have developed a next-generation in vitro cell-to-cell conduction assay, i.e., a quasi in vivo assay, in which the change in spatial cell-to-cell conduction is quantitatively evaluated from the change in waveforms of the convoluted electrophysiological signals from lined-up cardiomyocytes on a single closed loop of a microelectrode of 1 mm diameter and 20 mu m width in a cultivation chip. To evaluate the importance of the closed-loop arrangement of cardiomyocytes for prediction, we compared the change in waveforms of convoluted signals of the responses in the closed-loop circuit arrangement with that of the response of cardiomyocyte clusters using a typical human ether a go-go related gene (hERG) ion channel blocker, E-4031. The results showed that (1) waveform prolongation and fluctuation both in the closed loops and clusters increased depending on the E-4031 concentration increase. However, (2) only the waveform signals in closed loops showed an apparent temporal change in waveforms from ventricular tachycardia (VT) to ventricular fibrillation (VF), which is similar to the most typical cell-to-cell conductance abnormality. The results indicated the usefulness of convoluted waveform signals of a closed-loop cell network for acquiring reproducible results acquisition and more detailed temporal information on cell-to-cell conduction. (C) 2016 The Japan Society of Applied Physics

We herein examined the ability of a template matching algorithm to recognize particles with diameters ranging from 1 to 20 mu m in a microfluidic channel. The algorithm consisted of measurements of the distance between the templates and the images captured with a high-speed camera in order to search for the presence of the desired particle. The results obtained indicated that the effects of blur and diffraction rings observed around the particle are important phenomena that limit the recognition of a target. Owing to the effects of diffraction rings, the distance between a template and an image is not exclusively linked to the position of the focus plane; it is also linked to the size of the particle being searched for. By using a set of three templates captured at different Z focuses and an 800x magnification, the template matching algorithm has the ability to recognize beads ranging in diameter from 1.7 to 20 mu m with a resolution between 0.3 and 1 mu m. (C) 2016 The Japan Society of Applied Physics

We developed a microprocessing-assisted technique to select single-strand DNA aptamers that bind to unknown targets on the cell surface by modifying the conventional systematic evolution of ligands by exponential enrichment (cell-SELEX). Our technique involves 1) the specific selection of target-cell-surface-bound aptamers without leakage of intracellular components by trypsinization and 2) cloning of aptamers by microprocessing-assisted picking of single cells using magnetic beads. After cell-SELEX, the enriched aptamers were conjugated with magnetic beads. The aptamer-magnetic beads conjugates attached to target cells were collected individually by microassisted procedures using microneedles under a microscope. After that, the sequences of the collected magnetic-bead-bound aptamers were identified. As a result, a specific aptamer for the surface of target cells, e.g., human umbilical vein endothelial cells (HUVECs), was chosen and its specificity was examined using other cell types, e.g., HeLa cells. The results indicate that this microprocessing-assisted cell-SELEX method for identifying aptamers is applicable in biological research and clinical diagnostics. (C) 2016 The Japan Society of Applied Physics

We investigated electrophysiological properties of human induced-pluripotent-stem-cell-derived and embryonic-stem-cell-derived cardiomyocytes, and analyzed action potential parameters by plotting their frequency distributions. In the both cell lines, the distribution analysis revealed that histograms of maximum upstroke velocity showed two subpopulations with similar intersection values. Subpopulations with faster maximum upstroke velocity showed significant prolongation of action potential durations by application of E-4031, whereas others did not, which may be partly due to shallower maximum diastolic potentials. We described electrophysiological and pharmacological properties of stem-cell-derived cardiomyocytes in the respective sub-populations, which provides a way to characterize diverse electrical properties of stem-cell-derived cardiomyocytes systematically. (C) 2016 The Authors. Production and hosting by Elsevier B. V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

A series of studies aimed at developing methods and technologies of analyzing epigenetic information in cells and in those networks, as well as that of genetic information, was examined to expand our understanding of how living systems are determined. Technologies of analyzing epigenetic information was developed starting from the twin complementary viewpoints of cell regulation as an "algebraic" system (emphasis on temporal aspects) and as a "geometric" system (emphasis on spatial aspects). Exploiting the combination of latest microfabrication technologies and measurement technologies, which we call on-chip cellomics technology, we can select, control, and reconstruct the environments, interaction of single cells and cell networks from "algebraic" and "geometric" viewpoints. In this chapter, our developed technolgoeis and some results for spatial viewpoint of epigenetic information as a part of a series of cell-networkbased "geometric" studies of celluler systems in our research groups are summarized and reported. The knowlege and technolgies acquired from these viewpoints may lead to the use of cells that fully control practical applications like cell-network-based drug screening and the regeneration of organs from cells.

A combination of extracellular field potential (FP) and impedance measurement technologies for multielectrode array (MEA) chip architecture is developed for the simultaneous evaluation of information on the ion current and resistance of cells and microelectrodes. The simultaneous measurement system can not only evaluate the time course changes of characteristics in the MEAs but also clarification of the origin of the difference in the waveform of the field potentials of each cell on an microelectrode whether it is caused by the changes in the electrophysiological properties of cells or by the changes in the performance of an microelectrode (and cell-to-electrode contacts). The automatic impedance measurement technology in the system exploited the swiping of the wide frequency range of impedances of microelectrodes and calculated the true impedance of each microelectrode without significant effects on the cells on the MEA chip. Hence, the system can give us invisible cell-to-electrode contact information and its change, and also information on the degradation of the performance of microelectrodes during long-term cultivation and after the application of compounds into the MEA chip. The impedance spectrum measurement showed that (1) the increase in the impedance of microelectrodes correlated with its area decrease from 10% 7 to 10% 10m(2), (2) even the area of microelectrodes decreased from 10% 8 to 10% 10m(2), the noise level of field potential signals was independent and did not change, and (3) the attachment of cells on the microelectrode surface can be determined by a significant increase in impedance at 1 kHz corresponding to the width of the depolarization peak on the field potential recordings. These results indicate the potential to evaluate the cell-to-electrode contact and degradation of microelectrodes, which was not evaluated in conventional FP measurements only. These results also indicate that this method should be used for the evaluation of the changes in cell network conditions caused by various compounds. (C) 2015 The Japan Society of Applied Physics

The depletion effect on a concave microstructure for size-specific target particle collection was evaluated using a set of strictly size-controlled superparamagnetic hemispheres, "magcups", with mixtures of differently sized model target microbeads in the presence of dispersed polystyrene nanoparticles (PS-NPs), which are thought to enhance entropic depletion effects to capture model target beads having the size closest to those of the hemispherical microstructures of magcups. Magcups with diameters of 10 to 50 mu m were fabricated with the formation of three nickel layers (2nm thickness) inserted between silicon dioxide layers (15 nm) on polystyrene template spheres by vapor deposition and subsequent polystyrene removal by burning. Two different diameters of model target beads (10 and 20 mu m) were mixed with the magcups in order to evaluate the contribution of the depletion effect to the frequency of target bead capture by the concave microstructures of magcups of various sizes. To evaluate the depletion effect caused by the presence of dispersed PS-NPs, we compared the frequencies of target bead-magcup conjugations with and without PS-NPs in reaction solvents, and found that (i) the frequency of 10-mu m-diameter bead capture increased 3.0 times on average for 15- and 20-mu m-diameter magcups, and (ii) the frequencies of 10-mu m-diameter beads capture increased as almost the same level with that of 20-mu m bead for 30-, 40-, and 50-mu m-diameter magcups. These results suggest that conjugations of target beads with magcups were encouraged by the depletion effect due to the presence of PS-NPs. (C) 2015 The Japan Society of Applied Physics

A method of discriminating single cancer cells from whole blood cells based on their morphological visual characteristics (i.e., "imaging biomarker") was examined. Cells in healthy rat blood, a cancer cell line (MAT-LyLu), and cells in cancer-cell-implanted rat blood were chosen as models, and their bright-field (BF, whole-cell morphology) and fluorescence (FL, nucleus morphology) images were taken by an on-chip multi-imaging flow cytometry system and compared. Eight imaging biomarker indices, i.e., cellular area in a BF image, nucleus area in an FL image, area ratio of a whole cell and its nucleus, distance of the mass center between a whole cell and nucleus, cellular and nucleus perimeter, and perimeter ratios were calculated and analyzed using the BF and FL images taken. Results show that cancer cells can be clearly distinguished from healthy blood cells using correlation diagrams for cellular and nucleus areas as two different categories. Moreover, a portion of cancer cells showed a low nucleus perimeter ratio less than 0.9 because of the irregular nucleus morphologies of cancer cells. These results indicate that the measurements of imaging biomarkers are practically applicable to identifying cancer cells in blood. (C) 2015 The Japan Society of Applied Physics

We propose a new method of size separation of cells exploiting precisely size-controlled hemispherical superparamagnetic microparticles. A three-layered structure of a 2-nm nickel layer inserted between 15-nm silicon dioxide layers was formed on polystyrene cast spheres by vapor deposition. The polystyrene was then removed by burning and the hemispherical superparamagnetic microparticles, "magcups", were obtained. The standard target cells (CCRF-CEM, 12 +/- 62 mu m) were mixed with a set of different sizes of the fabricated magcups, and we confirmed that the cells were captured in the magcups having cavities larger than 15 mu m in diameter, and then gathered by magnetic force. The collected cells were grown in a culture medium without any damage. The results suggest that this method is quick, simple and non-invasive size separation of target cells.

An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as "imaging biomarkers'', with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 mu m(2) and (2) nucleus area larger than 90 mu m(2) were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 mu m(2) were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers-cluster area, nuclei area, nuclei number, and ratio of perimeter-can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.

Knowledge of life has expanded dramatically during the twentieth century and has produced the modern disciplines of genomics and proteomics. However, there remains the great challenge of discovering the integration and regulation of these living components in time and space within the cell. As we move into the postgenomic period, the complementarity between genomics and proteomics will become apparent, and the connections between them will be exploited, although, neither genomics, proteomics, nor, for that matter, their simple combination will provide the data necessary to interconnect molecular events in living cells in time and space. e cells in a group are dierent entities. Each of them respond to the perturbations dierently (Spudich and Koshland 1976). e dierences between cells arise even among those grown in homogeneous conditions and considered to have identical genetic information. Why and how do these dierences arise? ey might be caused by several factors like unequal distributions of biomolecules in cells, mutations, interactions between cells, and uctuations of environmental elements. A system that can observe interactions between specic cells continuously under fully controlled.

We have examined the ability of real-time simultaneous measurement of bright field/fluorescent images of cells in an on-chip bright field/fluorescent multi-imaging flow cytometer system. The system consists of (1) a disposable microfluidic hydrofocusing flow cytometry chip, (2) an optical microscopy module with splittable bright field/fluorescent multi-imaging optics, and (3) a real-time image-processing module with a 200 images/s high-speed digital camera. In the double "Y" shape three-way-inlet microfluidic pathways fabricated in the poly(dimethylsiloxane) (PDMS) microchip, we applied fluorescent polystyrene standard beads and HeLa cells stained with fluorescent dye, Hoechst 33258, and measured the z-axis (depth) dependence of the morphological index; the intensity profile of cells and nuclei. Then, we measured the tendency of the blur of bright field/fluorescent images in the simultaneous measurement of bright field/fluorescent images on a single light-receiving surface, and found that their blurs were similar within the same range of the depth of the microfluidic pathway for small cell cluster measurement, 25 m. Hence, the fluorescent images were applied as supporting information of the bright field images of cell clusters at the focal plane for the cell number counting. The result indicates the potential of precise identification of various types of cells by simultaneous morphological analysis of bright field and fluorescent images distributed with a single camera in a wider depth of microfluidic chip as a substitute for conventional biomarker detection. (C) 2014 The Japan Society of Applied Physics

A simple method for the fabrication of superparamagnetic particles was developed. Polystyrene spheres were used as templates, and their surfaces were coated with a magnetic element (Ni) by thermal deposition, controlling their thicknesses strictly. Magnetic properties of fabricated particles depended on the Ni layer thickness; the fabricated particles were typically superparamagnetic for a Ni layer thinner than 3 nm and ferromagnetic for a Ni layer thicker than 4 nm. For the improvement of the force generated on a particle in a magnetic field, the formation of multiple Ni layers on a particle was examined by sequential depositions of Ni and SiO2, isolating two Ni layers with a SiO2 layer. The SiO2 layer thickness should be larger than 10 nm for a sufficient isolation of two Ni layers to maintain the superparamagnetic properties of the particle, and the magnetic charge of the particle increased proportionally with the number of Ni layers on a particle. These results indicate that enhanced superparamagnetic particles with various diameters can easily be fabricated by the suggested method. (C) 2014 The Japan Society of Applied Physics

Polymerase chain reaction (PCR) is an essential technique for almost all life science fields that amplifies few target DNA copies. We developed a rapid real-time microdroplet PCR device by rapid switching of two types of circulation hot water, a mu L-sized droplet, and a thin film aluminum chip. Using this device, rapid PCR amplification was observed successfully and accomplished within 3min. By real-time PCR, many samples could be measured simultaneously. Moreover, melting curve analysis is essential for detecting differences of PCR amplicons in detail such as those related to nonspecific amplification, single nucleotide polymorphism, and genotyping methods. Thus, we developed a device that can be used for melting curve analysis using circulation water. From the obtained results, temperature homogeneity on the reaction plate was maintained during PCR and melting curve analysis using high-speed circulation water. The results indicate that this system can be applied to various life science fields. (C) 2014 The Japan Society of Applied Physics

To overcome the limitations and misjudgments of conventional prediction of arrhythmic cardiotoxicity, we have developed an on-chip in vitro predictive cardiotoxicity assay using cardiomyocytes derived from human stem cells employing a constructive spatiotemporal two step measurement of fluctuation (short-term variability; STV) of cell's repolarization and cell-to-cell conduction time, representing two origins of lethal arrhythmia. Temporal STV of field potential duration (FPD) showed a potential to predict the risks of lethal arrhythmia originated from repolarization dispersion for false negative compounds, which was not correctly predicted by conventional measurements using animal cells, even for non-QT prolonging clinical positive compounds. Spatial STV of conduction time delay also unveiled the proarrhythmic risk of asynchronous propagation in cell networks, whose risk cannot be correctly predicted by single-cell-based measurements, indicating the importance of the spatiotemporal fluctuation viewpoint of in vitro cell networks for precise prediction of lethal arrhythmia reaching clinical assessment such as thorough QT assay.

ヒトiPS細胞の出現によって創薬スクリーニング法もヒト細胞を用いたものが検討されている。特に最も分化誘導技術の開発が進んでいるヒト心筋細胞については,実際の致死性不整脈を引き起こす「興奮伝導異常」を計測するオンチップ計測技術を用いることで,(1)細胞のカリウムイオンチャネルの応答のゆらぎ解析による安定性変化の定量化(時間的観点),(2)心筋細胞ネットワークでの伝達状態のゆらぎ解析による伝達異常の定量化(空間的観点)によって,従来の計測法で偽陰性・偽陽性と評価されてきた候補薬の催不整脈リスクを正確に計測することが可能となりつつある。(著者抄録)

We examined the effect of a concave structure on DNA hybridization efficiency using an inner surface of hemispherical Janus nanocups in the range from 140 to 800 nm. Target DNA was specifically immobilized onto the inner cup surface, hybridized with complementary DNA-attached 20 nm Au probes, and the number of the hybridized probes was counted by scanning electron microscopy. The hybridization density of the attached Au probes on 800 nm nanocups was 255 mu m(-2), which was 0.57 times that on a flat surface, 449 mu m(-2), and increased to 394 mu m(-2) on a 140 nm cup, 0.88 times of a flat surface, as the cup size decreased. The local density of attached Au probes within the central 25% at the bottom of the 800 nm nanocups was 444 mu m(-2), which was closer to that on a flat surface, and the tendency was the same for all sizes of cups, indicating that the size dependency of DNA hybridization efficiency on the concave structures were mostly affected by the lower efficiency of side wall hybridization.

We have examined the contribution of temperature shift speed from denaturation to extension for the reduction of nonspecific amplification caused by the mismatched primer-target attachment. We have newly developed the photothermal quantitative polymerase chain reaction (qPCR) system, in which the direct absorption of a 1480nm infrared laser beam was controlled by a rotating gradient neutral density (ND) filter to acquire the precise control of the desired speed of temperature shift between 60 and 95°C up to 1 s. The results showed that a quick shift of the temperature during the qPCR procedure reduced nonspecific amplicons with a significant reduction of qPCR time when we have chosen proper primer sets, whereas the non-proper primer set amplified nonspecific amplicons in the fast qPCR. The results indicate that the potential of quick qPCR using proper primers can reduce nonspecific amplification and the required time for qPCR measurement, and the necessity of more precise check of the matching of the primer template adequate for the fast temperature shift and for quick qPCR analysis. © 2013 The Japan Society of Applied Physics.

