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 μm spatial resolution by a phase-contrast/fluorescent microscopy system with digital image processing. The core of the system is an n × n (n = 20–50) array of chambers, where each is 20–70 μm in diameter and 5–30 μm 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 μm min⁻¹ between cell divisions regardless of the chamber volume, and that the cell concentration reached 10¹² 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.

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 µ m in diameter when they are subjected to 2 J/m³ 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 µ m and 2 µ m diameter. We also applied this concentrating technique to fractionation of polystyrene spheres 10 µ m in diameter, and more than 90% of the particles in the laminar flow were successfully collected.

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.

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.