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 microm 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 microm in diameter and 5-30 microm 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 (<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 microm 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 new method for SNP analysis based on the detection of pyrophosphate (PPi) is demonstrated, which is capable of detecting small allele frequency differences between two DNA pools for genetic association studies other than SNP typing. The method is based on specific primer extension reactions coupled with PPi detection. As the specificity of the primer-directed extension is not enough for quantitative SNP analysis, artificial mismatched bases are introduced into the 3′-terminal regions of the specific primers as a way of improving the switching characteristics of the primer extension reactions. The best position in the primer for such artificial mismatched bases is the third position from the primer 3′-terminus. Contamination with endogenous PPi, which produces a large background signal level in SNP analysis, was removed using PPase to degrade the PPi during the sample preparation process. It is possible to accurately and quantitatively analyze SNPs using a set of primers that correspond to the wild-type and mutant DNA segments. The termini of these primers are at the mutation positions. Various types of SNPs were successfully analyzed. It was possible to very accurately determine SNPs with frequencies as low 0.02. It is very reproducible and the allele frequency difference can be determined. It is accurate enough to detect meaningful genetic differences among pooled DNA samples. The method is sensitive enough to detect 14 amol ssM13 DNA. The proposed method seems very promising in terms of realizing a cost-effective, large-scale human genetic testing system.

guidance of neuronal processes (neurites) is demonstrated by applying wet femtosecond-laser processing to an organosilane self-assembled monolayer (SAM) template. By scanning focused laser beam between cell adhesion sites, on which primary neurons adhered and extended their neurites, we succeeded in guiding the neurites along the laser-scanning line. This guidance was accomplished by multiphoton laser ablation of cytophobic SAM layer and subsequent adsorption of cell adhesion molecule, laminin, onto the ablated region. This technique allows us to arbitrarily design neuronal networks .

We demonstrate a simple and rapid method for SNP typing, allele frequency determination, and trace mutant analysis that works with even an inexpensive detection system. This method is based on microchip electrophoresis of tagged probes incorporated with one-colored ddNTP (METPOC). The assay uses dye terminator incorporation into a pair of probes of different lengths specific to wild- and mutant-type targets, respectively. They are hybridized to the targets prior to ddNTP-Cy-5 incorporation, which occurs only for a matched probe-target duplex. Because the extension reactions for the two probes are carried out simultaneously in one tube and the products from both probes are analyzed in one channel by one-color fluorescence detection, an accurate comparative analysis of SNPs is possible. SNP typing as well as allele frequency determination in the range above 0.1% can easily be carried out using a commercial microchip electrophoresis system in a few minutes.

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This article describes a novel laser-directed microfabrication method carried out in aqueous solution for the organization of cell networks on a platform. A femtosecond (fs) laser was applied to a platform culturing PC12, HeLa, or normal human astrocyte (NHA) cells to manipulate them and to facilitate mutual connections. By applying an fs-laser-induced impulsive force, cells were detached from their original location on the plate, and translocated onto microfabricated cell-adhesive domains that were surrounded with a cell-repellent perfluoroalkyl (R(f)) polymer. Then the fs-laser pulse-train was applied to the R(f) polymer surface to modify the cell-repellent surface, and to make cell-adhesive channels of several μm in width between each cell-adhesive domain. PC12 cells elongated along the channels and made contact with others cells. HeLa and NHA cells also migrated along the channels and connected to the other cells. Surface analysis by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) confirmed that the R(f) polymer was partially decomposed. The method presented here could contribute not only to the study of developing networks of neuronal, glial, and capillary cells, but also to the quantitative analysis of nerve function.

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We demonstrate an on-chip microparticle sorter with an ultrashort switching window using femtosecond laser pulses to overcome the fundamental limitation of the sorting performance described by Poisson statistics.