We have developed a non-destructive imaging flow cell-sorting system using an ultra-high-speed camera (shutter speed of 1/10,000 s) with a real-time image analysis unit and a poly(methyl methacrylate) (PMMA)-based disposable microfluidic chip for single-cell-based on-chip cellomics. It has a 3-D micropipetting device that supports fully automated sorting and collection of samples. The entire fluidic system is implemented in a disposable plastic chip, enabling biological samples to be lined up in a laminar flow using hydrodynamic focusing. Its optical system enables direct observation-based cell identification using specific image indexes and phase-contrast/fluorescence microscopy, real-time image processing. It has a non-destructive, wider dynamic range, sorting procedure using mild electrostatic force in a laminar flow; agarose gel electrodes are used to prevent electrode loss and electrolysis bubble formation. The microreservoir used for recultivating collected target cells is contamination-free. An integrated ultra-high-speed droplet polymerase chain reaction measurement module is used for DNA/mRNA analysis of the collected target cells. This system was used to separate cardiomyocyte cells from a mixture of various cells. All the operations were automated using the 3-D micropipetting device. The results demonstrate that this imaging flow cell-sorting system is practically applicable for biological research and clinical diagnosis.

To predict the risk of fatal arrhythmia induced by cardiotoxicity in the highly complex human heart system, we have developed a novel quasiin vivo electrophysiological measurement assay, which combines a ring-shaped human cardiomyocyte network and a set of two electrodes that form a large single ring-shaped electrode for the direct measurement of irregular cell-to-cell conductance occurrence in a cardiomyocyte network, and a small rectangular microelectrode for forced pacing of cardiomyocyte beating and for acquiring the field potential waveforms of cardiomyocytes. The advantages of this assay are as follows. The electrophysiological signals of cardiomyocytes in the ring-shaped network are superimposed directly on a single loop-shaped electrode, in which the information of asynchronous behavior of cell-to-cell conductance are included, without requiring a set of huge numbers of microelectrode arrays, a set of fast data conversion circuits, or a complex analysis in a computer. Another advantage is that the small rectangular electrode can control the position and timing of forced beating in a ring-shaped human induced pluripotent stem cell (hiPS)-derived cardiomyocyte network and can also acquire the field potentials of cardiomyocytes. First, we constructed the human iPS-derived cardiomyocyte ring-shaped network on the set of two electrodes, and acquired the field potential signals of particular cardiomyocytes in the ring-shaped cardiomyocyte network during simultaneous acquisition of the superimposed signals of wholecardiomyocyte networks representing cell-to-cell conduction. Using the small rectangular electrode, we have also evaluated the response of the cell network to electrical stimulation. The mean and SD of the minimum stimulation voltage required for pacing (VMin) at the small rectangular electrode was 166 ± 74 mV, which is the same as the magnitude of amplitude for the pacing using the ring-shaped electrode (179 33 mV). The results showed that the addition of a small rectangular electrode into the ring-shaped electrode was effective for the simultaneous measurement of whole-cell-network signals and single-cell/small-cluster signals on a local site in the cell network, and for the pacing by electrical stimulation of cardiomyocyte networks. © 2013 The Japan Society of Applied Physics.

We have evaluated the electrophysiological characteristics of a line-shaped network of a three-dimensionally controlled in vitro human cardiomyocyte assay (hCM line) against conventional cell clusters as the standard model (hCM cluster) from the viewpoint of quality control of sample variety and time-course stability. The beating intervals of the hCM line demonstrated a more stable uniformity of samples (846 +/- 130 ms, 15.3% fluctuation) and better time-course stability, whereas those of the hCM cluster showed a much larger variety of samples (2001 +/- 1127 ms, 56.3% fluctuation) and weaker time-course stability. The field potential amplitude of the hCM line also showed better uniformity of samples (629 +/- 428 mu V, 68.0% fluctuation) against those of the hCM cluster (1984 +/- 2288 mu V, 115.3% fluctuation). The results suggested the importance of the cell-network shape control for the uniformity and stability of the beating interval and the field potential amplitude. They also suggest that the hCM line can improve the reproducibility and accuracy of the samples, which is important for a functional human cardiotoxicity model. (C) 2013 The Japan Society of Applied Physics

Quantitative evaluation of mechanophysiological responses of cardiomyocytes has become more important for more precise prediction of cardiotoxicity. For the accurate detection of cardiomyocyte contraction, we have developed an on-chip single-cell-shape control technology on the basis of an agarose microchamber system and an on-chip optical image analysis system that records the contractile motions of cardiomyocytes with noninvasive/nondestructive measurement for long-term experiments. Using this on-chip single-cell-shape control technology, the shape of single cardiomyocytes was controlled by seeding the cells in 21-mu m-radius (circular) or 20 x 70 mu m(2) (rectangular) agarose microchambers. To detect the contractility of cardiomyocytes, the cells were labeled with microbeads attached onto the surface of target cells and the motion of beads was acquired and analyzed using a newly developed wider-depth-of-field optics equipped with a 1/100 s high-speed digital camera. Mechanophysiological properties such as displacement and direction of movement were obtained using a real-time processing system module at spatial and temporal resolutions of 0.15 mu m and 10 ms, respectively. Comparisons of displacement and direction of contraction between circular and rectangular cardiomyocytes indicated that the rectangular cardiomyocytes tended to contract along the longitudinal direction as in a real heart. This result suggests that the shape of cells affected the function of cells. The on-chip single-cell-shape control technology and optical image analysis system enable the detection of the motion of contraction of single-shape-controlled cardiomyocytes, and are expected to be applicable to the more precise prediction of cardiotoxicity. (C) 2013 The Japan Society of Applied Physics

The interface between engineering and molecular life sciences has been fertile ground for advancing our understanding of complex biological systems. Engineered microstructures offer a diverse toolbox for cellular and molecular biologists to direct the placement of cells and small organisms, and to recreate biological functions in vitro: cells can be positioned and connected in a designed fashion, and connectivity and community effects of cells studied. Because of the highly polar morphology and finely compartmentalized functions of neurons, microfabricated cell culture systems and related on-chip technologies have become an important enabling platform for studying development, function and degeneration of the nervous system at the molecular and cellular level. Here we review some of the compartmentalization techniques developed so far to highlight how high-precision control of neuronal connectivity allows new approaches for studying axonal and synaptic biology.

The present study focused on beating synchronization, and tried to elucidate the interlayer regulatory mechanisms between the cells and clump in beating synchronization with using the stochastic simulations which realize the beating synchronizations in beating cells with low cell-cell conductance. Firstly, the fluctuation in interbeat intervals (IBIs) of beating cells encouraged the process of beating synchronization, which was identified as the stochastic resonance. Secondly, fluctuation in the synchronized IBIs of a clump decreased as the number of beating cells increased. The decrease in IBI fluctuation due to clump formation implied both a decline of the electrophysiological plasticity of each beating cell and an enhancement of the electrophysiological stability of the clump. These findings were identified as the community effects. Because IBI fluctuation and the community effect facilitated the beating stability of the cell and clump, these factors contributed to the spontaneous ordering in beating synchronization. Thirdly, the cellular layouts in clump affected the synchronized beating rhythms. The synchronized beating rhythm in clump was implicitly regulated by a complicated synergistic effect among IBI fluctuation of each beating cell, the community effect and the cellular layout. This finding was indispensable for leading an elucidation of mechanism of emergence. The stochastic simulations showed the necessity of considering the synergistic effect, to elucidate the interlayer regulatory mechanisms in biological system. (C) 2013 Elsevier Ireland Ltd, All rights reserved.

We have developed methods and systems of analyzing epigenetic information in cells, as well as that of genetic information, to expand our understanding of how living systems are determined. A system of analyzing epigenetic information was developed starting from the twin complementary viewpoints of cell regulation as an 'algebraic' system (emphasis on temporal aspects) and as a 'geometric' system (emphasis on spatial aspects). As an example of the 'geometric' system, we have developed an quasi-in vivo hiPS cardiomyocyte network assay and confirmed that it can predict the risk of lethal arrythmia correctly in 22 compounds. The knowlege acquired from this study may lead to the use of cells that fully control practical applications like cell-based drug screening and the regeneration of organs. © 2013 IEEE.

We have developed methods and systems of analyzing epigenetic information in cells to expand our understanding of how living systems are determined. Because cells are minimum units reflecting epigenetic information, which is considered to map the history of a parallel-processing recurrent network of biochemical reactions, their behaviors cannot be explained by considering only conventional deonucleotide (DNA) information-processing events. The role of epigenetic information on cells, which complements their genetic information, was inferred by comparing predictions from genetic information with cell behaviour observed under conditions chosen to reveal adaptation processes and community effects. A system of analyzing epigenetic information, on-chip cellomics technology, has been developed starting from the twin complementary viewpoints of cell regulation as an "algebraic'' system (emphasis on temporal aspects) and as a "geometric'' system (emphasis on spatial aspects) exploiting microfabrication technology and a reconstructive approach of cellular systems not only for single cell-based subjects such as Escherichia coli and macrophages but also for cellular networks like the community effect of cardiomyocytes and plasticity in neuronal networks. One of the most important contributions of this study was to be able to reconstruct the concept of a cell regulatory network from the "local'' (molecules expressed at certain times and places) to the "global'' (the cell as a viable, functioning system). Knowledge of epigenetic information, which we can control and change during cell lives, complements the genetic variety, and these two types of information are indispensable for living organisms. This new knowlege has the potential to be the basis of cell-based biological and medical fields such as those involving cell-based drug screening and the regeneration of organs from stem cells. (C) 2012 The Japan Society of Applied Physics

A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES) cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

The contributions of metal thickness and the diameter of metal shell particles to quantitative backscattered electron (BSE) imaging in field emission scanning electron microscopy (FE-SEM) were studied to evaluate the potential of using these particles as simultaneously distinguishable labels of target molecules in FE-SEM studies. Gold spherical shells were fabricated with 200 or 300 nm diameter and 5, 10, 15 or 20 nm Au shell thickness, placed on silicon substrates, respectively, and observed in BSE imaging mode by FE-SEM. Flat Au films of the same thicknesses were formed on a Si substrate to evaluate the contribution of shell diameter to BSE imaging. The relationship between relative BSE intensity, which was calculated by setting the intensities of the Si substrate and 20 nm-thick Au layer as standards, and Au layer thickness was studied for all samples. With increasing Au layer thickness, BSE intensity also proportionally increased for all samples (R 2 &gt
0.93) in the range of these thicknesses. Gradients of the increase were 1.5 times different between the flat film and metal shells, which was caused by the presence of voids in shell particles. The difference in gradients for increasing shell thickness between 200 and 300 nm particles was 15%. This result indicated that 1.5 times difference in shell diameter contributed to the increase of BSE intensity against the increase of shell thicknesses as a 15% error, and strict control of both metal shell diameter and thickness in the fabrication process is essential when using these shells as labels of BSE measurements in FE-SEM. © 2012 The Surface Science Society of Japan.

A series of studies aimed at developing methods and systems of analyzing epigenetic information in cells and in cell networks, as well as that of genetic information, was examined to expand our understanding of how living systems are determined. Because cells are minimum units reflecting epigenetic information, which is considered to map the history of a parallel-processing recurrent network of biochemical reactions, their behaviors cannot be explained by considering only conventional DNA information-processing events. The role of epigenetic information on cells, which complements their genetic information, was inferred by comparing predictions from genetic information with cell behaviour observed under conditions chosen to reveal adaptation processes, population effects and community effects. A system of analyzing epigenetic information was developed starting from the twin complementary viewpoints of cell regulation as an "algebraic" system (emphasis on temporal aspects) and as a "geometric" system (emphasis on spatial aspects). Exploiting the combination of latest microfabrication technology and measurement technologies, which we call on-chip cellomics assay, we can control and re-construct the environments and interaction of cells from "algebraic" and "geometric" viewpoints. In this review, temporal viewpoint of epigenetic information, a part of the series of single-cell-based "algebraic" and "geometric" studies of celluler systems in our research groups, are summerized and reported. The knowlege acquired from this study may lead to the use of cells that fully control practical applications like cell-based drug screening and the regeneration of organs.

We have examined a method to address the defocusing problem on target samples in a microfluidic pathway by an optical approach and report our experiment. An imaging optics has been constructed for extension of the depth of focused field. This system consists of four parts: (1) a low numerical aperture (NA; i.e., large depth of field) objective lens; (2) a zoom lens; (3) a light-emitting diode (LED) illumination source; and (4) a charge-coupled device (CCD) camera. As a low NA objective lens contributes to the extension of the depth of field and a zoom lens contributes to the optimization of pixel resolution on an image sensor of a camera, the same resolution as that of a 40x objective lens was acquired by the combination of a 10x objective lens and a 4x zoom lens as the spatial resolution of the latter combination was within the size of pixels of the CCD camera. As a result, improved depth of field was obtained at any magnification from 10x to 40x, and it was indicated that an extended depth of field optics for image-based microfluidic pathways such as in flow cytometry can be constructed using a low NA objective lens and a zoom lens. (C) 2012 The Japan Society of Applied Physics

We have developed a novel cell-sorting system involving microscopic imaging using a poly(methyl methacrylate) (PMMA)-based microfluidic chip with a pair of gel electrodes and real-time image-processing procedures for the quantification of cell shapes. The features of this system are as follows. 1) It can recognize cells both by microscopic cell imaging with a 10,000 event/s high-speed camera and by the photodetection of fluorescence. 2) Multistage sorting is used to reduce errors to an infinitesimally low level by using a pair of wide agarose-gel electrodes. 3) Carry-over-free analysis can be performed using a disposable microfluidic chip. 4) An field programmable gate array (FPGA) 10,000 event/s real-time image analysis unit for quantifying the cell images in cell sorting. To separate the target cells from other cells on the basis of the cell shape, we adopted an index of roughness for the cell surface R, which compares the actual perimeter of cell surface and the estimated perimeter of cross-sectional view of cell shape by approximating the cell as a sphere. Sample cells flowing through microchannels on the chip were distinguished by the dual recognition system involving optical analysis and a fluorescence detector, and then separated. Target cells could be sorted automatically by applying an electrophoretic force, and the sorting ability depended on the precision with which cells were shifted within the laminar flow. These results indicate that the cell-sorting system with on-chip imaging is practically applicable for biological research and clinical diagnostics. (C) 2012 The Japan Society of Applied Physics

A method of gold nanoparticle (Au NP) labeling with backscattered electron (BE) imaging of field emission scanning electron microscopy (FE-SEM) was applied for specific detection of target biomolecules on a cell surface. A single-stranded DNA aptamer, which specifically binds to the target molecule on a human acute lymphoblastic leukemia cell, was conjugated with a 20 nm Au NP and used as a probe to label its target molecule on the cell. The Au NP probe was incubated with the cell, and the interaction was confirmed using BE imaging of FE-SEM through direct counting of the number of Au NPs attached on the target cell surface. Specific Au NP-aptamer probes were observed on a single cell surface and their spatial distributions including submicron-order localizations were also clearly visualized, whereas the nonspecific aptamer probes were not observed on it. The aptamer probe can be potentially dislodged from the cell surface with treatment of nucleases, indicating that Au NP-conjugated aptamer probes can be used as sensitive and reversible probes to label target biomolecules on cells. (C) 2012 The Japan Society of Applied Physics

We have developed a three-dimensionally controlled in vitro human cardiomyocyte network assay for the measurements of drug-induced conductivity changes and the appearance of fatal arrhythmia such as ventricular tachycardia/ fibrillation for more precise in vitro predictive cardiotoxicity. To construct an artificial conductance propagation model of a human cardiomyocyte network, first, we examined the cell concentration dependence of the cell network heights and found the existence of a height limit of cell networks, which was double-layer height, whereas the cardiomyocytes were effectively and homogeneously cultivated within the microchamber maintaining their spatial distribution constant and their electrophysiological conductance and propagation were successfully recorded using a microelectrode array set on the bottom of the microchamber. The pacing ability of a cardiomyocyte's electrophysiological response has been evaluated using microelectrode extracellular stimulation, and the stimulation for pacing also successfully regulated the beating frequencies of two-layered cardiomyocyte networks, whereas monolayered cardiomyocyte networks were hardly stimulated by the external electrodes using the two-layered cardiomyocyte stimulation condition. The stability of the lined-up shape of human cardiomyocytes within the rectangularly arranged agarose microchambers was limited for a two-layered cardiomyocyte network because their stronger force generation shrunk those cells after peeling off the substrate. The results indicate the importance of fabrication technology of thickness control of cellular networks for effective extracellular stimulation and the potential concerning thick cardiomyocyte networks for long-term cultivation. (C) 2012 The Japan Society of Applied Physics

Re-entry of excitation in the heart is one of the abnormal phenomena that causes lethal arrhythmia and is thought to be induced by the looped structure of the excitation conduction pathway. To evaluate the geometrical pattern dependence of electrophysiological results, we fabricated three models of cardiomyocyte networks and compared their beating frequencies (BFs), amplitudes of a depolarization peak, and field potential durations (FPDs). The set of different closed-loop-shaped network models from 3 to 8 mm in length showed the same BFs, amplitudes, and FPDs independent of their loop lengths, whereas the BFs and FPDs of 60 mu m small clusters, and the FPDs of the 2mm open-line-shaped network model were different from those of a closed-loop-shaped network model. These results indicate that the mm order larger size of clusters might create lower BFs, and the closed-loop-shaped model may generate longer FPDs. They also suggest the importance of spatial arrangement control of the cardoimyocyte community for reproducible measurement of electrophysiological properties of cardiomyocytes, especially control of the closed-loop formation, which might change the waveforms of FPDs depending on the difference in the geometry and conduction pathway of the cell network. (C) 2012 The Japan Society of Applied Physics

Cardiotoxicity testing with a multi-electrode array (MEA) system requires the stable beating of cardiomyocytes for the measurement of the field potential duration (FPD), because different spontaneous beating rates cause different responses of FPD prolongation induced by drugs, and the beating rate change effected by drugs complicates the FPD prolongation assessment. We have developed an on-chip MEA system with electrical stimulation for the measurement of the FPD during the stable beating of human embryonic stem (ES) cell-derived cardiomyocyte clusters. Using a conventional bipolar stimulation protocol, we observed such large artifacts in electrical stimulation that we could not estimate the FPD quantitatively. Therefore, we improved the stimulation protocol by using sequential rectangular pulses in which the positive and negative stimulation voltages and number of pulses could be changed flexibly. The balanced voltages and number of pulses for sequential rectangular pulses enabled the recording of small negative artifacts only, which hardly affected the FPD measurement of human-ES-cell-derived cardiomyocyte clusters. These conditions of electrical stimulation are expected to find applications for the control of constant beating for cardiotoxicity testing. (C) 2012 The Japan Society of Applied Physics

We have demonstrated the efficacy of a microfluidic medium exchange method for single cells using passive centrifugal force of a rotating microfluidic-chip based platform. At the boundary of two laminar flows at the gathering area of two microfluidic pathways in a Y-shape, the cells were successfully transported from one laminar flow to the other, without mixing the two microfluidic mediums of the two laminar flows during cell transportation, within 5 s with 1 g (150 rpm) to 36.3 g (900 rpm) acceleration, with 93.5% efficiency. The results indicate that this is one of the most simple and precise tools for exchanging medium in the shortest amount of time.

Backgrounds: Conventional in vitro approach using human ether-a-go-go related gene (hERG) assay has been considered worldwide as the first screening assay for cardiac repolarization safety. However, it does not always oredict the potential QT prolongation risk or pro-arrhythmic risk correctly. For adaptable preclinical strategiesto evaluate global cardiac safety, an on-chip quasi-in vivo cardiac toxicity assay for lethal arrhythmia (ventricular tachyarrhythmia) measurement using ring-shaped closed circuit microelectrode chip has been developed.
Results: The ventricular electrocardiogram (ECG)-like field potential data, which includes both the repolarization and the conductance abnormality, was acquired from the self-convolutied extracellular field potentials (FPs) of a lined-up cardiomyocyte network on a circle-shaped microelectrode in an agarose microchamber. When Astemisol applied to the closed-loop cardiomyocyte network, self-convoluted FP profile of normal beating changed into an early afterdepolarization (EAD) like waveform, and then showed ventricular tachyarrhythmias and ventricular fibrilations (VT/Vf). QT-prolongation-like self-convoluted FP duration prolongation and its fluctuation increase was also observed according to the increase of Astemizole concentration.
Conclusions: The results indicate that the convoluted FPs of the quasi-in vivo cell network assay includes both of the repolarization data and the conductance abnormality of cardiomyocyte networks has the strong potential to prediction lethal arrhythmia.

We have examined the orientation dependence of minimum electric field intensity for the stimulation of cardiomyocytes, which were cultivated in agarose chambers, using a lined-up cardiomyocyte network with different numbers of cells and orientations. When the cell network was arranged parallel to the electric field, the required minimum electric field intensity decreased to one-fourth as cell number increased, whereas that of the cell network arranged orthogonal to the electrical field did not decrease and was independent of cell number. The required electrical field intensity of the 100 mu m rod-shaped single cardiomyocyte in a microchamber arranged parallel to the electric field was also 40% lower than that of the cell network arranged orthogonal to the electric field. The results indicate that the gradient of the electric field potential between two ends of the cell network or rod-shaped single cell is important for their excitation. (C) 2011 The Japan Society of Applied Physics

A model for the quasi-in vivo heart electrocardiogram (ECG) measurement of the ST period has been developed. As the part of ECG data at the ST period is the convolution of the extracellular field potentials (FPs) of cardiomyocytes in a ventricle, we have fabricated a lined-up cardiomyocyte cell-network on a lined-up microelectrode array and a circular microelectrode in an agarose microchamber, and measured the convoluted FPs. When the ventricular tachyarrhythmias of beating occurred in the cardiomyocyte network, the convoluted FP profile showed similar arrhythmia ECG-like profiles, indicating the convoluted FPs of the in vitro cell network include both the depolarization data and the propagation manner of beating in the heart. (C) 2011 The Japan Society of Applied Physics

We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA)) based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e. g., microparticles 2.5 mu m and 3.0 mu m in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 mu L with 1 x 10(6) particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics.

A single-cell-based screening assay requires strict identification and isolation of particular target cells from a mixture of various kinds of cells. We have developed a visual-image-based on-chip microfluidic cell sorting method for the collection of neurons. One of the advantages of our method of purifying neurons is the direct monitoring and reorganization of neurons with specific image indexes, such as the cell size, shape, internal complexity, and spatial distribution of a fluorescent dye of a specific antibody marker by phase-contrast/fluorescence microscopy and image processing, which has not been realized using conventional diffraction-based cell sorting systems. First, we compared the differences of microscopic images (shapes) of neurons and glia cells, and found that only neurons have neurites extending from the cell body. We also found that the smooth surface shape indicates neurons, and the rough surface shape indicates glia cells. After picking the neuron cells manually chosen by observing their shapes as described above, we confirmed that the purified neurons can be cultivated and can keep their electrophysiological functions on the chip even after the purification procedure. The results indicate the potential of a nonlabel, noninvasive on-chip cell sorting procedure for neurons using micrograph images for an on-chip ultrahigh-speed camera-based imaging cell sorter. (c) 2011 The Japan Society of Applied Physics

We have developed a real-time imaging cell sorting system composed of a micrometer-sized gel-electrode-embedded microfluidic sorting chip and a real-time image analysis/recognition unit equipped with a high-speed camera and image processing circuits. For the microfluidic continuous cell sorting, we have examined the precise position and velocity control of flowing particles and the precise acquisition of microscopic images of flowing particles. The results showed that (1) hydrodynamic focusing can line up particles precisely within a range of 5 mu m particle size distribution, (2) active air pressure-driven flow velocity control can create the flow in the microfluidic pathways up to 160 mm/s with 0.15 MPa air pressure maintaining linear correlation between air pressure and flow velocity, and (3) 1 mu s flash illumination can prevent the blur even under 200 mm/s flow. Applying the above elements into the system, the recognition error of target particles was within 5% for 2 mu m particles with 2.5 mm/s flow. The experimental results demonstrate the potential of the image index-based on-chip cell sorter for practical application. (c) 2011 The Japan Society of Applied Physics

We have proposed and developed the novel principle of a V-shaped electrode array in a microfluidic pathway for continuous concentration and separation of particles by dielectrophoretic (DEP) force. The advantage of V-shape microelectrode arrays with a microfluidic flow for cell separation is that whole particles are concentrated into the center of a micropathway independent of the difference in their dielectric constants in the X-Y plane, while the particles are split between the top or bottom of the micropathway in the Z-axis direction depending on the differences in their dielectric constants and the applied AC frequency. After the application of a sinusoidal AC voltage of 1MHz and 20 V-pp, both polystyrene spheres and Bacillus subtilis spores were concentrated at the tip of the V-shaped electrode at the center of microfluidic flow in the X-Y plane independent of their dielectric constant differences. They were also split into two directions in the Z-axis, i.e., polystyrene spheres rose to the top, and spores went down to the bottom depending on their dielectric constant differences and were successfully separated in two layered downstreams. The results indicate the potential of V-shaped electrode arrays for simple continuous purification of mixed particles depending on their dielectric constants. (c) 2011 The Japan Society of Applied Physics

Nanocups (NCs), sub-micrometer semispherical bowls consisting of two different nanometer-thick metals on inner and outer layers, have been fabricated to mimic a localized nano-scale biochemical reaction environment for reactive biomolecules. Homogeneous polystyrene beads were used as a cast of the NCs, placed on a Si substrate, dried, and processed by oxygen plasma etching until the desired diameters and gaps among neighboring bead casts. For the fabrication of Au/Ni double-layered NCs, Au and Ni were sequentially deposited on upper halves of the bead surfaces by thermal evaporation with nanometer-order thickness control. The polystyrene casts were removed completely by UV-ozone oxidization reaction, and Au/Ni double-layered NCs were fabricated on a Si substrate. To orient the holes of the fabricated NCs to top for the substrate, poly(dimethylsiloxane) (PDMS) sol was dropped on the NCs placed on the Si substrate, hardened, and peeled off from the substrate, and then the NCs were placed on the PDMS surface with those holes turned-up. To examine the selective interaction of biomolecules on the inner layer of NCs as the artificial nanospace for biomolecular reactions, a thiolated target DNA was immobilized onto the inner layer of a Au/Ni NC as a model. The target DNA was labeled through hybridization reaction using small Au nanoparticles (NPs) on which a complementary probe DNA was immobilized. Both the surface-specific immobilization of the target DNA on the Au layer of the NC and the specific hybridization in NC nanospaces were confirmed by direct observations after those reactions using field emission scanning electron microscopy (FE-SEM), indicating that the inside of the fabricated NCs can be used as the artificial nanospace for studying localized biomolecular reactions. (C) 2011 The Japan Society of Applied Physics

Backgrounds: To clarify the role of cardiac fibroblasts in beating synchronization, we have made simple lined-up cardiomyocyte-fibroblast network model in an on-chip single-cell-based cultivation system.
Results: The synchronization phenomenon of two cardiomyocyte networks connected by fibroblasts showed (1) propagation velocity of electrophysiological signals decreased a magnitude depending on the increasing number of fibroblasts, not the lengths of fibroblasts; (2) fluctuation of interbeat intervals of the synchronized two cardiomyocyte network connected by fibroblasts did not always decreased, and was opposite from homogeneous cardiomyocyte networks; and (3) the synchronized cardiomyocytes connected by fibroblasts sometimes loses their synchronized condition and recovered to synchronized condition, in which the length of asynchronized period was shorter less than 30 beats and was independent to their cultivation time, whereas the length of synchronized period increased according to cultivation time.
Conclusions: The results indicated that fibroblasts can connect cardiomyocytes electrically but do not significantly enhance and contribute to beating interval stability and synchronization. This might also mean that an increase in the number of fibroblasts in heart tissue reduces the cardiomyocyte 'community effect', which enhances synchronization and stability of their beating rhythms.

Identification of double layered thin film elements by backscattered electron (BSE) contrast imaging by scanning electron microscope (SEM) was examined. A flat type sample was formed with an inner layer composed of five different element areas (Au, Ag, Ge, Cu and Fe) 50 nm thick and a thin gold outer layer 2, 5, or 10 nm thick on a silicon substrate. Dependence of BSE intensities of the sample on the acceleration voltage of incident electrons was measured from 3 to 30 kV, and the acceleration voltage sufficient to discriminate different elements shifted from 8.8 to 10.2 kV depending on the increase of the thickness of outer gold layer. Moreover, the dependence of the substrate shape was also evaluated using Ge/Au double layered spherical nano-shells. The contribution of the nano-shell diameter to the BSE intensity was tested using different sized nano-shells. The contribution was within 10% for 1.5 times difference of the diameter of nano-shells. Results between flat thin film and the nano-shell were compared and those differences were within 6% for 10 to 20 kV of acceleration voltage, which indicates that inner elements of double layered thin film objects can be identified using BSE observation of the SEM at appropriate acceleration voltage.

現在，薬物の心毒性を検証するための主要な計測手法は，常に偽陰性（false negative）の危険性を内在している．ヒト幹細胞からヒト心筋細胞の再生が可能になったため，再生された心筋細胞をチップ上に1細胞単位で構成的に配置して，ヒト臓器・組織の応答により近いin vitro細胞ネットワークモデルを構築することが可能になった．そのモデルを使用し，(1)細胞のK⁺イオンチャネルの応答のゆらぎ解析による安定性変化の定量化（時間的観点），(2)心筋細胞ネットワークにおける伝導状態のゆらぎ解析による伝導異常の定量化（空間的観点）のふたつの観点から，心筋細胞間の興奮伝導の異常発生を定量的に観測できる心毒性検査法を検討した．その結果， in vitro系であっても偽陰性の薬剤を「ゆらぎ」から効果的に識別できることを確認した．細胞間の興奮収縮の伝導異常である致死性不整脈の発生を計測するには，従来の細胞計測に加えて，細胞間の相互作用を理解するための細胞ネットワークの階層という，新しいin vitroプラットホームが重要であると考えられる．

We developed three techniques to make artificial neuronal networks constructed from rat hippocampal neurons. 1) a method of non-invasively collecting primary cultured neurons and their deposition, 2) a technique for microprocessing agarose for the purpose of assembling artificial neuronal networks, 3) a multi-electrode array system for measurement of the multi-point extracellular potential of neurons. The three techniques allow us to assemble and evaluate artificial neuronal networks constructed from particular cells. We can manipulate neuro-transmission pathways and investigate roles played by the innate period or stability information for each individual cell in the framework of physiological mechanism. It is thus possible to construct and demonstrated the actual neuronal networks simulated by the computed neural networks.

Discrimination of thin film elements by backscattered electron (BSE) imaging of field emission scanning electron microscope was examined. Incident electron acceleration voltage dependence on thin films' BSE intensities in five elements (Au, Ag, Ge, Cu and Fe) on a silicon substrate was experimentally measured from 3 to 30 kV. Normalization of BSE intensities using the difference between maximum and minimum brightness was proposed and allowed reproducible comparison among the elements. Measured intensities, which have correlation with electron backscattering coefficient against atomic number, indicated the existence of adequate acceleration voltage for improvement of resolution to discriminate different elements, showing the possibility of discriminating at least these six elements simultaneously by BSE imaging with nanometer-scale spatial resolution.

Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR) and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'.© 2010 Matsumura et al
licensee BioMed Central Ltd.

Various size-controlled metal nanoparticles (NPs) coated with probe DNAs have been developed Gold, silver, germanium, copper, or nickel was thermally deposited as the inner layer on the surface of a polystyrene bead, and gold was coated as the outer layer for immobilizing thiolated probe DNAs by Au-S covalent bonding The ultraviolet visible (UV-vis) spectra of NPs showed that an outer gold layer thickness of 2 nm was sufficient for the immobilization of probe DNAs having a signal/noise (S/N) ratio of specific attachment of NP probes on the DNA chips eight-times higher than that of fluorescent probes The size distributions of NPs were within the 6 7% coefficient of variation regardless of the type of metal and size The different metal layers of NPs were also discriminated successfully by measuring backscattered electron intensity by scanning electron microscopy (SEM) The results indicate that NPs can be used for a two-dimensional probe set for SEM observation of size differences and differences in the type of metal used (C) 2010 The Japan Society of Applied Physics

A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by treatment with Benzonase. Binding of the fluorescent-aptamers to the cells was evaluated by measuring fluorescence intensity and was further confirmed using flow cytometry. Removal of the aptamers can be achieved in ~10 min by the DNase nuclease digestion. Incubation of cells with aptamers or with the nucleases results in no apparent damage to the cells and does not affect their growth rates. The latter were equivalent to the rates measured for the untreated cells. Our method provides an alternative to traditional antibody-based techniques and could be especially suitable for non-invasive reversible cell labelling and cell separations where maintaining native cell activity is needed. © 2010 Terazono et al
licensee BioMed Central Ltd.

We have developed methods and systems for analyzing epigenetic information in cells, as well as that of genetic information, to expand our understanding of how living systems are determined. Because cells are minimum units reflecting epigenetic information, which is considered to map the history of a parallel-processing recurrent network of biochemical reactions, their behaviors cannot be explained by considering only conventional DNA informationprocessing events. The role of epigenetic information in cells, which complements their genetic information, was inferred by comparing predictions from genetic information with cell behavior observed under conditions chosen to reveal adaptation processes and community effects. A system for analyzing epigenetic information was developed starting from the twin complementary viewpoints of cell regulation as an 'algebraic' system (emphasis on temporal aspects) and as a 'geometric' system (emphasis on spatial aspects). The knowledge acquired from this study may lead to the use of cells that fully control practical applications like cell-based drug screening and the regeneration of organs. © Springer-Verlag Berlin Heidelberg 2010.

The detection sensitivity obtained when combining electron-microscopy counting with gold-nanoparticle labeling of target nucleic acids complementary to probe DNA microarrays with the detection sensitivity obtained when using optical-microscopy measurement of conventional fluorescent labeling. Target DNAs were reacted with probe DNAs on DNA microarrays on which micro-columns had been etched so the target DNA could be agitated. The targets were reacted with the other probe DNA immobilized onto the Surface of the nanoparticles. The labeled target DNAs were measured by using field-emission scanning electron microscopy (FE-SEM) imaging and counting the nanoparticles. The detection sensitivity of nanoparticle-labeling measurement was 1000 times that of fluorescent-labeling measurement, and the S/N ratio of nanoparticle-labeling measurement was 100 times that Of fluorescent-labeling measurement. The results indicate that the nanoparticle-labeling method using FE-SEM counting is sensitive enough that it has a possibility for measuring targets expressed in a cell without amplification such as polymerase chain reaction (PCR). (C) 2009 Elsevier B.V. All rights reserved.

Polymerase chain reaction (PCR) is a common method used to create copies of a specific target region of a DNA sequence and to produce large quantities of DNA. A few DNA molecules, which act as templates, are rapidly amplified by PCR into many billions of copies. PCR is a key technology in genome-based biological analysis, revolutionizing many life science fields such as medical diagnostics, food safety monitoring, and countermeasures against bioterrorism. Thus, many applications have been developed with the thermal cycling. For these PCR applications, one of the most important key factors is reduction in the data acquisition time. To reduce the acquisition time, it is necessary to decrease the temperature transition time between the high and low ends as much as possible. We have developed a novel rapid real-time PCR system based on rapid exchange of media maintained at different temperatures. This system consists of two thermal reservoirs and a reaction chamber for PCR observation. The temperature transition was achieved within 0.3 sec, and good thermal stability was achieved during thermal cycling with rapid exchange of circulating media. This system allows rigorous optimization of the temperatures required for each stage of the PCR processes. Resulting amplicons were confirmed by electrophoresis. Using the system, rapid DNA amplification was accomplished within 3.5 min, including initial heating and complete 50 PCR cycles. It clearly shows that the device could allow us faster temperature switching than the conventional conduction-based heating systems based on Peltier heating/cooling.

A method of producing precisely size-controlled metal nanoparticles is described. Polystyrene (PS) spheres placed on a substrate were used as a cast for the metal nanoparticles. The diameters of the PS spheres were processed into the desired sizes by oxygen plasma etching, and metal was deposited on the PS to the desired thickness by thermal evaporation. The PS casts were then removed by the UV-excited ozone oxidization reaction. The diameters of the obtained cap-shaped metal shells had a distribution within 5% of the coefficient of variation. These particles can be used as simultaneously applicable biological labels along with different-sized nanoparticles in immuno-electron microscopy. (C) 2010 The Japan Society of Applied Physics DOI: 10.1143/JJAP.49.048004

Weaponized biological agents are as great a threat as nuclear or chemical weapons. They must be detected at the earliest stage to prevent diffusion because once these agents are dispersed into the air, the rapidly decreasing concentration makes detection more of a challenge. Polymerase chain reaction (PCR) is a common method to create copies of a specific target region of a DNA sequence and to produce large quantities of DNA molecules. A few DNA molecules are rapidly amplified by PCR into billions of copies. While PCR is a powerful technique and is capable of countering new threats relatively easily, it is plagued by the number of processes necessary. Therefore, we have developed an integrated PCR system for rapid detection of biological agents captured from the air. Each processing function is performed by a dedicated module, and reduction in the process time has been made the top priority, without loss in the signal/noise ratio of the total system. Agents can be identified within 15 min from capture. A fully automated operation protects operators from exposure to potentially highly lethal samples.

We have developed an on-chip cell sorting system and microgel electrode for applying electrostatic force in microfluidic pathways in the chip. The advantages of agarose electrodes are 1) current-driven electrostatic force generation, 2) stability against pH change and chemicals, and 3) no bubble formation caused by electrolysis. We examined the carrier ion type and concentration dependence of microgel electrode impedance, and found that CoCl2 has less than 1/10 of the impedance from NaCl, and the reduction of the impedance of NaCl gel electrode was plateaued at 0.5 M. The structure control of the microgel electrode exploiting the surface tension of sol-state agarose was also introduced. The addition of 1% (w/v) trehalose into the microgel electrode allowed the frozen storage of the microgel electrode chip. The experimental results demonstrate the potential of our system and microgel electrode for practical applications in microfluidic chips. (C) 2010 The Japan Society of Applied Physics

Polymerase chain reaction (PCR) is a powerful technique to detect microorganisms, viruses, or cells by amplifying a single copy or a few copies of a fragment of a particular DNA sequence. To reduce acquisition time, it is necessary to decrease the temperature transition time between denaturation and extension. We have developed a simple rapid real-time microlitter-sample droplet PCR system accomplished by the rapid liquidbased heat-exchange of sample droplets by quick switching of two circulating hot waters of denaturation and extension, a microlitter-sized droplet and a thin-film aluminum chip. Using this system, rapid PCR amplification of a set of droplets lined up on an aluminum chip was conducted successfully as shown by the increase in fluorescence intensity, and was accomplished within 3.5 min in 40 cycles of 1 s denaturation and 3 s extension reaction, which is one magnitude faster than conventional fast PCR systems. This method allows the rapid detection of DNA fragments and has a possibility for measuring multiple samples simultaneously in a miniaturized microfluidic chip. (C) 2010 The Japan Society of Applied Physics

For the precise detection of the number of expressed biomarkers at the single-cell level, we have developed a method of quantifying and specifying target DNA fragments by using a set of gold nanoparticles as labels and field-emission scanning electron microscopy (FE-SEM) to measure the number and sizes of gold nanoparticles attached to target samples. One or more target DNAs on a substrate were labeled with a set of different-sized gold nanoparticle probes having complementary sequences to different target candidates. The type and number of the target DNAs having a specific sequence were identified by counting the attached nanoparticles of a specific size in FE-SEM images. The results evaluated using a DNA microarray showed high specificity and sensitivity, and a linear correlation between the number of attached particles and the target DNA concentration, indicating the feasibility of quantitative detection in the femtomolar to nanomolar concentration range. (C) 2010 The Japan Society of Applied Physics

We show here that the viscous drag and dielectrophoretic force generated in a V-shaped ladder electrode array in a microfluidic channel cause both attracted and repelled microparticles to move to the electrodes at the centre of the channel. Both Bacillus spores and 1-mu m polystyrene spheres in a flow concentrated at the edges of V-shaped electrodes to which a 20 V(pp) 1MHz AC voltage was applied. The results indicated the advantages of this simple setup for concentrating microparticles regardless of their dielectric constants, which is essential for highly precise cell separation and analysis. (C) 2010 The Japan Society of Applied Physics

Limitation of conventional hERG assay and QT prolongation testing for accurate prediction of Torsades de Pointes (TdP) by compounds unvailed us the necesity of new approach to evaluate global cardiac safety. On-chip re-entry cell network model assay has the potential to measure the TdP probability as pre-clinical testing for cardiac safety by the single cell-based optical/electrical measurement of the heart pressure, Na, K, Ca ion channel conditions and their fluctuations. The application of ion channel inhibitors resulted in dose-dependent changes to the field potential waveform, and these changes were identical to those induced in the native cardiomyocytes. This study shows that on-chip model represent a promising in vitro assay for cardiac toxicity and drug screening.

The simultaneous detection of multiple target biomolecules with scanning electron microscopy is difficult due to the lack of a suitable label set. Unlike the fluorescence dyes used for fluorescence microscopy, there are no readily available labels that can be distinguished. We propose using nanoscopic metal particles of different element species as labels because various metals scatter electrons differently, making them distinguishable in backscattered electron images. We have developed a universal method for forming such metal particles. They are prepared by thermal deposition of a metal onto surface-adsorbed latex spheres having the shape of a half-shell, creating exposed metal and latex surfaces. Either surface can be selectively modified with probe DNA to enable the half-shell label to react with target DNA. Half-shells prepared with different metals were clearly distinguished in the backscattered electron observation mode of a field emission scanning electron microscope. Target DNA was detected with these half-shells, indicating that this half-shell labeling can be used for simultaneous detection of specific target biomolecules. (c) 2009 Elsevier B.V. All rights reserved.

The lethal ventricular arrhythmia Torsade de pointes (TdP) is the most common reason for the withdrawal or restricted use of many cardiovascular and non-cardiovascular drugs. The lack of an in vitro model to detect pro-arrhythmic effects on human heart cells hinders the development of new drugs. We hypothesized that recently established human induced pluripotent stem (hiPS) cells could be used in an in vitro drug screening model. In this study, hiPS cells were driven to differentiate into functional cardiomyocytes, which expressed cardiac markers including Nkx2.5, GATA4, and atrial natriuretic peptide. The hiPS-derived cardiomyocytes (hiPS-CMs) were analyzed using a multi electrode assay. The application of ion channel inhibitors resulted in dose-dependent changes to the field potential waveform, and these changes were identical to those induced in the native cardiomyocytes. This study shows that hiPS-CMs represent a promising in vitro model for cardiac electrophysiologic studies and drug screening. (c) 2009 Elsevier Inc. All rights reserved.

The force generation and motion of muscle are produced by the collective work of thousands of sarcomeres, the basic structural units of striated muscle. Based on their series connection to form a myofibril, it is expected that sarcomeres are mechanically and/or structurally coupled to each other. However, the behavior of individual sarcomeres and the coupling dynamics between sarcomeres remain elusive, because muscle mechanics has so far been investigated mainly by analyzing the averaged behavior of thousands of sarcomeres in muscle fibers. In this study, we directly measured the length-responses of individual sarcomeres to quick stretch at partial activation, using micromanipulation of skeletal myofibrils under a phase-contrast microscope. The experiments were performed at ADP-activation (1 mM MgATP and 2 mM MgADP in the absence of Ca(2+)) and also at Ca(2+)-activation (1 mM MgATP at pCa 6.3) conditions. We show that under these activation conditions, sarcomeres exhibit 2 distinct types of responses, either "resisting" or "yielding," which are clearly distinguished by the lengthening distance of single sarcomeres in response to stretch. These 2 types of sarcomeres tended to coexist within the myofibril, and the sarcomere "yielding" occurred in clusters composed of several adjacent sarcomeres. The labeling of Z-line with anti-alpha-actinin antibody significantly suppressed the clustered sarcomere "yielding." These results strongly suggest that the contractile system of muscle possesses the mechanism of structure-based inter-sarcomere coordination.

We propose a method to attach probe DNAs with a homogeneous population for quantitative measurement of a minimum number of target DNA/RNA for the next generation of DNA chip. To control the homogeneous population of DNA probes, polymer films having neighboring reactive groups at the same distance, i.e., aromatic polyurea film formed with 3,5-diaminobenzonic acid (DBA) and methylenedi(p-phenylene) diisocyanate (MDI) and aliphatic polyurea film formed with DBA and hexamethylene diisocyanate (HDI), were polymerized directly on the surface of a chip substrate by vapor deposition polymerization. The spatial arrangement and homogeneity of the reactive groups on the surfaces of the substrates were examined by observing the immobilization of DNA fragments bound to 20-nm gold nano-particles on the polymer films. The results showed that physical adsorption of the DNA was effectively inhibited on the DBA/HDI film in contrast to the DBA/MDI film and the conventional aminosilane surface. Moreover, the distribution of the particles was homogeneous on the DBA/HDI film, unlike on the other surfaces. These results indicate that the aliphatic polyurea film could be used to control the selectivity and uniformity of reactive groups on the surface of substrates for more precise quantitative measurement of DNA chip technology. © 2009 The Surface Science Society of Japan.

We report the curvature size dependence of the density of attached single-stranded DNA (ssDNA) on the surface of gold nanoparticles. The densities of immobilized ssDNA on 10, 20,30, and 50 nm gold nanoparticles were examined, and we found that the maximum density of the immobilized ssDNA on 10 nm particles was 13 times larger than that on 50 nm particles, which was still 10 Limes larger than that on flat gold surfaces. This result indicates the importance of curvature in the nanometer-scale attachment of ssDNAs to nanoparticles.

We propose a method to attach probe DNAs with a homogeneous population for quantitative measurement of a minimum number of target DNA/RNA for the next generation of DNA chip. To control the homogeneous population of DNA probes, polymer films having neighboring reactive groups at the same distance, i.e., aromatic polyurea film formed with 3,5-diaminobenzonic acid (DBA) and methylenedi(p-phenylene) diisocyanate (MDI) and aliphatic polyurea film formed with DBA and hexamethylene diisocyanate (HDI), were polymerized directly on the surface of a chip substrate by vapor deposition polymerization. The spatial arrangement and homogeneity of the reactive groups on the surfaces of the substrates were examined by observing the immobilization of DNA fragments bound to 20-nm gold nano-particles on the polymer films. The results showed that physical adsorption of the DNA was effectively inhibited on the DBA/HDI film in contrast to the DBA/MDI film and the conventional aminosilane surface. Moreover, the distribution of the particles was homogeneous on the DBA/HDI film, unlike on the other surfaces. These results indicate that the aliphatic polyurea film could be used to control the selectivity and uniformity of reactive groups on the surface of substrates for more precise quantitative measurement of DNA chip technology. [DOI: 10.1380/ejssnt.2009.728]

The polymerase chain reaction (PCR) is a key technology used in genome-based biological analysis; however, requests have been made to shorten the operation time for emergency tests such as medical diagnostics, and countermeasures against bioterrorism. We have developed a novel rapid real-time PCR system using the direct absorption of an IR laser beam by water droplets as the heating device. The advantage of this system is that only the target water droplet was heated photothermally without transmitting any heat to the surroundings, which is important for the production of fast thermal cycle intervals. The system consists of a fluorescent microscope, an oil chamber with a set of water droplets lined up at the bottom, a 1480 nm IR laser unit, which is absorbed by water and can be focused on the droplets on the stage of the microscope, and an image intensifier to quantify the PCR reaction within a water droplet by measuring the change of fluorescent intensity. Using the system, we examined the PCR procedure under the following conditions: initial heating to 95 degrees C, maintaining this temperature for 10 s, and the suggested here and in similar places throughout 50 cycles of I s at 95 degrees C for denaturation and 3 s at 60 degrees C for annealing/extension. The temperature increase and decrease between the two temperatures 95 and 60 degrees C, were within 1 and 0.8 s respectively, i.e., 32 K/s, which is 1.5 times faster than the conventional heat conduction-based system. Rapid PCR amplification was observed successfully by the rise change in the sigmoidal curvature of fluorescent intensity, and the procedure was accomplished within 3.5 min, including the initial heating and complete 50 PCR cycles. The results indicate that the direct absorption-based heating of water droplets photothermally could give us a faster temperature chnage than the conventional heat-conduction-based systems such as Peltier heating/cooling.

An in vitro blood-brain barrier (BBB) model is useful for drug discovery and efficacy measurements because it is a simple and convenient model of the in vivo BBB. However, the conventional in vitro BBB model does not account for shear stress to endotherial cell (EC) layers although in vivo ECs are exposed by shear stress. To improve this deficiency, we applied a microfluidics technique to a conventional in vitro BBB model and constructed a new in vitro BBB model. First, we confirmed that ECs can survive and proliferate on a cross-linked collagen gel and on an agarose including microbeads decorated with collagen type IV (CIV). In addition, we found that the cross-linker 1-ethyl-3carbodiimide hydrochloride (EDC) with N-hydroxysuccinimide (NHS) is less effective for EC proliferation than glutaraldehyde (GA), ethyleneglycol diglycidyl ether (EGDE), and agarose with microbeads. Applying a focused infrared laser, we fabricated microtunnels within the collagen gel, and we successfully cultured ECs on the inner tunnel wall. The results indicate the potential of gel microstructures for a microfluidic in vitro BBB model.

Time-lapse video microscopic observation is useful for analysis of cell biology, especially in rapid response of immune cells. Dendritic cells (DCs) have multiple functions in the immune system, and DCs in peripheral blood play an especially important role at the front line of infection. We have developed a time-lapse video microscopic method for the evaluation of single myeloid DCs (MDC-1) from human peripheral blood. MDC-1 displayed generalized plasma membrane ruffling and phagocytosis of Pseudomonas aeruginosa. The morphological changes of MDC-1 increased following stimulation with P. aeruginosa but not after stimulation with supernatant from a P. aeruginosa culture. The activation of these morphological changes in MDC-1 could be quantitatively analyzed using the time-lapse video microscopy. This novel system may be useful for the evaluation of rapid response with human immune cells against bacterial infection.

We have developed a multielectrode array recording system for single-neurite-firing measurement using an artificially constructed neuronal network on a chip, which has a Wpm diameter array with electrodes spaced at 50 mu m, for noninvasive 64-channel 100 kHz multirecording and the stimulation of a plurality of neurites extending from a single neuron. To improve the signal/noise ratio, the ground plane was set on the multi-electrode-array plane and platinum black was set on each of the Wpm electrodes. Using this system, we performed a multisite recording of neurites of a single neuron of a rat hippocampal network in cases of both spontaneous firing and evoked responses to electrical stimulations, and estimated the velocity of action potential propagation among neurites of a single neuron from six recording sites. This demonstrated the potential use of our low-noise chip and our high-speed measurement system for the analysis of neuronal network activities at the single-neuron level.

One of the advantages of atomic force microscopy (AFM) is that it can accurately measure the heights of targets on flat substrates. It is difficult, however, to determine the shape of nanoparticles on rough surfaces. We therefore propose a curvature-reconstruction method that estimates the sizes of particles by fitting sphere curvatures acquired from raw AFM data. We evaluated this fitting estimation using 15-, 30-, and 50-nm gold nanoparticles on mica and confirmed that particle sizes could be estimated within 5% from 20% of their curvature measured using a carbon nanotube (CNT) tip. We also estimated the sizes of nanoparticles on the rough surface of dried cells and found we also can estimate the size of those particles within 5%, which is difficult when we only used the height information. The results indicate the size of nanoparticles even on rough surfaces can be measured by using our method and a CNT tip. (c) 2007 Elsevier B.V. All rights reserved.

To understand the topologically dependence of neural network function and its community effects, a constructive approach to forming a model culture system in which we can fully control the spatiotemporal pattern modification during cultivation is useful. We thus newly developed an on-chip multi-electrode array (MEA) system combined with an agarose microchamber (AMC) array to record the firing at multiple cells simultaneously over a long term and to topographically control the cell positions and their connections in order to form two linearly aligned micropatterned networks using additional photothermal etching during cultivation. The electrical connection through the additional neurite connection between two networks in both synchronized spontaneous firings and evoked responses to electrical stimulation was measured, and the localized synaptogenesis at the additional connection point and the propagation by chemical synapses were confirmed. The results show the advantages of AMC/MEA cultivation and measurement methods and indicate they will be useful for investigating community effects by pattern modification during cultivation.

We examined the origin of individuality of two daughter cells born from an isolated single Escherichia coli mother cell during its cell division process by monitoring the change in its swimming behavior and tumbling frequency using an on-chip single-cell cultivation system. By keeping the isolated condition of an observed single cell, we compared its growth and swimming property within a generation and over up to seven generations. It revealed that running speed decreased as cell length smoothly increased within each generation, whereas tumbling frequency fluctuated among generations. Also found was an extraordinary tumbling mode characterized by the prolonged duration of pausing in predivisional cells after cell constriction. The observed prolonged pausing may imply the coexistence of two distinct control systems in a predivisional cell, indicating that individuality of daughter cells emerges after a mother cell initiates constriction and before it gets physically separated into two new cell bodies.

To elucidate the role of the community effect in cardiomyocytes, we developed an on-chip single-cell-based culture system with which the dynamics of the change of beat rate and beat rate fluctuation of cultured cardiomyocytes can be measured at the single-cell level before and after the formation of a cell network. We examined the dependence of the community effect on cell network pattern by culturing cardiomyocytes in differently shaped microchambers and investigated the relation between the network pattern and the stability of the beat rate. We found that beat rate fluctuation tends to decrease as cell-community size increases, irrespective of cell network pattern. This indicates that on-chip single-cell-based cardiomyocyte communities might be able to model a heart tissue accurately enough to be used in practical applications such as drug screening. (c) 2007 Elsevier Inc. All rights reserved.

Knowing how individual cells respond to environmental changes helps one understand phenotypic diversity in a bacterial cell population, so we simultaneously monitored the growth and motility of isolated motile Escherichia coli cells over several generations by using a method called on-chip single-cell cultivation. Starved cells quickly stopped growing but remained motile for several hours before gradually becoming immotile. When nutrients were restored the cells soon resumed their growth and proliferation but remained immotile for up to six generations. A flagella visualization assay Suggested that deflagellation underlies the observed loss of motility. This set of results demonstrates that single-cell transgenerational study under well-characterized environmental conditions can provide information that will help us understand distinct functions within individual cells. (c) 2007 Elsevier Inc. All rights reserved.

One of the best approaches to understanding the mechanism of information acquisition and storage is to characterize the plasticity of network activity by monitoring and stimulating individual neurons in a topologically defined network and doing this for extended periods of time. We therefore previously developed an on-chip multi-electrode array (MEA) system combined with an array of agarose microchambers (AM,Cs). It is possible to record the firing at multiple cells simultaneously for long term and topographically control the cells position and their connections. In our present study, we demonstrated the effect of tetanic stimulation in a linearly lined-up patterned network on the AMC/MEA chip. We detected reproducible activity changes that were induced by tetanic stimulation and saw that these changes were maintained for 6-24 h. The results show the advantage of our ANIC/MEA cultivation and measurements methods and suggest they will be useful for investigating the long-term plasticity depending on network topology and size. @ 2007 Elsevier Inc. All rights reserved.

<p>We have developed a series of single-cell-based analysis called 'on-chip cellomics', consisting of singlecell-based sorting for purifying target cells, single-cell-based cultivation on chip for fully geometric/temporal control of target cells, and single-cell-based genome/proteome analysis for identifying target biomarkers of epigenetic phenomenon. For 'on-chip cellomics' study, non-destructive cell sorting is the most important for the following re-cultivation of target cells on the cultivation chips keeping their intact condition and information. We thus have proposed and examined four approaches of non-destructive cell purification; aptamermagnet microbeads method exploiting digestable DNA/RNA-based aptamer probes, apoptosis method using spot irradiation of focused laser beam to promote apoptosis on cells except for target cells, removable fluorescent probe detection method with disposable microchip, and ultra high-speed camera image detection method with disposable microchip. The results indicated those four methods are non-destructive and can be used for the sufficient selective cell purification for the following on-chip cell cultivation. In this paper, we introduce briefly about our results and potential of those cell purification methods and compare the advantages and defects of those methods.</p>

We have developed a curvature reconstruction method (CRM) for the size estimation of nanoparticles attached and sunk into the rough surface of cells by fitting the curvature of such particles' outer shapes measured using sectional height images obtained by atomic force microscopy (AFM). A dried cell surface, on which a mixture of 30 and 50 nm gold particles was placed, was traced by AFM using a carbon nanotube (CNT) tip to estimate particle size distribution using CRM, and particle size distribution was also measured by field-emission scanning electron microscopy (FE-SEM) for confirmation. The results showed the CRM-estimated diameters of the nanoparticles were within an error,of 7.57 +/- 5.41 nm independent of the difference in the sizes of particles, indicating that (1) a set of nanometer-sized marker probes with 20 nm differences in diameter can be distinguished even on the rough surface of cells, and that (2) errors might be caused by the offset of traces of particle curvature indicated by the shape of the CNT tip used.

Formation of simple neuronal networks in vitro is one of the promising methods to study biological information processing. Agarose microchambers have several advantages to form and maintain simple network structures. Here, in this work, a novel method for fabricating microwells in an agarose-layer is reported. Chaotropic effects of sodium iodide (NaI) is applied for etching agarose films. A conventional glass micropipette filled with NaI solution was aligned and a small amount of NaI was ejected to surface of the film. The agarose was denatured by the soaked NaI. The denatured agarose was washed out by distilled water. The size of the well was determined by the quantity of ejected NaI solution and its diffusion time. Conditions for fabricating wells of 100 to 600 μm diameters were established. Multiple wells up to 100 were formed on a single surface sequentially by programmed movement of the microscope stage. Rat hippocampal neurons were successfully cultured in the wells. Combining this method with microelectrode-array substrates will enable us of recording neuronal activity from simple neuronal networks as well as co-culture systems of heterogeneous tissues.

We fabricated micro flow channel based on the collagen tunnel for in vitro BBB blood capirary model using collagen gel tunnel, PDMS and infrared laser. This flow channel system can add the influence of shear stress to EC and evaluate the permeability by continuous microfluidic flow during cultivation. This system is expected to become a novel tool for emulating in vivo permeability of drug candidates available only in small quantities.

We investigate the effect of haloperidol on a four-cell and nine-cell cardiomyocyte network on an agarose microchamber array chip to evaluate a cell-based model for drug screening. Using a network of cardiomyocytes whose beating intervals were stable and relatively uniform ( they only fluctuated 10% from the mean beating interval), we easily observed the effect of haloperidol on the cell network beating interval 5 min after administering it. We also observed the beating interval returned to its original state 10 min after the haloperidol was washed out of the chip. Although the four-cell network showed the unstable recovery of its beating rhythm after washout of haloperidol, the nine-cell network recovered completely to the stable original beating rhythm even after a second administration of haloperidol. The results indicate the importance of the community size in cell networks used in the stable cell-based screening model. Moreover, they indicate the advantage of using direct cell-based measurements in which the amount of drug administered and the time course over which it is administered are strictly controlled for evaluating the quantitative chemical effects of drugs on cells.

A cellular phenotype is considered to be determined not only by genetic information but also by convoluted information on past states of a cell and its ancestors, i.e. hysteresis. This 'hysteretic effect' forms the basis of epigenetic phenomena. To understand these phenomena by which cells transmit certain phenotypes to descendants, it is necessary to observe individual cells and compare the phenotypes of each between generations under stringently controlled environmental conditions. We, therefore, did an individual-cell-based differential assay using Escherichia coli as a model organism. We observed normal-sized isolated cells change into elongated phenotypes, and subsequently measured the transmission of their characteristics between generations. This change occurred when the final length of the normal cells exceeded their cell-length boundary, i.e., 10 mu m with 5% probability. Once a cell became elongated, it divided unequally, producing two daughter cells; one was elongated and the other was normal. The elongated daughter transmitted the elongated phenotype to one lineage of the descendants by repeating unequal cell divisions with an average interdivision time half that of the normal phenotype, whereas the normal daughter retained normal phenotypic characteristics. The results suggest one possible non-genetic inheritance of cellular characteristics where phenotypic differences can only be inherited by geometrical information, independent of specific gene regulation.

To investigate the roles that the community effect and entrainment function of cultured cardiomyocyte play in decreasing beating fluctuation and reestablishing synchronized beating, we developed a single-cell-based two-dimensional network culture assay to measure and compare the dynamics of beating rhythm synchronization of individual cells before and after they form networks. Studying the formation of two-cell networks, we found that their synchronized beating tended to be determined by the cardiomyocyte whose beat rate fluctuated less than that of the other cardiomyocyte. We further found that the strength of this tendency increased with the number of cells in the network. These results indicate that (1) beating fluctuation is one of the important factors influencing the reestablishment of a stable synchronous beating rhythm, (2) the larger networks reduce fluctuation, and (3) the formation of a spatial network can itself stabilize cardiomyocyte beat rates. (c) 2006 Elsevier Inc. All rights reserved.

Ion current rectification with quartz nanopipette electrodes was investigated through the control of the surface charge. The presence and absence of a positively charged poly-L-lysine (PLL) coating resulted in the rectified current with opposite polarity. The results agreed with the theories developed for current-rectifying conical nanopores, suggesting the similar underlying mechanism among asymmetric nanostructure in general. This surface condition dependence can be used as the fundamental principle of multi-purpose real-time in vivo biosensors.

We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive. (c) 2006 Elsevier Inc. All rights reserved.

Cell-to-cell communication is considered to underlie the coordinated behavior and the multicellularity of cell group class, which cannot be explained only by the knowledge of lower class of life system from molecule to individual cell, because they are determined by at least two different ways: diffusible chemical signals and their direct physical contacts. We show in this paper a new method of individual-cell-based cell observation that can estimate the role of cell-to-cell communication, diffusible chemical signals, and physical contacts as separated properties, by applying an on-chip individual-cell-based cultivation system. The exchange of stationary phase medium on isolated individual Escherichia coli from exponential phase medium and the control of physical contacts indicated that the cell-to-cell direct contact did not affect the growth rate; only the communication through diffusible signals affects the growth rates as Hill's equation manner. (c) 2006 Elsevier Inc. All rights reserved.

Prions are propagating proteins that are ordered protein aggregates, in which the phenotypic trait is retained in the altered protein conformers. To understand the dynamics of the prion aggregates in living cells, we directly monitored the fate of the aggregates using an on-chip single-cell cultivation system as well as fluorescence correlation spectroscopy (FCS). Single-cell imaging revealed that the visible foci of yeast prion Sup35 fused with GFP are dispersed throughout the cytoplasm during cell growth, but retain the prion phenotype. FCS showed that [PSI+] cells, irrespective of the presence of foci, contain diffuse oligomers, which are transmitted to their daughter cells. Single-cell observations of the oligomer-based transmission provide a link between previous in vivo and in vitro analyses of the prion and shed light on the relationship between the protein conformation and the phenotype.

To understand the control mechanism of innate immune response in macrophages, a series of phagocytic responses to plural stimulation of antigens on identical cells was observed. Two zymosan particles, which were used as antigens, were put on different surfaces of a macrophage using optical tweezers in an on-chip single-cell cultivation system, which maintains isolated conditions of each macrophage during their cultivation. When the two zymosan particles were attached to the macrophage simultaneously, the macrophage responded and phagocytosed both of the antigens simultaneously. In contrast, when the second antigen was attached to the surface after the first phagocytosis had started, the macrophage did not respond to the second stimulation during the first phagocytosis
the second phagocytosis started only after the first process had finished. These results indicate that (i) phagocytosis in a macrophage is not an independent process when there are plural stimulations
(ii) the response of the macrophage to the second stimulation is related to the time" delay from the first stimulation. Stimulations that occur at short time intervals resulted in simultaneous phagocytosis, while a second stimulation that is delayed long enough might be neglected until the completion of the first phagocytic process. © 2006 Matsumura et al
licensee BioMed Central Ltd.

We have developed a new three-dimensional (3D) microfabrication method for agarose gel, photothermal microneedle etching (PTMNE), by means of an improved photothermal spot heating using a focused 1064 nm laser beam for melting a portion of the agarose layer at the tip of the microneedle, where a photoabsorbent chromium layer is coated to be heated. The advantage of this method is that it allows the 3D control of the melting topography within the-thick agarose layer with a 2 Pin resolution, whereas conventional photothermal etching can enable only two-dimensional (2D) control on the surface of the chip. By this method, we can form the spheroid clusters of particular cells from isolated single cells without any physical contact with other cells in other chambers, which is important for measuring the community effect of the cell group from isolated single cells. When we set single cancer cells in microchambers of 100 mu m in diameter, formed in a 50-mu m-thick agarose layer, we observed that they grew, divided, and formed spheroid clusters of cells in each microchamber. The result indicates the potential of this method to be a fundamental technique in the research of multicellular spherical clusters of cells for checking the community effect of cells in 3D structures, such as the permeabilities of chemicals and substrates into the cluster, which is complementary to conventional 2D dish cultivation and can contribute to the cell-based screening of drugs.

Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy. (c) 2006 Elsevier B.V. All rights reserved.

Adding an artificial bolaamphiphile to a dispersion of giant multilamellar vesicles (GMVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) induced a cup-shaped deformation in GMVs accompanied by partial extrusion of the inner vesicle; thereafter, the deformed vesicles returned to their original shape. On the other hand, when the artificial bolaamphiphile together with a surfactant was added to the vesicular dispersion, these deformation and reformation dynamics were transmitted from the outer membranes in GMVs to the inner membranes until an intact inner vesicle was extruded out of the outer membrane. The microscopic aspects of these processes were investigated using amphiphiles tagged with individual fluorophores.

We began a series of studies aimed at developing methods and systems of analyzing epigenetic information in cells, as well as that of genetic information, to expand our understanding of how living systems are determined. The role of epigenetic information on cells, which complements their genetic information, was inferred by comparing predictions from genetic information with cell behaviour observed under conditions chosen to reveal adaptation processes and community effects. A system of analyzing epigenetic information was developed starting from the twin complementary viewpoints of cell regulation as an 'algebraic' system (emphasis on temporal aspects) and as a 'geometric' system (emphasis on spatial aspects). The knowlege acquired from this study may lead to the use of cells that fully control practical applications like cell-based drug screening and the regeneration of organs. © 2006 Society for Chemistry and Micro-Nano Systems.

To understand the contribution of community effect on the stability of beating frequency in cardiac myocyte cell groups, the stepwise network formation of cells as the reconstructive approach using the on-chip agarose microchamber cell microcultivation system with photo-thermal etching method was applied. In the system, the shapes of agarose microstructures were changed step by step with photo-thermal etching of agarose-layer of the chip using a 1064-nm infrared focused laser beam to increase the interaction of cardiac myocyte cells during cultivation. First, individual rat cardiac myocyte in each microstructure were cultivated under isolated condition, and then connected them one by one through newly-created microchannels by photo-thermal etching to compare the contribution of community size for the magnitude of beating stability of the cell groups. Though the isolated individual cells have 50% fluctuation of beating frequency, their stability increased as the number of connected cells increased. And finally when the number reached to eight cells, they stabilized around the 10% fluctuation, which was the same magnitude of the tissue model cultivated on the dish. The result indicates the importance of the community size of cells to stabilize their performance for making cell-network model for using cells for monitoring their functions like the tissue model. © 2005 Kojima et al
licensee BioMed Central Ltd.

The properties of individual cells are thought to depend on the time course change of the number and the spatial distribution of regulatory molecules in cells. To understand the strict meaning of those roles of some particular sets of regulatory proteins, we need to measure both the phenotypical characteristics of cells and the quantitative amount of mRNAs in each of those individual cells. For that purpose, we have developed a novel method to measure the quantitative amount of mRNA expression in individual cells keeping their spatial distributions in the cell without any amplification process like PCR. In this method, a set of different sizes of gold nano-particles attached with different probe-DNA respectively were used as a set of probes to detect different mRNAs existing in a cell. At first, the optimum condition of the immobilization of probe-DNA onto the gold nano-particle surface was examined. Next, the selectivity of the probe-DNA immobilized onto the nano-particle was tested using complementary oligonucleotide molecules. We confirmed the several different kinds of gold nano-particle probes were hybridized with target oligonucleotides having complementary sequences with almost 100% selectively. For the counting and distinguishing each of the gold nano-particles, we used two different methods and compared: one is Atomic Force Microscopy and the other is Scanning Electron Microscopy. Quantitative detection of mRNAs in individual cells keeping their spatial distributions was then examined using the gold nano-particle-based detection system. In this meeting, we will present the results and will discuss about the potential and problems of this method for the single-cell-based quantitative expression analysis.

For the single-molecule level quantitative measurement of target mRNAs attached on the DNA chip, one of the most important things is how to maintain the homogeneous, desirable-spaced arrangement of DNA-probes on the surface of the chip. Thus we have developed the novel method for decorating DNA-probes on the chip with desired intervals and homogeneous concentration applying the vapor deposition polymerization (VDP) technique, in which hexamethylene diisocyanate (HDI) and 3,5-diaminobenzonic acid (DBA) molecules were polymerized directly on the chip to form the polymer thin layer. The interval of the reactive carboxyl-group (-COOH) on the side-chains of DBA was controlled to become 2.0 nm by choosing another molecule having this length (HDI). After we made 10-nm-thick HDI-DBA polymer film on the chip, we attached amine-group of the thiol-, amine-decorated DNA-probe to the carboxyl-group of DBA. Then we checked the spatial arrangement and homogeneity of DNA-probes attached on the polymer film using 20-nm gold (Au) nano-particles. By the scanning electric microscopy observation, we first confirmed the spatial distribution of Au nano-particles on the chip was 254 particles/μ m2, whereas the less than 1 particles/μ m2 for the control sample, in which no DNA-probe was attached on the film. Moreover, the standard deviation of localization of particle distribution was less than 8 particles differences / 254 particles/μ m2, which is a magnitude better than the conventional silane-coupling method. The result indicates the potential of our method to form the homogeneous DNA-probe surface on the.

We have developed a procedure for stepwise topographical control of network patterns and neurite connection directions between adjacent living neurons using an individual-cell-based on-chip multi-electrode array ( MEA) cell cultivation system with an agarose microchamber (AMC) array. This procedure enables flexible and precise control of the cell positions and easy and flexible control of the pattern modification of connections between the cells in AMCs through stepwise photo-thermal etching in which a portion of the agarose layer on the chip is melted with a 1480 nm infrared laser beam even during cultivation. With adequate laser power and this stepwise procedue, we can fabricate narrow micrometer-order grooves (microchannels) during cultivation in a stepwise manner. Using this procedure, we controlled the direction of elongation of axons and dendrites selectively and confirmed the direction by immunostaining. We also demonstrated electrophysiological one-way transmission of signals among aligned hippocampal neurons in which the directions of the neurite connections were controlled using this stepwise photo-thermal etching procedure. These results demonstrate the potential of full direction control of neurite connections between neurons using stepwise photo-thermal etching to form microchannels one by one in an on-chip AMC/MEA cell cultivation system. We can thus better understand the meaning of neuronal network patterns and connection directions.

The emergence of variation and subsequent inheritance of the emergent characteristics in a clonal population of bacteria is considered as evidence for epigenetic processes in the cell. We report here the results of experiments in which we quantitatively examined variations in single Escherichia coli cells with an identical genetic endowment in order to establish whether certain characteristics of single cells were inherited by their descendants maintained in a uniform environment. Significantly large variations of interdivision time, initial length, and final length were observed from generation to generation. Comparing the generations shows that interdivision time had no correlation with that of the consecutive generations, whereas those of initial length and final length were positively correlated with those of neighbouring generations.

A series of studies aimed at developing methods and systems for analyzing epigenetic information in cells are presented. The role of the epigenetic information of cells, which is complementary to their genetic information, was inferred by comparing the predictions of genetic information with the cell behaviour observed under conditions chosen to reveal adaptation processes and community effects. Analysis of epigenetic information was developed starting from the twin complementary viewpoints of cells regulation as an 'algebraic' system (emphasis on the temporal aspect) and as a 'geometric' system (emphasis on the spatial aspect). The knowlege acquired from this study will lead to the use of cells for fully controlled practical applications like cell-based drug screening and the regeneration of organs. © 2004 Yasuda
licensee BioMed Central Ltd.

The cytoplasmically inherited genetic determinant [PSI+} of the yeast Saccharomyces cerevisiae is presumed to be a manifestation of the prionlike properties of the chromosome-encoded Sup35 protein. Here, we show the relationship between the cell growth and the inheritance of Sup35p-GFP aggregation using on-chip single-cell observation assay. When Sup35 was expressed by induction, an aggregation was started-after soluble Sup35-GFP in the cytoplasm reached to the critical concentration. Once the aggregation was generated, the concentration of free Sup35-GFP remained constant at a critical value. After stopping the Sup35-GFP induction by changing the cultivation buffer, the concentration of soluble Sup35-GFP remained constant, whereas the size of aggregation decreased. This result indicates that the aggregation of Sup35-GFP is a reversible growth/shrinking phenomenon with the same velocity constant as that for a forward/reverse reaction.

We have developed a new method that enables agar microstructures to be used to cultivate cardiac myocyte cells in a manner that allows their connection patterns to be controlled. Non-contact three-dimensional photo-thermal etching with a 1064-nm infrared focused laser beam was used to form the shapes of agar microstructures. This wavelength was selected as it is not absorbed by water or agar. Identical rat cardiac myocytes were cultured in adjacent microstructures connected by microchannels and the interactions of asynchronous beating cardiac myocyte cells observed. Two isolated and independently beating cardiac myocytes were shown to form contacts through the narrow microchannels and by 90 minutes had synchronized their oscillations. This occurred by one of the two cells stopping their oscillation and following the pattern of the other cell. In contrast, when two sets of synchronized beating cells came into contact, those two sets synchronized without any observable interruptions to their rhythms. The results indicate that the synchronization process of cardiac myocytes may be dependent on the community size and network pattern of these cells. © 2004 Kojima et al
licensee BioMed Central Ltd.

Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. We have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-cell-based on-chip cell-cultivation system with an agarose microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal cells. We were able to control elongation of individual axons during cultivation (from cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA cell cultivation system for the controlled development of individual cell-based neural networks. © 2004 Suzuki et al
licensee BioMed Central Ltd.

Studying cell functions for cellomics studies often requires the use of purified individual cells from mixtures of various kinds of cells. We have developed a new non-destructive on-chip cell sorting system for single cell based cultivation, by exploiting the advantage of microfluidics and electrostatic force. The system consists of the following two parts: a cell sorting chip made of polydimethylsiloxane (PDMS) on a 0.2-mm-thick glass slide, and an image analysis system with a phase-contrast/fluorescence microscope. The unique features of our system include (i) identification of a target from sample cells is achieved by comparison of the 0.2-μm-resolution phase-contrast and fluorescence images of cells in the microchannel every 1/30 s
(ii) non-destructive sorting of target cells in a laminar flow by application of electrostatic repulsion force for removing unrequited cells from the one laminar flow to the other
(iii) the use of agar gel for electrodes in order to minimize the effect on cells by electrochemical reactions of electrodes, and (iv) pre-filter, which was fabricated within the channel for removal of dust contained in a sample solution from tissue extracts. The sorting chip is capable of continuous operation and we have purified more than ten thousand cells for cultivation without damaging them. Our design has proved to be very efficient and suitable for the routine use in cell purification experiments. © 2004 Takahashi et al
licensee BioMed Central Ltd.

We have developed an on-chip single-cell based microcultivation method for analyzing the variability of genetic information stored in single cells and their epigenetic correlations. The method uses four systems: an on-chip cell sorter for purifying the cells in a non-destructive manner; an on-chip single-cell cultivation chip for isolating single cells; an on-chip agarose microchamber system for constructive cell-cell network formation during cultivation; and an on-chip single-cell-based expression analysis system. Using these systems, we could measure the variability of prokaryotic cells and eukaryotic cells having the same DNA and found that, although prokaryotic cells have a large variability in their interdivision times, sister eukaryotic cells having the same DNA synchronized well. We also measured the dynamics of synchronization of beating cardiac myocytes and found that two isolated cells synchronize by one cell following the other after a short pause in beating. These results showed the potential of the on-chip microcultivation method's constructive approach to analyzing cell systems. Copyright (C) 2004 John Wiley Sons, Ltd.

We have developed a new type of non-contact three-dimensional photo-thermal etching method for agar microculture chips exploiting the characteristics of two different wavelengths of infrared laser beams. We used two different wavelengths of infrared (1480 and 1064 nm) focused laser beam as a heat source to melt and remove a portion of 200 mum high agar gel layer on the 5 nm thick chromium-coated glass slide. As the 1480 nm infrared beam is absorbed by water, the agar gel on the light pathway is heated and melted. On the other hand, as the 1064 nm infrared beam is not absorbed by water and agar, the melting of the agar occurred just near the chromium thin layer that absorbs 1064 nm infrared light. Using this non-contact etching, we can easily make microstructures in agar-layer using infrared laser beam only within a few minutes; i.e. cell-culture holes are melted by 100 mW, 1480 nm laser and tunnels by 100 mum/s, 40 mW, 1064 nm laser, respectively. The size of holes and tunnels were also controlled by choosing the irradiation power and time of infrared lasers. Those results indicate that we can make and use microstructures for biological use without any expensive microfablication facilities nor a series of complicated procedure and time. (C) 2004 Elsevier B.V. All rights reserved.

Measurement of the correlation between sensor-protein expression, motility and environmental change is important for understanding the adaptation process of cells during their change of generation. We have developed a novel assay exploiting the on-chip cultivation system, which enabled us to observe the change of the localization of expressed sensor-protein and the motility for generations. Localization of the aspartate sensitive sensor protein at two poles in Escherichia coli decreased quickly after the aspartate was added into the cultivation medium. However, it took more than three generations for recovering the localization after the removal of aspartate from the medium. Moreover, the tumbling frequency was strongly related to the localization of the sensor protein in a cell. The results indicate that the change of the spatial localization of sensor protein, which was inherited for more than three generations, may contribute to cells, motility as the inheritable information. © 2004 Inoue et al
licensee BioMed Central Ltd.

We have developed a new type of single-cell based on-chip cell-cultivation system with an agarose microchamber (AMC) array and a photo-thermal etching module for step-by-step topographical control of the network patterns of living neural cells during long-term cultivation The advantages of this system are that (1) it can control positions and numbers of cells for cultivation by using agar-based microchambers, and (2) it can change the neural network complexity during cultivation by photo-thermal melting a portion of agar at the focal point of a 1064 nm infrared laser beam. This laser wavelength is permeable with respect to water and agarose, and it is only absorbed at the thin chromium layer on the chromium-coated glass slide surface at the bottom of the agarose layer. With adequate laser power, we can easily fabricate narrow tunnel-shaped channels between. the microchambers at the bottom of the agar layer without the complicated steps conventional microfabrication processes entail even during cultivation; we demonstrated that rat hippocampal cells in two adjacent chambers formed fiber connections through new connections between chambers after these had been photo-thermally fabricated. We also verified the fiber connection between those cells by using calcium-based fluorescent microscopy. These results indicate that this system can potentially be used for studying the complexity of neural network patterns for epigenetic memorization. (C) 2003 Elsevier B.V. All rights reserved.

We have developed a new type of individual-cell-based electrophysiological measurement method using an on-chip multielectrode array (MEA) cell-cultivation system with an agarose microchamber (AMC) array for topographical control of the network patterns of a living neuronal network. The advantages of this method are that it allows the recording of the firing of multiple cells simultaneously for weeks without contamination using the MEA, and that it allows control of the cell positions and numbers, and their connections for cultivation using AMCs with microchannels fabricated by photothermal etching where a portion of the agarose layer is melted with a 1480 nm infrared laser beam. Using this method, we formed an individual-cellbased neural network pattern of Rat,hippocampal cells within the AMC array without cells escaping from the electrode positions in the microchamber during a thirteen-day cultivation, and could record the cell firing of lined-up hippocampal cells in response to 20muA, 5 kHz stimulation via an electrode. This demonstrated the potential of our on-chip AMC/MEA cell cultivation method for long-term single-cell-based electrophysiological measurement of a neural network system for understanding the topographical meaning of neuronal network patterns.

We have developed an on-chip single-cell microcultivation assay as a means of simultaneously observing the growth and movement of single bacterial cells during long-term cultivation. This assay enables the direct observation of single cells captured in microchambers fabricated on thin glass slides and having semipermeable membrane lids, in which the cells can swim within the space without escape for the long periods. Using this system, the relationship between the cell cycle and the tendency of movement was observed and it was found that the mean free path length did not change during the cell cycle, and that the growth and the swimming were not synchronized. The result indicates that the ability of movement of the cells was independent of the cell cycle.

We have developed a novel method for on-chip cultivation of neural cells in a flexible agarose-microchamber array on a glass slide. The agarose microchamber is a micrometer-order cavity constructed on the surface of an agarose layer by molding a 50-mum-high square/circular micro-cast of thick SU-8 photoresist. In addition, the shape of the agarose microchamber was rearranged by using the photothermal etching method, in which we used an infrared (1064-nm) focused laser beam as the heat source to melt and remove a portion of agarose gel at the heating spot. With the photothermal etching method, we can also manufacture narrow tunnel-shaped channels between microchambers. When nerve cells were cultured on the agar-microchamber array chip, the nerve cells in two adjacent microchambers connected through the photothermal-etched channel after 48 hours of cultivation. Those results suggest the potential of an agarose-microchamber array integrated with the photothermal etching method for the next stage of single cell cultivation and measurement of nerve cells, such as real-time control of cell interactions during cultivation. (C) 2003 Wiley Periodicals, Inc.

A new type of individual-cell-based on-chip multielectrode array (MEA) cell-cultivation system with an agarose microchamber (AMC) array for topographical control of the network patterns of a living neuronal network has been developed. The advantages of this system are that it allows control of the cell positions and numbers for cultivation using AMCs, as well as easy and flexible control of the pattern of connections between the AMCs through photothermal etching where a portion of the agarose layer is melted with a 1480nm infrared laser beam. With adequate laser power, narrow micrometer-order grooves (microchannels) can easily be fabricated that can be used to combine neighbouring AMCs to enable topographical control of the neural network pattern. Using this system, an individual-cell-based neural network pattern was formed of rat hippocampal cells within the AMC array without cells escaping from the electrode positions in the microchamber during an eight-day cultivation, and could record cell firing in response to 1.5V, 500kHz stimulation through an electrode. This demonstrated the potential of the on-chip AMC/MEA cell cultivation system for long-term single-cell-based electrophysiological measurement of a neural network system.

We have investigated non-destructive, non-contact single-cell based differential cell screening method using on-chip microcultivation and optical tweezers. The four-room microchamber for cultivating four single cells consists of 5 mum high microstructure on the glass slide and is covered with semipermeable membrane to cultivate those cells under contamination-free condition. The number of cells in microchambers is controlled by using optical tweezers to pick out excess cells. For studying the actual changes occurring in cells under isolated condition, we measured and compared four cells in four microchambers simultaneously for more than 10 generations. To examine the potential of this method, we observed single cells of Escherichia coli for more than 10 generations. The results of these observations showed that they keep their mean values even though there exist large dynamic variations in interdivision time and growth speed. The simultaneous observations of four cells also confirmed that these variations in cells are not derived from the time-course changes of environmental conditions because those variations in four cells did not synchronized. The results suggested the potential use of our method for cell assay to predict whether the cells' variation is in permissible range or is caused by drugs or other environmental effects. (C) 2003 Elsevier B.V. All rights reserved.

We have developed a new type of on-chip microcultivation chamber made of poly(dimethylsiloxane) (PDMS) for long-term cultivation of swimming cells. The advantages of this chamber are that (1) the microfluidic channel width of the valve can be controlled according to air pressure while monitoring the microscopic image of the channel width, and that (2) a simple single-step procedure is required for the fabrication of the valve structure and microfluidic pathway. Using this chamber, we can control the passage of swimming Chlamydomonas cells, through the channel while visualizing the opening and closing of the valve as the medium buffer passes any time. Thus the long-term observation of the behavior of a particular single cell is accomplished.

We have developed a method for flexible changing the shape of the electrode layer on a cultivation chip by nondestructive optical etching with a focused 1064 nm infrared laser during cultivation of cells on it. When the etched width of the scratch on the electrode layer of cultivation chip reached 10 mum, the conductance decreased to 0 S. The method was then examined for the area-specific stimulation of cardiac myocytes cultivated on the chip, and was founnd to stimulated the cells in the area successfully. The result indicates the ability of flexible-area-specific electric stimulation to change the shape of an electrode during cultivation.

To estimate the role that time and size had in controlling the Chlamydomonas cell cycle, we used a new on-chip single-cell microcultivation system, which involved the direct observation of single cells captured in microchambers made on a thin glass slide. The dependence of the pattern of energy supply for cells on its cell cycle was examined through a series of different intensities of continuous illumination in a minimal medium, and we found that cell division occurred when cells reached the critical size, which was 2.2 times larger than that of the newly created cells. When illumination stopped before cells reached the critical size, even though growth had stopped, they continued dividing during the delay time, which was shorter when cells were larger. With re-illumination after darkness, cells began to grow again and the timing of cell division was again controlled by the critical size. This indicates that the co-existence of two cell cycle regulation mechanisms and the sizer mechanism had a stronger influence than the timer. (C) 2003 Elsevier Science (USA). All rights reserved.

We have developed an on-chip microcultivation system for differential analysis of photosynthesizing cells of Chlamydomonas as a means of comparing the difference in interdivision time between sister cells and cells of adjacent generations having the same DNA and environment. The system enabled us to compare the change in the interdivision time of four sister cells simultaneously for several days, we found that the interdivision times of the four sister cells synchronize well, while, those of cells of subsequent generations do not. The result showed the potential of the system for measuring how much epigenctic information of eukaryotic cells can be conserved between sister cells. and cells of subsequent generations.

We have developed an on-chip single-cell microcultivation assay as a means of continuously observing certain single swimming cells in order to trace their movement. The single cells were captured in microchambers fabricated on thin glass slides and having semipermeable membrane lids, in which cells can swim within the space for a long term without escaping. This assay enables the direct measurement of the reflection of certain cells against the microchamber walls depending on their incidence angles. Using this assay, the reflection was examined. We found that the ratio of reflection of cells to those of non-reverse was almost the same, though most of cells reflected when their incident angle was perpendicular to the wall.

We have developed a on-chip single-cell microcultivation assay as a means of observing the adaptation process of single bacterial cells during nutrient concentration changes. This assay enables the direct observation of single cells captured in microchambers made on thin glass slides and having semipermeable membrane lids, in which cells were kept isolated with optical tweezers. After changing a medium of 0.2% (w/v) glucose concentration to make it nutrient-free 0.9% NaCl medium, the growth of all cells inserted into the medium stopped within 20 min, irrespective of their cell cycles. When a nutrient-rich medium was restored, the cells started to grow again, even after the medium had remained nutrient-free for 42 h. The results indicate that the cell's growth and division are directly related to their nutrient condition. The growth curve also indicates that the cells keep their memory of what their growth and division had been before they stopped growing. (C) 2003 Elsevier Science (USA). All rights reserved.

We have developed a new type of on-chip cell cultivation system using an agarose microchamber (AMC) array and a photo-thermal etching method, thus enabling topographical control of neuronal network pattern step-by-step during cell cultivation. By using photo-thermal etching (micro melting) method, the number of microtunnel connecting microchambers can be easily increased, even during cell cultivation, according to the progress of the neuronal network formation. To demonstrate the capability of this system for topographical control of network formation, we cultured hippocampal neurons in this AMC array. We found that the cells in microchambers made fiber connections through microtunnels. Furthermore the cells even made fiber connections through additional microtunnels fabricated during cultivation by photo-thermal etching. The results showed that the photo-thermal etching could be used during cultivation without damaging cells.

In this paper, we have developed a newly developed on-chip cell sorting system that offers impact-free sorting for single-cell based observation analysis or regenerative medicine.

Knowledge about life processes has expanded dramatically during the 20th century and produced the modern disciplines of genomics and proteomics. However, there remains the great challenge of discovering the integration and regulation of living components in time and space within the cell. As we move into the postgenomic period, the complementarity between genomics and proteomics will become apparent, and the connections between them will be exploited, although genomics, proteomics, or their simple combination will not provide the data necessary to interconnect molecular events in living cells in time and space. The cells in a group are different entities, and differences arise even among cells grown in homogeneous conditions considered to have identical genetic information. These cells respond differently to perturbations. The question is why and how these differences arise. They might be caused by unequal distributions of biomolecules in cells, mutations, interactions between cells, fluctuations of environmental elements that affect the cell, etc. To understand the principle underlying the differences, keeping in mind the possibilities mentioned above, a system is necessary that would allow for the continuous observation of specific cells under fully controlled circumstances such as interactions between cells.

We have developed a new method that enables agar microstructures to be used to cultivate cells and that allows cell network patterns to be controlled. The method makes use of non-contact three-dimensional photo-thermal etching with a 1480 nm infrared focused laser beam, which is strongly absorbed by water and agar gel, to form the shapes of agar microstructures. It allows microstructures to be easily formed in an agar layer within a few minutes, with cell-culture holes formed by the spot heating of a 100 mW laser and tunnels by the tracing of a 100 mm s(-1), 40 mW laser. We cultivated rat cardiac myocytes in adjacent microstructures and observed synchronized beating in them 90 min after they had made physical contact. Our results indicate that the system can make and use microstructures for cell-network cultivation in a minimal amount of time without any expensive microfabrication facilities or complicated procedures.

A giant vesicle composed of an amphiphile with a reactive site exhibited a morphological change when the vesicular amphiphile reacts with an added amphiphilic reaction-partner within its membrane.

A new type of cell-cultivation system based on photo-thermal etching has been developed for the on-chip cultivation of living cells using an agarose microchamber array. The method can be used to flexibly change the chamber structure by photo thermal etching, even during the cultivation of cells, depending upon the progress in cell growth. We used an infrared (1064 nm) focused laser beam as a heat source to melt and remove agar gel at the heated spot on a thin chromium layer. The melting of the agar occurred just near the chromium thin layer, and the size of the photo thermally etched area depended almost linearly on the power of the irradiated laser beam from 2 m m to 50 m m. Thus by using photo-thermal etching with adequate laser power we could easily fabricate narrow tunnel-shaped channels between the microchambers at the bottom of the agar-layer even during cell cultivation. After 48 h of cultivation of nerve cells, the nerve cells in two adjacent chambers made fiber connections through the fabricated narrow tunnel-shaped channels. These results suggest that photo-thermal etching occurred only in the area where an absorbing material was used, which means that it is possible to photo-thermally etch lines without damaging the cells in the microchambers. The results also suggest that the agar-microchamber cell cultivation system in combination with photo-thermal etching can potentially be used for the next stage of single cell cultivation including the real-time control of the interaction of cells during cell cultivation.

To investigate the non-gene tic variability of the division cycle and growth of single cells, we compared pairs of Escherichia coli daughter cells' growth and division time under isolated conditions using a newly developed on-chip culture system. The advantages of the system are: (i) continuous cultivation of isolated single cells or a group of cells in pm-sized micro-cages under isolated contamination-free and exchangeable medium conditions, and (ii) continuous observation and comparison of those floating daughter cells at a spatial resolution of 0.2 mum by phase-contrast/fluorescence microscopy and digital image processing. Using this system, cell growth and division time of daughter cells from an isolated mother cell were measured. A broad distribution of the cell division time and the division time differences of two daughter cells from the same mother cells were found. They are not attributable to a difference in DNA. The same tendency of broad distribution of the division time and the division time differences suggests the involvement of a probabilistic process to start the division.

In order to provide total systems for single cell expression analysis, we have been developing a set of technology including cell cultivation, cell sorting, gene analysis from a specific cell, etc., using microfabrication technology. In this paper, we present the development of a cell sorter chip used as the core technology for establishing the cell sorting system that can separate and collect an individual cell according to its biological or biochemical information.

Molecular biology is moving rapidly towards the stage of functional genomics in which rapid preparations of expressed messages (mRNA) will be required. If a DNA library is constructed on a solid support (DNA probe array) and any kind of expressed messages can be individually recovered from the probe array, DNA probe array will become a very useful method. Consequently, we developed a DNA preparation method using a DNA probe array that utilizes photo-thermal denaturation to recover specific DNA. The protocol for preparing DNA probe array was investigated to realize the stable immobilization of DNA probes on the surface of solid support. We used a glass plate coated with Cr (5-10 nm thick). The Cr surface was modified with 3-glysidoxypropyltrimethoxysilane to introduce active residues that can couple with amino residues at the 5' termini of DNA probes. The Cr surface acts as a photo-thermal transducer. The preparation method of DNA uses infrared (1053-nm) laser irradiation to thermally denature and release DNA immobilized in a specific area of a DNA probe array. Different DNA fragments fixed in place on the DNA probe array could be separately recovered. There were enough quantities of recovered DNA that can be amplified by using PCR. © 2001, The Institute of Electrical Engineers of Japan. All rights reserved.

A method is described for continuous observation of isolated single cells that enables genetically identical cells to be compared
it uses an on-chip microculture system and optical tweezers. Photolithography is used to construct microchambers with 5-μm-high walls made of thick photoresist (SU-8) on the surface of a glass slide. These microchambers are connected by a channel through which cells are transported, by means of optical tweezers, from a cultivation microchamber to an analysis microchamber, or from the analysis microchamber to a waste microchamber. The microchambers are covered with a semi-permeable membrane to separate them from nutrient medium circulating through a "cover chamber" above. Differential analysis of isolated direct descendants of single cells showed that this system could be used to compare genetically identical cells under contamination-free conditions. It should thus help in the clarification of heterogeneous phenomena, for example unequal cell division and cell differentiation. © Springer-Verlag 2001.

To investigate the properties of isolated single cells with their environment, we developed the differential analysis method for single cells using an on-chip microculture system. The advantages of the system are, (i) continuous cultivation of a series of isolated single cells or a group of cells under contamination free conditions, (ii) continuous observation and comparison of those cells with 0.2 mum spatial resolution by a phase-contrast/fluorescent microscopy system with digital image processing. The core of the system is an n x n (n = 20-50) array of chambers, where each is 20-70 mum in diameter and 5-30 mum deep holes etched into a biotin-coated 0.17 mm thick glass slide. The biotin-coated glass slide is covered with the streptavidin coated cellulose semipermeable membrane, which is fixed on the surface of the glass slide by streptavidin-biotin attachment, separating those holes from the nutrient medium circulating through a 'cover chamber' above. A single cell or group of cells can thus be isolated from environment perfused with the same medium, and the medium in each chamber can be changed within the diffusion time (&lt;1/30 s). In addition, the microchamber volumes of specific cells or cell groups can be controlled by the sizes of the chambers. By using this system we found that the length of isolated Escherichia coli increased at 0.06 &mu;m min(-1) between cell divisions regardless of the chamber volume, and that the cell concentration reached 10(12) cells ml(-1) under contamination free conditions. The system is thus particularly useful for one cell level analysis because the direct descendants of single cells can be cultured and compared in the isolated microchambers, and the physical properties of the cells in each microchamber can be continuously observed and compared.

A photothermal method to recover specific DNA fragments fixed in place on a DNA chip is described. This method uses infrared (IR) laser irradiation to thermally denature and release specific DNA immobilized in a specific area of a chip. A 1053-nm IR laser beam with an intensity of 10-100; mW is focused on the target area at a resolution of 10 mu m, and the DNA fragments are released from the chip surface. We have demonstrated that DNA fragments containing different numbers of base pairs (231-799 bp) fixed in place on the DNA chip can be separately recovered. There are enough quantities of recovered DNA fragments that can be amplified by using polymerase chain reaction (PCR). The photothermal method coupled with the DNA chip can therefore be used in highly sensitive purification of DNA and will have many applications in the DNA chip technology. (C) 2000 Elsevier Science S.A. All rights reserved.

We investigated the non-contact handling of micrometer-sized samples in the micro-chamber using acoustic radiation force for lining up the microparticles and for mixing solutions. The chamber for handling samples consists of a fused quartz cell attaching a pair of 3.5 MHz lead zirconate titanate (PZT) transducers both sides. For lining up the particles, pure water containing 7-mu m polystyrene spheres was introduced into the chamber. When 3.5 MHz ultrasound was irradiated into the chamber, the particles lined up at the pressure node of ultrasound less than 0.3 mu m of the spatial distribution. For mixing, samples such as erythrocytes and fluorescent dye were introduced into the chamber from the inlet which arranged at the side wall of the chamber and the laminar flow of the two different kinds of solutions keeping their boundaries is observed. When the 3.5 MHz ultrasound irradiation into the chamber started, the boundaries of the two sample's flow were broken and the erythrocytes spread and mixed into all the span of the chamber. The possible damage caused by the 3.5 MHz ultrasound during the mixing process was also measured, and no significant release of the erythrocyte's component was detected even without the degas process, which was pre-processed for preventing cavitation generation. The results suggested the potential use of acoustic radiation force for non-contact, non-destructive and time-resolved handling method for micro-chamber as the sample preparation process. (C) 2000 Elsevier Science S.A. All rights reserved.

High-throughput, selective extraction of a particular DNA fragment from a mixture of DNA before PCR amplification is becoming increasingly important in the DNA analysis field. Although the latest microchip technology has enabled real-time DNA expression analysis using hybridization between surface-bound probe DNA and sample DNA, the potential of this technology in purification of a small amount of DNA has not been demonstrated. We report here a method for area-selective release and collection of specific DNA, in which an IR laser beam is focused onto surface-bound sample DNA at the target-spotted area to denature hybridized DNA. First, sample DNA labeled with a fluorescent dye was hybridized to a probe DNA immobilized on a chromium-coated chip. A 1053-nm IR laser beam with an intensity of 10-100 mW was then focused on the target area with a spatial resolution of 10 mu m, causing the release of the fluorophore-labeled sample DNA as a result of photo-thermal denaturation. Confirmation of the amount of eluted DNA by PCR amplification after collection indicated that more than 10(-20) mol DNA/mu m(2) area was eluted from the microchip, representing more than 70% of the chip-bound sample DNA. These results indicate that this method can be applied to the highly sensitive purification of DNA ill microchip technology.

A simple method for measuring the microscopic spatial distribution of the acoustic radiation force by exploiting the competition between the acoustical radiation force and the electrostatic force on charged microspheres is investigated. First, the effective charge of polystyrene spheres is measured by monitoring the mobility of the particles in the 1 Hz, 13.4 V-pp/mm alternating electric field. Next, the shift of the particles away from the sound pressure node with an increase in intensity of the electrostatic force is observed by optical microscopy, and finally the acoustic radiation force is estimated. In the experiment, the spatial distribution of the acoustic radiation force in the 500 kHz, 80 Vpp standing wave was proportional to the sine curve, and had the same tendency as the result obtained from calculation. The intensity was 0.5 pN for 7 mu m polystyrene spheres.

The generation mechanism of acoustic streaming and radiation pressure are discussed along with the KZK equation, which explains successfully the propagation of finite-amplitude sound waves. The energy loss of a sound beam in a viscous fluid generates the driving force of streaming which induces mass flow in the beam. The radiation pressure acts on the surface of an object whose acoustic impedance is different from the fluid. The effect of flow generated around the object on the radiation pressure is greater when the object is smaller. The main purpose of this paper is to describe the unified relationships among waveform distortion, acoustic streaming, and radiation pressure. © 1998 Scripta Technica.

We observed the interference effect of electron-capture X-rays emitted by the nuclear transformations in radioisotopes. This interference is between the direct monochromatic emission from the radioactive atoms and the emission totally reflected by the substrate surface. Nanometer-level structural information about the radioactive atoms can be obtained by analyzing the measured interference fringes because the period of these fringes depends on the position of the radioactive atoms relative to the substrate surface. In this work, we used the functional protein molecules (myosin subfragment 1(S1)) which were radioiodinated with no carrier added 125I to observe the conformational changes in aqueous solutions.

This paper describes a novel surface processing technique aimed at the chemical fixation of proteins on substrate surfaces. The essential feature of this newly developed technique is a combination of chemical and enzymatic processes. A simple technique using chemical modification and selective enzymatic digestion for immobilizing biomembrane-embedded proteins on inorganic solid bases, while strictly regulating their vectorial orientation, was developed. These processes are applicable to a wide range of membrane-embedded and individual proteins, because they exploit the most fundamental principle of proteins, that any protein has at least one N-terminus and one C-terminus. After thorough protection of the carboxyl and amino groups on the molecular surface of bacteriorhodopsin (bR) embedded in purple membrane (PM), in which the bR molecules are uniformly oriented, the derivatized bR was subjected to successive enzymatic digestions to regenerate the unique N-terminal amino group on the molecular surface. The derivatized bR was anchored on an inorganic base by the regenerated amino groups and formed an oriented layered structure. This was proved by analyzing the distance from the base to the gold clusters marking the enzymatically exposed C-termini, which was 4.4 nm, as measured by the fluorescent X-ray interference pattern. This thickness coincides well with that of native PM with embedded bR (4 nm). From the viewpoint of its simplicity, this immobilization process might have an advantage over the former multistep processes for surface functionalization and those for the regulation of the molecular orientation of proteins.

The acoustic radiation force on micrometer-sized polystyrene spheres was measured through observation of the sphere movement in a 500 kHz ultrasonic standing wave. The known spatial distribution of the force allowed verification of the correlation between the sphere velocity and the force. It was found that the linear dependency of the force on the cube of the sphere radius, as predicted by Yosioka, began to fail when the sphere radius was below 5 mu m. This can be accounted for by the presence of a shell layer surrounding the sphere, which increased the effective radius of the sphere. This may point to applications of the acoustic radiation force in the handling of moicrospheres smaller than hitherto thought possible. (C) 1997 American Institute of Physics.

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine distances between probes attached to the most reactive sulfhydryl (SH1) group on individual myosin ''heads.'' We measured intramolecular and intermolecular interhead distances as well as the distance between one head of heavy meromyosin (HMM) mixed with subfragment-1 (S1) heads attached to F-actin under rigor conditions. The SH1 cysteine was specifically labeled with either a donor (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid) or an acceptor probe (5-iodoacetamidofluorescein). In free solution, the distance between these probes was too large to allow significant FRET, but in the rigor complex with F-actin, intermolecular interhead distances between S1 molecules, HMM molecules, or S1 and HMM were determined to be 6.0-6.3 nm, The radial coordinate of the labels relative to F-actin was 5.0-6.4 nm. However, the intramolecular interhead distance in HMMs in which the two heads were labeled with D and A probes was estimated to be larger. The binding affinity of the second head of HMM(D/A) to F-actin may be reduced because of heterogeneous modification of the SH1 groups, such that the probability of single-head binding is increased.

We investigated the potential damage inflicted on erythrocytes by acoustic radiation force when the cells are concentrated by a 500-kHz ultrasonic standing wave at the pressure node, The extent of the damage was estimated from the concentrations of potassium ions, iron complexes, and lactate dehydrogenase released from the cells, After 2 min of ultrasound irradiation at 12.8 mJ/m(3), the cells concentrated on the pressure node, with a cell distribution half-width of 138 mu m: no significant release of intracellular components was detected, even after 15 min of irradiation, The results indicate that even small ions like potassium are not released as a result of ultrasound irradiation on cell membranes without cavitation, and they demonstrate the potential use of acoustic radiation force for concentrating living cells in biomedical applications. (C) 1997 Acoustical Society of America.

A method for concentrating cells in blood using an acoustic radiation force generated by superposition of the higher harmonics on fundamental ultrasound has been investigated. From the theoretical estimation, the efficiency of concentration of blood cells in a chamber based on a rectangular acoustic radiation force slope calculated by integration of the higher harmonics of ultrasound was 17% better than that based on a sine wave, while the peak pressure was reduced to 32% of that in the sine wave case. Concentration of blood cells was also performed experimentally using superposition of higher harmonics on a 500 kHz sine wave. At a 1.2 ml/min fluid flow rate, the blood cells were concentrated on the pressure node within 9 s, with a cell distribution half-width of 348 mu m (i.e., 76% of blood cells concentrated within 23% of the chamber width), which is 5% narrower than that of the sine wave. Though the experimental result was insufficient for the realization of the theoretical estimation because of the incomplete synthesis of the desired waveform, these results suggest the potential usefulness of this superposition method for improving the concentration efficiency with maintenance of the width of the potential slope and reduction of peak pressure.

We have characterized the functional protein, myosin subfragment 1 (S1), attached to a gold substrate by the sulfhydryl groups of cysteine in proteins. The amino groups of the regulatory light chain (RLC) isolated from myosin were labeled with a radioisotope (I-125), and the labeled RLC was incorporated into S1 from which the RLC had been removed. The radiation from I-125 showed that S1 molecules had attached to the gold and, through the interference effect of the monochromatic radiation from I-125, provided information about the position of labeled RLC sites in the S1 monolayer. The interference fringes showed that the RLC was located close to the gold surface and that all of the adsorbed S1 molecules had the same orientation. We confirmed that the motor function of S1 on the gold surface is maintained by observing sliding movement at low ionic strength and by observing the detachment at high ionic strength of fluorescent actin filaments in the presence of ATP. We also found that the adsorbed S1 molecules were not removed from the Au surface by a reducing agent. Thus the Au-S bond is more stable than the S-S bond.

A method for noncontact continuous concentration of biological materials such as erythrocytes could be incorporated in a fully automated analysis system providing contamination-free, real-time measurement. In this paper, I investigated the extent of possible damage inflicted on erythrocytes by acoustic radiation force when the cells are concentrated by a 5O0 kHz ultrasonic standing wave. After 2min of ultrasound irradiation at 12.8 mJ/m^3, the cells concentrated at the pressure node with a cell distribution halfwidth of 138 μm (i.e.,76% of erythrocytes were concentrated within 9.2% of the chamber width); no significant release of erythrocyte components was detected, even after 15 min irradiation. The results indicate that in the absence of cavitation even small ions like potassium are not released as a result of ultrasound irradiation, and they demonstrate the potential use of ultrasound for concentrating and separating living cells.

The muscle contractile apparatus has a highly ordered liquid crystalline structure. The molecular mechanism underlying the formation of this apparatus remains, however, to be elucidated. Selective removal and reconstitution of the components are useful means of examining this mechanism. In addition, this approach is a powerful technique for examining the structure and function of a specific component of the contractile system. In this study we have achieved the structural and functional reconstitution of thin filaments in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with gelsolin. Under these conditions no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached 135 +/- 64% of the original level. The augmentation of tension was attributable to the elongation of reconstituted filaments. As another possibility for augmented tension generation, we suggest the presence of an inhibitory system that was not reconstituted. In any case, the thin filaments of the cardiac contractile apparatus are considered to be assembled so as not to develop the highest degree of tension. Incorporation of the tropomyosin-troponin complex fully restored Ca2+ sensitivity without affecting maximum tension. The present results indicate that a muscle contractile apparatus with a higher order structure and function can be constructed by the self-assembly of constituent proteins.

The structural change of a 111In-labelled azobenzene derivative was examined using the interference effect of electron-capture X-rays emitted by nuclear transformations in radioactive atoms. Interference fringes were generated between the direct monochromatic emission from the radioactive atoms and the emission totally reflected by the substrate surface. The site of a radioactive atom can be determined by analysing the measured interference fringes, because the period of these fringes depends on the position of the radioactive atoms relative to the substrate surface.

A method for separating particles in liquid by exploiting the competition between acoustic radiation force and electrostatic force has been investigated. To elucidate the size and charge dependence of the shift of the particles away from the sound pressure node, the effective charges of polystyrene spheres and alumina particles, which have opposite signs of electric charge on the surface, were measured for particles of various diameters. The shift of the particles from the pressure node due to the electrostatic force was also measured. Polystyrene spheres smaller than 10 mu m in diameter showed same mobility in a 1 Hz, 13.4V(pp)/mm alternating electric field, which indicates that the effective charge of the particle is directly proportional to the radius of the particle. Using 500 kHz ultrasound and a 0.5 V/mm electric field, 7 mu m polystyrene spheres and 10 mu m alumina particles were shifted in opposite directions, according to the sign of the effective charge on the particles.

An isotonic control system for studying dynamic properties of single myofibrils was developed to evaluate the change of sarcomere lengths iii glycerinated skeletal myofibrils under conditions of spontaneous oscillatory contraction (SPOC) in the presence of inorganic phosphate and a high ADP-to-ATP ratio, Sarcomere length oscillated spontaneously with a beak-to-peak amplitude of about 0.5 mu m under isotonic conditions in which the external loads were maintained constant at values between 1.5 x 10(4) and 3.5 x 10(4) N/m(2). The shortening and yielding of sarcomeres occurred in concert, in contrast to the previously reported conditions (isometric or auxotonic) under which the myofibrillar tension is allowed to oscillate, This synchronous SPOC appears to be at a higher level of synchrony than in the organized state of SPOC previously observed under auxotonic conditions, The period of sarcomere length oscillation did not largely depend on external load. The active tension under SPOC conditions increased as the sarcomere length increased fi-om 2.1 to 3.2 mu m, although it was still smaller than the tension under normal Ca2+ contraction (which is on the order of 10(5) N/m(2)). The synchronous SPOC implies that there is a mechanism fbr transmitting information between sarcomeres such that the state of activation of sarcomeres is affected by the state of adjacent sarcomeres. We conclude that the change of myofibrillar tension is not responsible for the SPOC of each sarcomere but that it affects the level of synchrony of sarcomere oscillations.

A method for separating particles in liquid by exploiting the competition between acoustic radiation force and electrostatic force is investigated. The displacement of particles from the pressure node of an ultrasound standing wave varies according to the effective charges, radii, and stiffness of the particles, and the particles of different radii can thus be separated even when they consist of the same material. When the particles are of different materials, they can also be separated in relations to their effective electric charges or their stiffness. In this study, the efficacy of this method was theoretically estimated and demonstrated by an experiment, with a mixture of polystyrene spheres of different radii in water, using 500-kHz ultrasound and a 3.3-V/mm electric field. The measured ratio of the displacements of polystyrene spheres 10 and 20 mu m in diameter from the pressure node was 2:3, which is consistent with the value predicted theoretically. Radiation forces on the polystyrene spheres were also measured by using this technique. (C) 1996 Acoustical Society of America.

A deoxyribonucleic acid (DNA) concentration method using acoustic radiation force is proposed. Ethanol was added to an aqueous DNA sample in order to initiate clustering of the DNA molecules of 2686 bps. The change in the distribution of the DNA clusters induced by a standing wave ultrasound irradiation was observed with an optical microscope. The observable DNA clusters gathered on the pressure node in the ultrasonic standing wave field and formed larger clusters that did not break apart after the irradiation was stopped. Without ultrasound irradiation, no significant change in the cluster distribution was observed. This method may have a potential use for concentrating and collecting biomaterials such as nucleic acids and proteins. (C) 1996 Acoustical Society of America.

We have developed a method of measuring the isometric tension in glycerinated stalks of Vorticella convallaria. Using this method, we measured tension vs. pCa relations in glycerinated V. convallaria stalks. The maximum isometric tension was 4 x 10(-8) N on average. The Hill's parameter, n, which is the number of calcium ions bound simultaneously and cooperatively to a contractile element (a force generating element), is approximately 3.2 when the Ca2+ concentration is increased and 2.5 when it is decreased. In order to estimate the efficiency of the energy conversion of Ca2+ binding to mechanical work, we measured the Ca2+ induced Carnot cycle in the Vorticella stalk. The energy efficiency was tentatively estimated to be about 7%. With this method, we have also succeeded in measuring the isometric tension of isolated spasmoneme, the rubber-like contractile fibrous organelle in the stalk. The maximum tension of spasmoneme was approximately one tenth that of the glycerinated stalk. We speculate that the isolated spasmoneme was only partially functional due to damage sustained when it was pulled out Of the stalk. (C) 1996 Wiley-Liss, Inc.

The efficacy of the ultrasonic standing plane wave in concentrating small particles was theoretically evaluated and compared with experimental results. Acoustic energy density was estimated by measuring the ultrasonic absorption, and particle distribution was observed by dark-field microscopy. The theory predicts that diffusion is negligible in concentrating polystyrene spheres larger than 5 mu m in diameter when they are subjected to 2J/m(3) ultrasound. The half-width of the steady-state particle distribution was of the same order of magnitude as the theoretical value for the particles of 1 mu m and 2 mu m diameter. We also applied this concentrating technique to fractionation of polystyrene spheres 10 mu m in diameter, and more than 90% of the particles in the laminar flow were successfully collected.

The elastic properties of nebulin were studied by measuring the elasticity of single skeletal myofibrils, from which the portion of the thin filament located at the I band had been selectively removed by treatment with plasma gelsolin under rigor conditions. In this myofibril model, a portion of each nebulin molecule at the I band was expected to be free of actin filaments and exposed. The length of the exposed portion of the nebulin molecule was controlled by performing the gelsolin treatment at various sarcomere lengths. The relation between the passive tension and extension of the exposed portion of the nebulin showed a convex curve starting from a slack length, apparently in a fashion similar to that of wool. The slack sarcomere length shifted depending on the length of the exposed portion of the nebulin, however, the relation being represented by a single master curve. The elastic modulus of nebulin was estimated to be two to three orders of magnitude smaller than that of an actin filament. Based on these results, we conclude that nebulin attaches to an actin filament in a side-by-side fashion and that it does not significantly contribute to the elastic modulus of thin filaments. The relation between the passive tension and extension of connectin (titin) was obtained for a myofibril from which thin filaments had been completely removed with gelsolin under contracting conditions; this showed a concave curve, consistent with the previous results obtained in single fibers.

: Recent work has suggested that the length of thin filaments in skeletal muscles is determined by a protein ruler, nebulin, located along the long axis of thin filaments. To examine the function of nebulin, the length distribution of the thin filaments was investigated by staining actin filaments with fluorescent rhodamine-phalloidin in rabbit cardiac muscles, which do not contain nebulin, and in skeletal muscles, which contain nebulin, by laser scanning confocal microscopy. The microscopic observation showed a difference in staining patterns between cardiac and skeletal muscle fibers when the staining was done without chemical fixation. This suggests that nebulin suppresses the attachment of phalloidin to actin filaments. Analysis of fluorescence distribution showed that the length deviation of thin filaments in the cardiac muscle was as small as that in the skeletal muscle. This indicates that the length of the thin filaments is regulated even without nebulin. © 1994, The Japan Academy. All rights reserved.

We have devised a simple method for measuring tension development of single myofibrils by micromanipulation with a pair of glass micro-needles. The tension was estimated from the deflection of a flexible needle under an inverted phase-contrast microscope equipped with an image processor, so that the tension development is always accompanied by the shortening of the myofibril (auxotonic condition) in the present setup. The advantage of this method is that the measurement of tension (1/30 s for time resolution and about 0.05-mu-g for accuracy of tension measurement; 0.05-mu-m as a spatial resolution for displacement of the micro-needle) and the observation of sarcomere structure are possible at the same time, and the technique to hold myofibrils, even single myofibrils, is very simple. This method has been applied to study the tension development of glycerinated skeletal myofibrils under the condition where spontaneous oscillation of sarcomeres is induced, i.e., the coexistence of MgATP, MgADP and inorganic phosphate without free Ca2+. Under this condition, we found that the tension of myofibrils spontaneously oscillates accompanied by the oscillation of sarcomere length with a main period of a few seconds; the period was lengthened and shortened with stretch and release of myofibrils. A possible mechanism of the oscillation is discussed.

A new heterogeneous "sandwich" immunoassay utilizing microparticles as labels to realize high sensitivity is described. In this method, antibody fixed on the microparticles reacts with antigen previously trapped on a microplate surface, which makes the antigen molecules visible and countable with an inverted optical microscope. The method is highly sensitive because the reacted single microparticle, therefore single antigen molecule, can be detected. The sensitivity depends both on the reaction efficiency of the immunoreaction and on nonspecific adsorption of the microparticles on the microplate surface. Therefore, the protocol for preparing microparticle having antibody on the surface and a microplate having capture antibody was investigated to realize high sensitivity. Carboxylated microparticles of 0.76 μm in diameter were conjugated with affinity-purified antibody using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. It was determined that 1 g microparticles had 880 μg antibody (approximately 1100 antibody molecules per 1 microparticle). The immunoreaction efficiency reached 18% at 1 × 10-13 mol/liter antigen concentration. The lower detection limit was 3.1 × 10-14 mol/liter (1.6 amol) using human α-fetoprotien as a model antigen. © 1992.